methods for detecting the presence of a nucleic acid analyte and a species of candida in a liquid sa

专利摘要:
METHODS FOR THE QUICK DETECTION OF ANALYTICS, SYSTEM AND REMOVABLE CARTRIDGE. The present invention relates to systems and methods for detecting analytes. Specifically, the present invention relates to methods for detecting the presence of an analyte in a sample, for example, a liquid sample, for detecting the presence of a pathogen, or a virus, or even a target nucleic acid in a whole blood sample, and also to detect the presence of a Candida species in a liquid sample. The present invention also relates to a system for detecting one or more analytes, as well as a removable cartridge sized to facilitate insertion into and removal of a system of the invention.
公开号:BR112013010952B1
申请号:R112013010952-1
申请日:2011-10-19
公开日:2020-08-25
发明作者:Tomas Jay Lowery；Mark John Audeh；Matthew Blanco；James Franklin Chepin；Vasiliki Demas；Rahul Dhand；Marilyn Lee Fritzemeier；Isaac Koh；Sonia Kumar；Lori Anne Neely；Brian Mozeleski；Daniella Lynn Plourde；Charles William Ritter-Shaus；Parris Wellman
申请人:T2 Biosystems, Inc.；
IPC主号:

专利说明:

[0001] [0001] This invention features tests and devices for the detection of analytes, and their use in the treatment and diagnosis of disease.
[0002] [0002] Magnetic sensors have been designed to detect molecular interactions in a variety of media, including biofluids, food products, and soil samples, among other media. Under target binding, these sensors cause changes in the properties of neighboring water molecules (or any solvent molecules with free hydrogens) in a sample, which can be detected by magnetic resonance (NMR / MRI) techniques. Thus, using these sensors, in a liquid sample, it is possible to detect the presence, and potentially quantify the amount, of an analyte (small molecules, DNA, RNA, proteins, carbohydrates, organisms, metabolites, and pathogens (for example, viruses )) at very low concentration using magnetic sensors.
[0003] [0003] In general, magnetic sensors are magnetic particles that bind or otherwise stick to their intended molecular target to form clusters (aggregates). It is believed that when magnetic particles assemble in clusters and the effective cross-sectional area becomes larger (and the density of the number of clusters is less), interactions with water or other solvent molecules are altered, leading to a change in measured relaxation rates (for example, T2, T1, T2 *), susceptibility, frequency of precession, among other physical changes. In addition, the formation of agglomerate can be designated to be reversible (for example, by temperature change, chemical cleavage, pH change, etc.) so that "sensible" or "antisense" (competitive and inhibition) assays can be developed to the detection of specific analytes. The sense (agglomeration) and antisense (de-agglomeration) types of assays can be used to detect a wide variety of biologically relevant materials. The MRS (magnetic resonance imaging) phenomenon has been previously described (see, United States Patent Publication No. 20090029392).
[0004] [0004] Many diagnostic tests require sensitivity in the picomolar or subpicomolar range. Current detection of infectious agents, nucleic acids, small molecules, biological warfare agents, and molecular targets (biomarkers) or the combination of molecular targets and immunoassay targets generally requires early sample preparation, time to analyze the sample, and single tests for each of the individual analyzed. There is a need for a commercially achievable, fast NMR-based analyte detection device suitable for use with magnetic nanosensors having four unique qualities and characteristics: 1) little to no sample preparation, 2) multiple detection across multiple molecular types, 3) rapid acquisition of diagnostic information, and 4) accurate information for clinical decision-making at the point of care. Summary of the Invention
[0005] [0005] The invention features systems and methods for detecting analytes.
[0006] [0006] The invention features a method for detecting the presence of an analyte in a liquid sample, the method including: (a) contacting a solution with magnetic particles to produce a liquid sample of 1 × 106 to 1 × 1013 of magnetic particles per milliliter of the liquid sample (for example, 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 699 nm (for example, from 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or from 500 to 699 nm), a T2 relaxation per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or from 1 × 1010 to 1 × 1012 mM-1s-1), and bonding fractions on its surface, the bonding fractions operative to alter particle aggregation magnetic in the presence of the analyte or a multivalent binding agent; (b) placing the liquid sample in a device, the device including a holder defining a cavity holding the liquid sample including the magnetic particles, the multivalent binding agent, and the analyzed, and having an RF spiral arranged over the cavity, the spiral RF configured to detect a signal produced by exposing the liquid sample to a magnetic polarization field created using one or more magnets and an RF pulse sequence; (c) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (d) following step (c), measure the signal; and (e) based on the result of step (d), detect the analyzed. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more than one or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. For example, the analyte can be creatinine, a liquid sample can include a multivalent binding agent containing a plurality of creatinine conjugates, and the magnetic particles can include a surface decorated with creatinine antibodies. In another embodiment, the analyte may be tacrolimus, a liquid sample may include a multivalent binding agent containing a plurality of tacrolimus conjugates, and the magnetic particles may include a surface decorated with tacrolimus antibodies. In particular modalities of the method, step (d) includes measuring the T2 relaxation response of the liquid sample, and where the increase in agglomeration in the liquid sample produces an increase in the T2 relaxation rate observed in the sample. In certain embodiments, the analyzed is a target nucleic acid (for example, a target nucleic acid extracted from a leukocyte, or a pathogen).
[0007] [0007] The invention features a method for detecting the presence of an analyte in a liquid sample, the method including (a) contacting a solution with magnetic particles to produce a liquid sample including from 1 × 106 to 1 × 1013 magnetic particles per milliliter of the liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 * 1011, or 1 * 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter from 700 nm to 1200 nm (for example, from 700 to 850, 800 to 950, 900 to 1050, or from 1000 to 1200 nm) , a relaxation of T2 per particle of 1 * 109 to 1 * 1012 mM-1s-1 (for example, from 1 * 109 to 1 * 1010, 1 * 109 to 1 * 1011, or from 1 * 1010 to 1 * 1012 mM-1s-1), and have fractions of bond on their surface, the fractions of bond operative to alter an aggregation of magnetic particles in the presence of the analyzed; (b) placing the liquid sample in a device, the device including a holder defining a cavity holding the liquid sample including the magnetic particles, the multivalent binding agent, and the analyzed, and having an RF spiral arranged over the cavity, the spiral RF configured to detect a signal produced by exposing the liquid sample to a magnetic polarization field created using one or more magnets and an RF pulse sequence; (c) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (d) following step (c), measuring the signal; and (e) based on the result of step (d), detecting the presence or concentration of an analysand. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, a liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. For example, the analyte can be creatinine, a liquid sample can include a multivalent binding agent containing a plurality of creatinine conjugates, and the magnetic particles can include a surface decorated with creatinine antibodies. In another embodiment, the analyte may be tacrolimus, the liquid sample may include a multivalent binding agent containing a plurality of tacrolimus conjugates, and the magnetic particles may include a surface decorated with tacrolimus antibodies. In particular modalities of the method, step (d) includes measuring the T2 relaxation response of the liquid sample, and where the increase in agglomeration in the liquid sample produces an increase in the T2 relaxation rate observed in the sample. In certain embodiments, the analyzed is a target nucleic acid (for example, a target nucleic acid extracted from a leukocyte, or a pathogen).
[0008] [0008] The invention also features a method for detecting the presence of a pathogen in a whole blood sample, the method including: (a) providing an individual's whole blood sample; (b) mixing the whole blood sample with an erythrocyte lysis agent solution to produce the disrupted red blood cells; (c) following step (b), centrifuge the sample to form a supernatant and pellet, discard some or all of the supernatant, and resuspend the pellet to form an extract, optionally wash the pellet (for example, with buffer TE) before resuspending the pellet and optionally repeating step (c); (d) lyse the extract cells to form a lysate; (e) placing the lysate from step (d) in a detection tube and amplifying a target nucleic acid in the lysate to form an amplified lysate solution including the target nucleic acid, where the target nucleic acid is characteristic of the pathogen to be detected; (f) after step (e), add the detection tube of 1 × 106 to 1 × 1013 magnetic particles per milliliter of the amplified lysate solution (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 700 nm to 1200 nm (for example, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), and bonding portions on their surface, the bonding fractions operative to alter the aggregation of magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (g) placing the detection tube in a device, the device including a holder defining a cavity to hold the detection tube including the magnetic particles and the target nucleic acid, and having an RF spiral arranged over the cavity, the configured RF spiral to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (h) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (i) after step (h), measure the signal from the detection tube; and (j) based on the result of step (i), detect the pathogen. In certain embodiments, steps (a) through (i) are completed in 4 hours (for example, in 3.5 hours, 3.0 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour) . In another embodiment, step (i) is carried out without any prior purification of the amplified lysate solution (i.e., the lysate solution is unfractionated after being formed). In particular embodiments, step c includes washing the sediment before resuspending the sediment to form the extract. In particular embodiments, step (d) includes combining the extract with beads to form a mixture and stirring the mixture to form a lysate. The magnetic particles can include one or more populations having a first probe and a second probe conjugated to their surface, the first probe operating to bind to a first segment of the target nucleic acid and the second probe operating to bind to a second segment of the target nucleic acid, where magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay can be a disintegration assay in which the magnetic particles include a first population having a first binding portion on its surface and a second population having a second binding portion on its surface, and the multivalent binding portion including a first probe and second probe, first operating probe to attach to the first binding portion and second operating probe to attach to a second binding portion, the binding portions and multivalent binding portion operative to alter an aggregation of the particles magnetic fields in the presence of the target nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the lysate also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7% , 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% of nonionic surfactant ( for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0 , 4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In yet other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The lysate can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold.
[0009] [0009] The invention features a method for detecting the presence of a target nucleic acid in a whole blood sample, the method including: (a) providing one or more cells from an individual's whole blood sample; (b) lyse the cells to form a lysate; (c) amplifying a target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid; (d) following step (c), add the amplified lysate solution and 1 × 106 to 1 × 1013 magnetic particles per milliliter of the amplified lysate solution to a detection tube, where the magnetic particles have an average diameter of 700 nm to 1200 nm and binding portions on its surface, the binding portions operative to alter the aggregation of magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (e) placing the detection tube in a device, the device including a holder defining a cavity to hold the detection tube including the magnetic particles and the target nucleic acid, and having an RF spiral arranged over the cavity, the configured RF spiral to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (h) following step (f), measure the signal from the detection tube; and (i) based on the result of step (h), detecting the target nucleic acid. In particular embodiments, the target nucleic acid is purified before step (d). In particular embodiments, step (b) includes combining the extract with the beads to form a mixture and stirring the mixture to form a lysate. The magnetic particles can include one or more populations having a first probe and a second probe conjugated to their surface, the first probe operating to bind to a first segment of the target nucleic acid and the second probe operating to bind to a second segment of the target nucleic acid, where magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay can be a disintegration assay in which the magnetic particles include a first population having a first binding portion on its surface and a second population having a second binding portion on its surface, and the multivalent binding portion including a first probe and second probe, first operating probe to attach to the first binding portion and second operating probe to attach to a second binding portion, the binding portions and multivalent binding portion operative to alter an aggregation of the particles magnetic fields in the presence of the target nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the lysate also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7% , 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% of nonionic surfactant ( for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0 , 4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In yet other embodiments, the magnetic particles optionally include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) one or more proteins per milligram of the magnetic particles. The lysate can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold.
[0010] [00010] The invention also features a method for detecting the presence of a target nucleic acid in a whole blood sample, the method including: (a) providing an extract produced by lysis of red blood cells in an individual's whole blood sample, centrifuge the sample to form a supernatant and pellet, discard some or all of the supernatant, and resuspend the pellet to form an extract, optionally wash the pellet (eg with TE buffer) before resuspending the pellet and optionally repeat the centrifuging, discarding, and washing step (a); (b) lyse the cells in the extract to form a lysate; (c) placing the lysate from step (b) in a detection tube and amplifying the nucleic acids in it to form an amplified lysate solution including from 40% (weight / weight) to 95% (weight / weight) of the target nucleic acid (for example, from 40 to 60%, from 60 to 80%, or from 85 to 95% (weight / weight) of target nucleic acid) and from 5% (weight / weight) to 60% (weight / weight) of non-target nucleic acid (for example, from 5 to 20%, from 20 to 40%, or from 40 to 60% (weight / weight) of non-target nucleic acid); (d) after step (c), add 1 × 106 to 1 × 1013 magnetic particles per milliliter of the amplified lysate solution to the detection tube, where the magnetic particles have an average diameter of 700 nm to 1200 nm and portions of binding on its surface, the binding portions operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (e) placing the detection tube in a device, the device including a holder defining a cavity to hold the detection tube including the magnetic particles and the target nucleic acid, and having an RF spiral arranged over the cavity, the configured RF spiral to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (g) following step (f), measure the signal from the detection tube; and (h) based on the result of step (g), detecting the target nucleic acid, where step (g) is carried out without any previous purification of the amplified lysate solution. In particular embodiments, step (b) includes combining the extract with beads to form a mixture and stirring the mixture to form a lysate. The magnetic particles can include one or more populations having a first probe and a second probe conjugated to their surface, the first probe operating to bind to a first segment of the target nucleic acid and the second probe operating to bind to a second segment of the target nucleic acid, where magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay can be a disintegration assay in which the magnetic particles include the first population having a first binding portion on its surface and a second population having a second binding portion on its surface, and the multivalent binding portion including a first probe and second probe, first operating probe to attach to the first binding portion and second operating probe to attach to a second binding portion, the binding portions and multivalent binding portion operative to alter an aggregation of the particles magnetic fields in the presence of the target nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the lysate also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7% , 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% of nonionic surfactant ( for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0 , 4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The lysate can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold.
[0011] [00011] The invention features a method for detecting the presence of a Candida species in a liquid sample, the method including: (a) lysing the Candida cells in the liquid sample; (b) amplifying a nucleic acid to be detected in the presence of a sense primer and an antisense primer, each of which is universal for multiple Candida species to form a solution including a Candida amplicon; (c) contacting the solution with magnetic particles to produce a liquid sample including from 1 × 106 to 1 * 1013 magnetic particles per milliliter of the liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108 , 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 700 nm at 1200 nm (for example, from 700 to 850, 800 to 950, 900 to 1050, or from 1000 to 1200 nm), a relaxation of T2 per particle from 1 * 109 to 1 * 1012 mM-1s-1 (for example , from 1 * 108 to 1 * 109, 1 * 108 to 1 * 1010, 1 * 109 to 1 * 1010, 1 * 109 to 1 * 1011, or from 1 * 1010 to 1 * 1012 mM-1s-1), and binding portions on its surface, the binding portions operative to alter aggregation of the magnetic particles in the presence of Candida amplicon or a multivalent binding agent; (d) placing the liquid sample in a device, the device including a holder defining a cavity to hold the liquid sample including the magnetic particles and Candida's amplicon, and having an RF spiral arranged over the cavity, the RF spiral configured to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (f) after step (e), measure the signal; and (g) based on the result of step (f), determine if the Candida species were present in the sample. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. The sense primer can include, for example, the 5'-GGC ATG CCT GTT TGA GCG TC-3 'sequence (SEQ ID NO. 1). The antisense primer can include, for example, the sequence 5'-GCT TAT TGA TAT GCT TAA GTT CAG CGG GT-3 '(SEQ ID NO. 2). In certain embodiments, (i) the Candida species is Candida albicans, the first probe includes the 5'-ACC CAG CGG TTT GAG GGA GAA AC-3 'oligonucleotide sequence (SEQ ID NO. 3), and the second probe includes the 5'-AAA GTT TGA AGA TAT ACG TGG TGG ACG TTA-3 'oligonucleotide sequence (SEQ ID NO. 4); (ii) the Candida species is Candida krusei and the first probe and the second probe include an oligonucleotide sequence selected from: 5'-CGC ACG CGC AAG ATG GAA ACG-3 '(SEQ ID NO. 5), 5'- AAG TTC AGC GGG TAT TCC TAC CT-3 '(SEQ ID NO. 6), and 5'-AGC TTT TTG TTG TCT CGC AAC ACT CGC-3' (SEQ ID NO. 15); (iii) the Candida species is Candida glabrata, the first probe includes the oligonucleotide sequence: 5'-CTA CCA AAC ACA ATG TGT TTG AGA AG-3 '(SEQ ID NO. 7), and the second probe includes the sequence oligonucleotide: 5'-CCT GAT TTG AGG TCA AAC TTA AAG ACG TCT G-3 '(SEQ ID NO. 8); and (iv) the Candida species is Candida parapsilosis or Candida tropicalis and the first probe and the second probe include an oligonucleotide sequence selected from: 5'-AGT CCT ACC TGA TTT GAG GTCNitIndAA-3 '(SEQ ID NO. 9) , 5'-CCG NitIndGG GTT TGA GGG AGA AAT-3 '(SEQ ID NO. 10), AAA GTT ATG AAATAA ATT GTG GTG GCC ACT AGC (SEQ ID NO. 16), ACC CGG GGGTTT GAG GGA GAA A (SEQ ID NO. 17), AGT CCT ACC TGA TTT GAG GTC GAA (SEQ ID NO. 18), and CCG AGG GTT TGA GGG AGA AAT (SEQ ID NO. 19). In certain embodiments, steps (a) through (h) are completed in 4 hours (for example, in 3.5 hours, 3.0 hours, 2.5 hours, 2 hours, 1.5 hours, or 1 hour or any less). In particular embodiments, the magnetic particles include two populations, a first population containing the first probe on its surface, and the second population containing the second probe on its surface. In another embodiment, the magnetic particles are a single population containing both the first probe and the second probe on the surface of the magnetic particles. The magnetic particles can include one or more populations having a first probe and a second probe conjugated to its surface, the first operative probe to attach to a first segment of Candida's amplicon and a second operative probe to attach to a second segment of the amplicon of Candida, where the magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay can be a disintegration assay in which the magnetic particles include the first population having a first binding portion on its surface and a second population having a second binding portion on its surface, and the multivalent binding portion including a first probe and second probe, first operative probe to attach to the first attachment portion and a second operative probe to attach to a second attachment portion, the attachment portions and multivalent attachment portion operative to alter an aggregation of the particles magnetic fields in the presence of Candida amplicon. In particular modalities, the method can produce (i) a coefficient of variation in the T2 value of less than 20% in positive Candida samples; (ii) at least 95% of correct detection in less than or equal to 5 cells / mL in samples that peaked in 50 blood samples from an individual healthy patient; (iii) at least 95% of correct detection less than or equal to 5 cells / mL in samples that reached the maximum in 50 blood samples from an individual unhealthy patient; and / or (iv) greater than or equal to 80% correct detection in samples from a clinically positive patient (i.e., Candida positive by another technique, such as by cell culture) starting with 2mL of blood.
[0012] [00012] The invention features a method for detecting the presence of a Candida species in a whole blood sample, the method including: (a) providing an extract produced by lysis of red blood cells in an individual's whole blood sample; (b) centrifuge the sample to form a supernatant and sediment, discard some or all of the supernatants; (c) washing the pellet (for example, with TE buffer) by mixing the pellet with a buffer, shaking the sample (for example, by swirling) centrifuge the sample to form a supernatant and pellet, discard some or all of the supernatants; (d) optionally repeating steps (b) and (c); (e) forming a bead by tapping the pellet to form a lysate in the presence of a buffer (e.g., TE buffer); (f) centrifuge the sample to form a supernatant containing the lysate; (g) amplifying the nucleic acids in the lysate of step (f) to form a Candida amplicon; and (h) detect the presence of Candida amplicon, where, the method can produce (i) at least 95% of correct detection in less than or equal to 5 cells / mL in samples that reached the maximum in 50 blood samples healthy individual patient; (ii) at least 95% of correct detection less than or equal to 5 cells / mL in samples that reached the maximum in 50 blood samples from an individual unhealthy patient; and / or (iii) greater than or equal to 80% correct detection in clinically positive patient samples (i.e., Candida positive by cell culture) starting with 2 mL of blood from step (a).
[0013] [00013] The invention features a method for detecting the presence of a pathogen in a whole blood sample, the method including the steps of: (a) providing 0.05 to 4.0 ml of the whole blood sample (for example , from 0.05 to 0.25, 0.25 to 0.5, 0.25 to 0.75, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0, 65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 to 4.0 mL of whole blood); (b) placing an aliquot of the sample from step (a) in a container and amplifying a target nucleic acid in the sample to form an amplified solution including the target nucleic acid, where the target nucleic acid is characteristic of the pathogen to be detected; (c) placing the amplified liquid sample in a detection device; (d) based on the result of step (c), detect the pathogen, where the pathogen is selected from bacteria and fungi, and where the method is capable of detecting a concentration of pathogen of 10 cells / mL (for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cells / ml) in the whole blood sample. The detection device can detect the pathogen through an optical, fluorescent, mass, density, magnetic, chromatographic, and / or electrochemical measurement of the amplified liquid sample. In certain embodiments, steps (a) through (d) are completed in 3 hours (for example, in 3.2; 2.9; 2.7; 2.5; 2.3; 2.2; 2.1 ; 2.0; 1.9; 1.8; 1.7; 1.6; or 1.5 hour or 1 hour). In still other embodiments, step (c) is carried out without any prior purification of the amplified solution, and / or the liquid sample from step (c) includes whole blood proteins and non-target oligonucleotides. In certain embodiments, the pathogen is selected from bacteria and fungi. The pathogen can be any bacterial or fungal pathogen described here.
[0014] [00014] The invention also features a method for detecting the presence of a pathogen in a whole blood sample, the method including the steps of: (a) providing an individual's whole blood sample; (b) mixing 0.05 to 4.0 mL of the whole blood sample (for example, 0.05 to 0.25, 0.25 to 0.5, 0.25 to 0.75, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 to 4.0 mL of whole blood) with an erythrocyte lysis agent solution to produce ruptured red blood cells; (c) following step (b) centrifuge the sample to form a supernatant and pellet, discard some or all of the supernatants, and resuspend the pellet to form an extract, optionally wash the pellet (for example, with TE buffer) before resuspending the sediment and optionally repeating step (c); (d) lyse the extract cells to form a lysate; (e) placing the lysate from step (d) in a container and amplifying a target nucleic acid in the lysate to form an amplified lysate solution including the target nucleic acid, where the target nucleic acid is characteristic of the pathogen to be detected; (f) following step (e), mix the amplified lysate solution with 1 × 106 to 1 × 1013 magnetic particles per milliliter of the amplified lysate solution to form a liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 1200 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), a relaxation of T2 per particle from 1 × 108 to 1x1012 mM-1s-1 (for example, from 1x108 to 1x109, 1x108 to 1x1010, 1x109 to 1x1010, 1x109 to 1x1011, or 1x1010 to 1x1012 mM-1s-1), and the binding portions on their surface, the binding portions operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (g) placing the liquid sample in a device, the device including a holder defining a cavity to hold the detection tube including the magnetic particles and the target nucleic acid, and having an RF spiral arranged over the cavity, the RF spiral configured for detecting a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (h) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (i) after step (h), measure the signal of the liquid sample; and (j) based on the result of step (i), detect the pathogen, where the pathogen is selected from bacteria and fungi, and where the method is able to detect a concentration of pathogen of 10 cells / mL (for example, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 cells / ml) in the whole blood sample. In certain embodiments, steps (a) to (i) are completed in 3 hours (for example, in 3.2; 2.9; 2.7; 2.5; 2.3; 2.2; 2.1 ; 2.0; 1.9; 1.8; 1.7; 1.6; 1.5; or 1 or less hours). In still other embodiments, step (i) is carried out without any prior purification of the amplified lysate solution, and / or the liquid sample from step (i) includes whole blood proteins and non-target oligonucleotides. In certain embodiments, the pathogen is selected from bacteria and fungi. The pathogen can be any bacterial or fungal pathogen described here. In particular modalities the method is capable of measuring a pathogen concentration of 10 cells / mL in the whole blood sample with a coefficient of variation of less than 15% (for example, 10 cells / mL with a coefficient of variation of less than than 15%, 10%, 7.5%, or 5%; or 25 cells / mL with a coefficient of variation of less than 15%, 10%, 7.5%, or 5%; or 50 cells / mL with a coefficient of variation of less than 15%, 10%, 7.5%, or 5%; or 100 cells / mL with a coefficient of variation of less than 15%, 10%, 7.5%, or 5%). In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 μg to 100 μg (for example, 40 μg to 60 μg, 50 μg to 70 μg, 60 μg to 80 μg, or 80 μg to 100 μg) of one or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. The monitoring method can include any of the magnetic assisted agglomeration methods described here. The magnetic particles can include one or more populations having a first probe and a second probe conjugated to its surface, the first probe operating to bind to a first segment of the target nucleic acid and a second probe operating to bind to a second segment of the target nucleic acid, where magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay can be a disintegration assay in which the magnetic particles include the first population having a first binding portion on its surface and a second population having a second binding portion on its surface, and the multivalent binding portion including a first probe and second probe, first operative probe to attach to the first attachment portion and a second operative probe to attach to a second attachment portion, the attachment portions and multivalent attachment portion operative to alter an aggregation of the particles magnetic fields in the presence of the target nucleic acid.
[0015] [00015] The invention also features a method for detecting the presence of a virus in a whole blood sample, the method including the steps of: (a) providing a plasma sample from an individual; (b) mixing 0.05 to 4.0 mL of the plasma sample (for example, 0.05 to 0.25, 0.25 to 0.5, 0.25 to 0.75, 0.4 to 0.8, 0.5 to 0.75, 0.6 to 0.9, 0.65 to 1.25, 1.25 to 2.5, 2.5 to 3.5, or 3.0 to 4 , 0 ml of whole blood) with a lysis agent to produce a mixture comprising the disrupted viruses; (c) placing the mixture from step (b) in a container and amplifying a target nucleic acid in the filtrate to form an amplified filtered solution including the target nucleic acid, where the target nucleic acid is characteristic of the virus to be detected; (d) following step (c), mix the amplified filtered solution with 1 × 106 to 1 * 1013 magnetic particles per milliliter of the amplified filtered solution to form a liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 11013 magnetic particles per milliliter), where the magnetic particles have a average diameter from 150 nm to 1200 nm (e.g. 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and bonding portions on its surface, the bonding portions operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (e) placing the liquid sample in a device, the device including a holder defining a cavity to hold the detection tube including the magnetic particles and the target nucleic acid, and having an RF spiral arranged over the cavity, the RF spiral configured for detecting a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing the liquid sample to a polarizing magnetic field and an RF pulse sequence; (g) after step (f), measure the signal of the liquid sample; and (h) based on the result of step (g), detecting the virus, where the method is capable of detecting less than 100 copies of viruses (for example, less than 80, 70, 60, 50, 40, 30 , 20, or 10 copies) in the whole blood sample. In certain embodiments, steps (a) to (g) are completed in 3 hours (for example, in 3.2; 2.9; 2.7; 2.5; 2.3; 2.2; 2.1 ; 2.0; 1.9; 1.8; 1.7; 1.6; 1.5 hour, or 1 hour or less). The virus can be any viral pathogen described here. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. The monitoring method can include any of the magnetic assisted agglomeration methods described here. Magnetic particles can include one or more populations having a first probe and a second probe conjugated to its surface, a first operative probe to bind to a first segment of the target nucleic acid and a second operative probe to bind to a second segment of the target nucleic acid, where magnetic particles form aggregates in the presence of the target nucleic acid. Alternatively, the assay can be a disintegration assay in which the magnetic particles include the first population having a first binding portion on its surface and a second population having a second binding portion on its surface, and the multivalent binding portion including a first probe and second probe, first operative probe to attach to the first attachment portion and a second operative probe to attach to a second attachment portion, the attachment portions and multivalent attachment portion operative to alter an aggregation of the particles magnetic fields in the presence of the target nucleic acid.
[0016] [00016] In any of the systems and methods of the invention in which a PCR amplification is performed, the PCR method can be real-time PCR to quantify the amount of a target nucleic acid present in a sample.
[0017] (a) realizar um ou mais ciclos de amplificação; (b) expor a mistura de reação de amplificação, ou uma alíquota do mesmo, às condições permitindo a agregação ou desagregação das partículas superparamagnéticas, (c) expor a amostra a um campo magnético de polarização e uma sequência de pulso RF; (d) seguinte à etapa (c), medir o sinal do tubo de detecção; (e) repetir as etapas (a)-(d) até que uma quantidade desejada de amplificação seja obtida; e (f) com base no resultado da etapa (d), quantificar os amplicons presentes no ciclo correspondente de amplificação. [00017] The invention also features a method for quantifying a target nucleic acid molecule in a sample by amplifying the target nucleic acid molecule (for example, using PCR or isothermal amplification) in an amplification reaction mixture in a tube detection resulting in the production of amplicons corresponding to the target nucleic acid molecule. In this method, the amplification reaction mixture includes (1) a target nucleic acid molecule, (2) specific amplification primers for the target nucleic acid molecule, and (3) superparamagnetic particles. In this method, amplification is performed on a device including a support defining a cavity to hold the detection tube including the superparamagnetic particles and the target nucleic acid molecule, and having an RF spiral arranged over the cavity, the RF spiral configured to detect a signal produced by exposing a sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence. The amplification of this method included the following steps: (a) perform one or more amplification cycles; (b) exposing the amplification reaction mixture, or an aliquot thereof, to conditions allowing the aggregation or disaggregation of superparamagnetic particles, (c) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (d) following step (c), measure the signal from the detection tube; (e) repeat steps (a) - (d) until a desired amount of amplification is obtained; and (f) based on the result of step (d), quantify the amplicons present in the corresponding amplification cycle.
[0018] [00018] In this method, the initial amount of target nucleic acid molecule in the sample is determined based on the amount of amplicons determined in each cycle of amplification.
[0019] [00019] In any of the aforementioned methods of quantifying a target nucleic acid molecule, the detection tube can remain sealed throughout the amplification reaction. The superparamagnetic particles of these methods can be larger or smaller than 100 nm in diameter (for example, 30 nm in diameter).
[0020] [00020] Furthermore, in any of the aforementioned methods of quantifying a target nucleic acid molecule, the methods may also include applying a magnetic field to the detection tube following the measurement of the detection tube signal, resulting in sequestration of the superparamagnetic particles to the side of the detection tube, and releasing the magnetic field following the completion of one or more additional amplification cycles.
[0021] [00021] In addition, in any of the aforementioned methods of quantifying a target nucleic acid molecule, the sample may, for example, not include the isolated nucleic acid molecules prior to step (a) (for example, the sample may be whole blood or do not contain a target nucleic acid molecule prior to step (a)).
[0022] [00022] The invention features a method of monitoring one or more analyzed in a liquid sample derived from a patient for the diagnosis, control, or treatment of a medical condition in a patient, the method including (a) combining with the liquid sample 1 × 106 to 1x1013 magnetic particles per milliliter of the liquid sample (for example, 1x106 to 1x108, 1x107 to 1x108, 1x107 to 1x109, 1x108 to 1x1010, 1x109 to 1x1011, or 1x1010 to 1x1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 1200 nm (e.g. 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), and a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and where the magnetic particles have bonding portions on their surfaces, the portions operating connections to change the specific aggregation of the magnetic particles in the presence of one or more analyzed or a multivalent binding agent; (b) placing the liquid sample in a device, the device including a holder defining a cavity to hold the liquid sample including the magnetic particles and the one or more analyzed, and having an RF spiral arranged over the cavity, the RF spiral configured for detecting a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (c) exposing the sample to the polarizing magnetic field and the RF pulse sequence; (d) after step (c), measure the signal; (e) based on the result of step (d), monitor the one or more analyzed; and (f) using the result of step (e) to diagnose, control, or treat the medical condition. In one embodiment, the one or more analyzed includes creatinine. In another embodiment, the patient is immunocompromised, and the one or more analysts includes a selected analysand associated with the pathogen, antibiotic agents, antifungal agents, and antiviral agents (for example, the one or more analysts may include Candida spp., Tacrolimus, fluconazole, and / or creatinine). In yet another modality, the patient has cancer, and the one or more analyzed are selected for anticancer agents, and genetic markers present in a cancer cell. The patient may have, or be at risk for, an infection, and the one or more test subjects include a test subject selected from pathogen-associated subjects, antibiotic agents, antifungal agents, and antiviral agents. The patient may have an immunoinflammatory condition, and the one or more analyzed includes a selected one of anti-inflammatory agents and TNF-alpha. The patient may have heart disease, and the one or more analyzed may include a cardiac marker. The patient may have HIV / AIDS, and the one or more analyzed may include CD3, viral load, and AZT. In certain modalities, the method is used to monitor the patient's liver function, and where the one or more analyzed are selected from albumin, aspartate transaminase, alanine transaminase, alkaline phosphatase, gamma glutamyl transpeptidase, bilirubin, alpha fetoprotein, lactase dehydrogenase, antibodies mitochondria, and cytochrome P450. For example, the one or more analyzed includes cytochrome P450 polymorphisms, and the patient's ability to metabolize a drug is assessed. The method may include identifying the patient as a weak metaboliser, a normal metaboliser, an intermediate metaboliser, or an ultra fast metaboliser. The method can be used to determine an appropriate dose of a therapeutic agent in a patient (i) by administering the therapeutic agent to the patient; (ii) following step (i), obtaining a sample including the patient's therapeutic agent or metabolite; (iii) contacting the sample with the magnetic particles and exposing the sample to the magnetic polarization field and the RF pulse sequence and detecting the signal produced by the sample; and (iv) based on the result of step (iii), determine the concentration of the therapeutic agent or its metabolite. The therapeutic agent can be an anticancer agent, antibiotic agent, antifungal agent, or any therapeutic agent described herein. In any of the above monitoring methods, monitoring can be intermittent (for example, periodic), or continuous. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. The monitoring method can include any of the magnetic assisted agglomeration methods described here.
[0023] [00023] The invention features a method for diagnosing sepsis in an individual, the method including (a) obtaining a liquid sample derived from a patient's blood; (b) prepare a first test sample by combining with a portion of the liquid sample from 1 × 106 to 1 × 1013 magnetic particles per milliliter of the liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter from 150 nm to 1200 nm (for example, from 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), and a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and where the magnetic particles have bonding portions on their surfaces, the bonding portions operative to alter the specific aggregation of magnetic particles in the presence of one or more analytes associated with the pathogen or a multivalent binding agent; (c) prepare a second test sample by combining with a portion of the liquid sample of 1 * 106 to 1 * 1013 magnetic particles per milliliter of the liquid sample (for example, from 1 * 106 to 1 * 108, 1 * 107 to 1 * 108, 1 * 107 to 1 * 109, 1 * 108 to 1 * 1010, 1 * 109 to 1 * 1011, or 1 * 1010 to 1 * 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter from 150 nm to 1200 nm (for example, from 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), and a relaxation of T2 per particle of 1 * 108 to 1 * 1012 mM-1s-1 (for example, from 1 * 108 to 1 * 109, 1 * 108 to 1 * 1010, 1 * 109 to 1 * 1010, 1 * 109 to 1 * 1011, or from 1 * 1010 to 1 * 1012 mM-1s-1), and where the magnetic particles have bonding portions on their surfaces, the bonding portions operative to change the specific aggregation of magnetic particles in the presence of one or more analyzed characteristics of sepsis selected from GRO-alpha, High mob group 1 protein ility (HMBG-1), IL-1 receptor, IL-1 receptor antagonist, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL -13, IL-18, macrophage inflammatory protein (MIP-1), macrophage migration inhibiting factor (MIF), osteopontin, RANTES (regulated on activation, expressed and secreted normal T cell; or CCL5), TNF-α, C-reactive protein (CRP), CD64, monocyte chemotactic protein 1 (MCP-1), adenosine deaminase binding protein (ABP-26), inducible nitric oxide synthase (iNOS), protein binding of lipopolysaccharide, and procalcitonin; (d) placing each test sample in a device, the device including a holder defining a cavity to hold the liquid sample including the magnetic particles and the one or more analyzed, and having an RF spiral arranged over the cavity, the configured RF spiral to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing each test sample to the polarizing magnetic field and the RF pulse sequence; (f) following step (e), measure the signal produced by the first test sample and the signal produced by the second test sample; (g) based on the result of step (f), monitor the one or more analyzed from the first test sample and monitor the one or more analyzed from the second test sample; and (h) use the results from step (g) to diagnose the individual. In one embodiment, the one or more analyzed associated with the pathogen of the first test sample are derived from a pathogen associated with the selected sepsis of Acinetobacter baumannii, Aspergillus fumigatis, Bacteroides fragilis, B. fragilis, blaSHV, Burkholderia cepacia, Campylobacter jejuni / coli , Candida guilliermondii, C. albicans, C. glabrata, C. krusei, C. lusitaniae, C. parapsilosis, C. tropicalis, Clostridium pefringens, Coagulase negative Staph, Enterobacter aeraogenes, E. cloacae, Enterobacteriaceae, Enterococcus faecalis, E. faeciumis , Escherichia coli, Haemophilus influenzae, Kingella Kingae, Klebsiella oxytoca, K. pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), Morganella morgana, Neisseria meningitidis, Neisseria spp. non meningitidis, Prevotella buccae, P. intermedia, P. melaninogenica, Propionibacterium acnes, Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, S. haemolyticus, S. maltophilia, S. malprophus, S. saprophytic , S. maltophilia, Streptococcus agalactie, S. bovis, S. dysgalactie, S. mitis, S. mutans, S. pneumoniae, S. pyogenes, and S. sanguinis. The one or more analyzed associated with the pathogen can be derived from strains resistant to the treatment of bacteria, such as bacterial strains resistant to penicillin, resistant to methicillin, resistant to quinolone, resistant to macrolide, and / or resistant to vancomycin (for example, Staphylococcus methicillin-resistant aureus or vancomycin-resistant enterococci). In certain embodiments, the one or more analyzed from the second test sample is selected from GRO-alpha, High Mobility Group 1 Protein (HMBG-1), IL-1 receptor, IL-1 receptor antagonist, IL -1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage (MIF), osteopontin, RANTES (regulated on activation, expressed and secreted normal T cell; or CCL5), TNF-α, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1). In a particular embodiment, the method also includes preparing a third test sample to monitor the concentration of an antiviral agent, antibiotic agent, or antifungal agent circulating in the individual's bloodstream. In certain embodiments, the individual may be an immunocompromised individual, or an individual at risk of becoming immunocompromised. In any of the above monitoring methods, monitoring can be intermittent (for example, periodic), or continuous. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit non-specific reversibility in the absence of the analyzed and the multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. The monitoring method can include any of the magnetic assisted agglomeration methods described here.
[0024] [00024] The invention also features a method for monitoring one or more analyzed in a liquid sample derived from a patient for the diagnosis, control, or treatment of sepsis or SIRS in a patient, the method including: (a) combining with liquid sample of 1 × 106 to 1 × 1013 magnetic particles per milliliter of the liquid sample (for example, from 1 × 106 to 1 * 108, 1 * 107 to 1 * 108, 1 * 107 to 1 * 109, 1 * 108 to 1 * 1010, 1 * 109 to 1 * 1011, or 1 * 1010 to 1 * 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 1200 nm (for example, 150 to 250, 200 at 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or from 1000 to 1200 nm), and a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and where the magnetic particles have bonding portions on their surfaces, the bonding portions operative for the alter the specific aggregation of the magnetic particles in the presence of one or more analyzed or a multivalent binding agent; (b) placing the liquid sample in a device, the device including a holder defining a cavity to hold the liquid sample including the magnetic particles and the one or more analyzed, and having an RF spiral arranged over the cavity, the RF spiral configured for detecting a produced signal by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (c) exposing the sample to the polarizing magnetic field and the RF pulse sequence; (d) after step (c), measure the signal; (e) based on the result of step (d), monitor the one or more analyzed; and (f) use the result of step (e) to diagnose, control, or treat sepsis or SIRS. The method may include (i) monitoring an assay associated with the pathogen, and (ii) monitoring a second assay characteristic of sepsis selected from GRO-alpha, high mobility group 1 protein (HMBG-1), IL-1 receptor , IL-1, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18 receptor antagonist, inflammatory macrophage protein ( MIP-1), macrophage migration inhibitor (MIF), osteopontin, RANTES (regulated on activation, expressed and secreted normal T cell; or CCL5), TNF-α, C-reactive protein (CRP), CD64, protein 1 monocyte chemotactic (MCP-1), adenosine deaminase binding protein (ABP-26), inducible nitric oxide synthase (iNOS), lipopolysaccharide binding protein, and procalcitonin. In certain modalities, the analyte associated with the pathogen is derived from a pathogen associated with sepsis selected from Acinetobacter baumannii, Aspergillus fumigatis, Bacteroides fragilis, B. fragilis, blaSHV, Burkholderia cepacia, Campylobacter jejuni / coli, Candida guilliermondii, C. albicans, glabrata, C. krusei, C. Lusitaniae, C. parapsilosis, C. tropicalis, Clostridium pefringens, Coagulase negative Staph, Enterobacter aeraogenes, E. cloacae, Enterobacteriaceae, Enterococcus faecalis, E. faecium, Escherichia coli, Haemophilus influenzae, Kingem , Klebsiella oxytoca, K. pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), Morganella morgana, Neisseria meningitidis, Neisseria spp. non-meningitidis, Prevotella buccae, P. intermedia, P. melaninogenica, Propionibacterium acnes, Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, S. haemolyticus, S. maltophomophys, S. saprophytis, S. maltophilia, S. maltophilia, Streptococcus agalactie, S. bovis, S. dysgalactie, S. mitis, S. mutans, S. pneumoniae, S. pyogenes, and S. sanguinis. The analyte associated with the pathogen can be derived from a strain resistant to the treatment of bacteria, such as bacterial strains resistant to penicillin, resistant to methicillin, resistant to quinolone, resistant to macrolide, and / or resistant to vancomycin (for example, Staphylococcus aureus resistant methicillin or vancomycin-resistant enterococci). In particular modalities, the second analyzed is selected from GRO-alpha, High mobility group 1 protein (HMBG-1), IL-1 receptor, IL-1 receptor antagonist, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, macrophage inflammatory protein (MIP-1), macrophage migration inhibitor (MIF) factor, osteopontin, RANTES (regulated on activation, expressed and secreted normal T cell; or CCL5), TNF-α, C-reactive protein (CRP), CD64, and monocyte chemotactic protein 1 (MCP-1). In a particular embodiment, the method also includes preparing a third test sample to monitor the concentration of an antiviral agent, antibiotic agent, or antifungal agent circulating in the individual's bloodstream. In certain embodiments, the individual may be an immunocompromised individual, or an individual at risk of becoming immunocompromised. In any of the above monitoring methods, monitoring can be intermittent (for example, periodic), or continuous. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. The monitoring method can include any of the magnetic assisted agglomeration methods described here.
[0025] [00025] The invention also features a system for detecting one or more analyzed, the system including: (a) a first unit including (a1) a permanent magnet defining a magnetic field; (a2) a support defining a cavity to hold a liquid sample including the magnetic particles and the one or more analyzed and having an RF spiral arranged over the cavity, the RF spiral configured to detect a signal produced by exposing the liquid sample to a magnetic polarization field created using the permanent magnet and an RF pulse sequence; and (a3) one or more electrical elements in communication with the RF spiral, the electrical elements configured to amplify, rectify, transmit, and / or digitize the signal; and (b) a second unit including a removable cartridge sized to facilitate insertion into and removal from the system, where the removable cartridge is a modular cartridge including (i) a reagent module to retain one or more assay reagents; and (ii) a detection module including a detection chamber to hold a liquid sample including the magnetic particles and the one or more analyzed, where the reagent module and the detection module can be mounted on the modular cartridge before use, and where the detection chamber is removable from the modular cartridge. The modular cartridge can also include an input module, where the input module, the reagent module, and the detection module can be mounted on the modular cartridge before use, and where the input module is sterilizable. In certain embodiments, the system also includes a computer system with a processor to implement an assay protocol and store assay data, and where the removable cartridge also includes (i) a readable label indicating the analyte to be detected, (ii) a readable label indicating the test protocol to be implemented, (iii) a readable label indicating a patient identification number, (iv) a readable label indicating the position of test reagents contained in the cartridge, or (v) a readable label including instructions for the programmable processor. The system can include a cartridge unit, a stirring unit, a centrifuge, or any other component of the system described here.
[0026] [00026] The invention also features a system for detecting one or more analyzed, the system including: (a) a disposable sample holder defining a cavity to hold a liquid sample and having an RF spiral contained within the disposable sample holder and arranged over the cavity, the RF spiral configured to detect a signal produced by exposing the liquid sample to a magnetic polarization field created using the permanent magnet and an RF pulse sequence, where the disposable sample holder includes one or more connections fused; and (b) an MR reader including (b1) a permanent magnet defining a magnetic field; (b2) an RF pulse sequence and detection spiral; (b3) one or more electrical elements in communication with the RF spiral, the electrical elements configured to amplify, rectify, transmit, and / or digitize the signal; and (b4) one or more electrical elements in communication with the fused connection and configured to apply excess current to the fused connection, causing the connection to break and become the inoperable spiral following a predetermined useful life. In certain embodiments, the electrical element in communication with the RF coil is inductively coupled to the RF coil.
[0027] [00027] The invention features a system for the detection of creatinine, tacrolimus, and Candida, the system including: (a) a first unit including (a1) a permanent magnet defining a magnetic field; (a2) a support defining a cavity to hold a liquid sample including magnetic particles and creatinine, tacrolimus, and Candida and having an RF spiral arranged over the cavity, the RF spiral configured to detect the signal produced by exposing the liquid sample to a polarizing magnetic field created using the permanent magnet and an RF pulse sequence; and (a3) an electrical element in communication with the RF spiral, the electrical element configured to amplify, rectify, transmit and / or digitize the signal; and (b) a second unit including a removable cartridge sized to facilitate insertion and removal of the system, where the removable cartridge is a modular cartridge including (i) a plurality of reagent modules for retaining one or more test reagents; and (ii) a plurality of detection module including a detection chamber to hold a liquid sample including magnetic particles and creatinine, tacrolimus, and Candida, where the plurality of reagent modules includes (i) a first population of magnetic particles having an average diameter of 150 nm to 699 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 699 nm), a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and the creatinine antibodies conjugated to its surface; (ii) a multivalent linker containing a plurality of creatinine conjugates designed to form aggregates with the first population of magnetic particles in the absence of creatinine; (iii) a second population of magnetic particles having an average diameter of 150 nm to 699 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 699 nm ), a relaxation of T2 per particle from 1 * 108 to 1 * 1012 mM-1s-1 (for example, from 1 * 108 to 1 * 109, 1 * 108 to 1 * 1010, 1 * 109 to 1 * 1010, 1 * 109 to 1 * 1011, or from 1 * 1010 to 1 * 1012 mM-1s-1), and tacrolimus antibodies conjugated to its surface; (iv) a multivalent linker containing a plurality of tacrolimus conjugates designed to form aggregates with the second population of magnetic particles in the absence of tacrolimus; (v) a third population of magnetic particles has an average diameter of 700 nm to 1200 nm (for example, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), a relaxation of T2 per particle 1 × 109 to 1 × 1012 mM-1s-1 (for example, 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and having a first probe and a second probe conjugated to its selected surface to form aggregates in the presence of a Candida nucleic acid, a first operative probe to bind to a first segment of the Candida nucleic acid and a second probe operative to bind to a second segment of the Candida nucleic acid. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold. In another embodiment, the liquid sample includes from 1 × 106 to 1 × 1013 of the magnetic particles per milliliter of the liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter).
[0028] [00028] The invention features a method for measuring the concentration of creatinine in a liquid sample, the method including: (a) contacting a solution with (i) magnetic particles to produce a liquid sample including from 1 × 106 to 1 × 1013 particles magnetic per milliliter of the liquid sample (for example, from 1106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 1200 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1012 mM-1s-1), and creatinine antibodies conjugated to its surface, and (ii) a multivalent binding agent containing a plurality of creatinine conjugates designated p to form aggregates with magnetic particles in the absence of creatinine; (b) placing the liquid sample in a device, the device including a holder defining a cavity to hold the liquid sample including the magnetic particles, the multivalent binding agent, and the creatinine, and having RF spiral arranged over the cavity, the spiral RF configured to detect a signal produced by exposing the liquid sample to a magnetic polarization field created using one or more magnets and an RF pulse sequence; (c) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (d) after step (c), measure the signal; and (e) based on the result of step (d), determine the creatinine concentration in the liquid sample. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, the liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold.
[0029] [00029] The invention features a multivalent linker including two or more portions of creatinine covalently attached to a scaffold. In certain embodiments, the multivalent binding agent is a compound of the formula (I): (A) n- (B) (I) where (A) is
[0030] [00030] The invention features a solution including from 1 × 106 to 1 × 1013 magnetic particles per milliliter of the solution (for example, from 1x106 to 1x108, 1x107 to 1x108, 1x107 to 1x109, 1x108 to 1x1010, 1x109 to 1x1011, or 1x1010 to 1x1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 600 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 at 600 nm), a relaxation of T2 per particle from 1x108 to 1x1012 mM-1s-1 (for example, from 1x108 to 1x109, 1x108 to 1x1010, 1x109 to 1x1010, 1x109 to 1x1011, or from 1x1010 to 1x1012 mM-1s- 1), and a surface containing creatinine conjugate (A), where (A) is selected from:
[0031] [00031] The invention also features the solution including from 1x106 to 1x1013 magnetic particles per milliliter of the solution (eg, 1x106 to 1x108, 1x107 to 1x108, 1x107 to 1x109, 1x108 to 1x1010, 1x109 to 1x1011, or 1x1010 to 1x1013 particles magnetic per milliliter), where the magnetic particles have an average diameter of 150 nm to 600 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or 500 to 600 nm ), a relaxation of T2 per particle from 1x108 to 1x1012 mM-1s-1 (for example, from 1x108 to 1x109, 1x108 to 1x1010, 1x109 to 1x1010, 1x109 to 1x1011, or from 1x1010 to 1x1012 mM-1s-1), and a surface containing antibodies having an affinity for a creatinine conjugate (for example, a creatinine conjugate described here).
[0032] [00032] The invention also features a method for measuring the concentration of tacrolimus in a liquid sample, the method including: (a) contacting a solution with (i) magnetic particles to produce a liquid sample including from 1 × 106 to 1 × 1013 magnetic particles per milliliter of the liquid sample (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 1200 nm (for example, 150 to 250, 200 to 350, 250 to 450, 300 to 500 , 450 to 650, 500 to 700 nm, 700 to 850, 800 to 950, 900 to 1050, or 1000 to 1200 nm), a T2 relaxation per particle of 1 × 108 to 1 × 1012 mM-1s-1 ( for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 * 1010, 1 * 109 to 1 * 1010, 1 * 109 to 1 * 1011, or from 1 * 1010 to 1 * 1012 mM-1s-1 ), and tacrolimus antibodies conjugated to its surface, and (ii) a multivalent binding agent containing a plurality of conjugated tacrolimus designated those to form aggregates with the magnetic particles in the absence of tacrolimus; (b) placing the liquid sample in a device, the device including a holder defining a cavity to hold the liquid sample including the magnetic particles, the multivalent binding agent, and the tacrolimus, and having an RF spiral arranged over the cavity, the spiral RF configured to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (c) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (d) after step (c), measure the signal; and (e) based on the result of step (d), determine the concentration of tacrolimus in the liquid sample. In certain embodiments, the magnetic particles are substantially monodisperse; exhibit nonspecific reversibility in the absence of the analyzed and multivalent binding agent; and / or the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran). In particular embodiments, a liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7 %, 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% non-ionic surfactant (for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0.4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In still other embodiments, the magnetic particles include a surface decorated with 40 µg to 100 µg (for example, 40 µg to 60 µg, 50 µg to 70 µg, 60 µg to 80 µg, or 80 µg to 100 µg,) of a or more proteins per milligram of the magnetic particles. The liquid sample can include a multivalent linker containing a plurality of conjugate analytes for a polymeric scaffold.
[0033] [00033] The invention features a multivalent linker including two or more portions of tacrolimus, including tacrolimus metabolites described herein or structurally similar compounds for which the antibody has covalently attached affinity to a scaffold. In certain embodiments, the multivalent binding agent is a compound of the formula (II): (A) n- (B) (II) where (A) is
[0034] [00034] The invention features the solution including from 1 × 106 to 1 × 1013 magnetic particles per milliliter of the solution (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles per milliliter), where the magnetic particles have an average diameter of 150 nm to 600 nm (for example, and 150 to 250, 200 to 350, 250 to 450, 300 to 500, 450 to 650, or from 500 to 600 nm), a relaxation of T2 per particle of 1 × 108 to 1 × 1012 mM-1s-1 (for example, 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1010, 1 × 109 to 1 * 1011, or 1 * 1010 to 1 * 1012 mM-1s-1) , and a surface containing antibodies having an affinity with the tacrolimus conjugate:
[0035] [00035] In an embodiment of any of the above solutions, (i) the magnetic particles are substantially monodispersed; (ii) the magnetic particles exhibit non-specific reversibility in the plasma; (iii) the magnetic particles also include a surface decorated with a blocking agent selected from albumin, fish skin gelatin, gamma globulin, lysozyme, casein, peptidase, and an amine-containing portion (e.g., amino polyethylene glycol, glycine, ethylenediamine , or amino dextran); (iv) a liquid sample also includes a buffer, from 0.1% to 3% (weight / weight) of albumin (for example, from 0.1% to 0.5%, 0.3% to 0.7% , 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% of nonionic surfactant ( for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0 , 4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof; and / or (iv) the magnetic particles include a surface decorated with 40 μg to 100 μg (for example, 40 μg to 60 μg, 50 μg to 70 μg, 60 μg to 80 μg, or 80 μg to 100 μg) or more proteins per milligram of the magnetic particles. The solutions can be used in any of the systems or methods described here.
[0036] [00036] The invention features a removable cartridge sized to facilitate insertion into and removal of a system of the invention, where the removable cartridge includes one or more chambers for holding a plurality of reagent modules for holding one or more test reagents, where reagent modules include (i) a chamber for holding 1 × 106 to 1 × 1013 magnetic particles (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 * 108, 1 * 107 to 1 * 109 , 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, or 1 × 1010 to 1 × 1013 magnetic particles) having an average diameter of 100 nm to 699 nm (for example, 150 to 250, 200 to 350 , 250 to 450, 300 to 500, 450 to 650, or from 500 to 699 nm), a relaxation of T2 per particle from 1 × 108 to 1 × 1012 mM-1s-1 (for example, from 1 × 108 to 1 × 109, 1 × 108 to 1 × 1010, 1 * 109 to 1 * 1010, 1 * 109 to 1 * 1011, or 1 * 1010 to 1 * 1012 mM-1s-1), and bonding portions on their surfaces , the connecting portions operative to alter the specific aggregation of the magnetic particles in the presence of the one or more analyzed or a multivalent binding agent; and (ii) a chamber for retaining a buffer. In a related aspect, the invention features a removable cartridge sized to facilitate insertion into and removal of a system of the invention, where the removable cartridge comprises one or more chambers for holding a plurality of reagent modules for holding one or more test reagents , where the reagent modules include (i) a chamber to hold 1 × 106 to 1 × 1013 magnetic particles (for example, 1 × 106 to 1 × 108, 1 × 107 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 * 1010, 1 × 109 to 1χ1011, or 1x1010 to 1x1013 magnetic particles) having an average diameter of 700 nm to 1200 nm (for example, 700 to 850, 800 to 950, 900 to 1050 , or 1000 to 1200 nm), a relaxation of T2 per particle from 1x109 to 1x1012 mM-1s-1 (for example, from 1x109 to 1x1010, 1x109 to 1x1011, or from 1x1010 to 1x1012 mM-1s-1), and oligonucleotide binding portions on their surfaces, the oligonucleotide binding portions operative to alter the specific aggregation of magnetic particles in the presence of the um or m s analyzed; and (ii) a chamber for retaining a buffer. The magnetic particles can be any described here, decorated with any binding portion described here, to detect any analyte described here. In particular modes of the removable cartridges, the magnetic particles and buffer are together in a single chamber inside the cartridge. In still other embodiments, the buffer includes 0.1% to 3% (weight / weight) albumin, 0.01% to 0.5% non-ionic surfactant, a lysis agent, or a combination thereof. The removable cartridge can also include a chamber including beads to lyse the cells; a chamber including a polymerase; and / or a chamber including a primer.
[0037] [00037] The invention features a removable cartridge sized to facilitate insertion into and removal of a system of the invention, where the removable cartridge includes one or more chambers to hold a plurality of reagent modules to hold one or more test reagents, where the reagent modules include (i) a chamber for holding from 1x108 to 1x1010 magnetic particles having an average diameter of 100 nm to 350 nm, a relaxation of T2 per particle of 5x108 to 1x1010 mM-1s-1, and binding portions on its surface s (for example, antibodies, conjugate analyzed), the binding portions operative to alter the specific aggregation of the magnetic particles in the presence of the one or more analyzed or a multivalent binding agent; and (ii) a chamber for retaining a buffer including 0.1% to 3% (weight / weight) albumin (for example, 0.1% to 0.5%, 0.3% to 0.7% , 0.5% to 1%, 0.8% to 2%, or 1.5% to 3% (weight / weight) of albumin), 0.01% to 0.5% of nonionic surfactant ( for example, 0.01% to 0.05%, 0.05% to 0.1%, 0.05% to 0.2%, 0.1% to 0.3%, 0.2% to 0 , 4%, or 0.3% to 0.5% non-ionic surfactant), or a combination thereof. In one embodiment, the magnetic particles and buffer are together in a single chamber inside the cartridge.
[0038] [00038] In any of the systems, kits, cartridges, and methods of the invention, the liquid sample can include from 1 × 108 to 1 * 1010 magnetic particles having an average diameter of 100 nm to 350 nm, a relaxation of T2 per particle of 5 * 108 to 1 * 1010 mM-1s-1, and binding portions on its surface (eg, antibodies, conjugate analyzed), the binding portions operative to alter the specific aggregation of magnetic particles in the presence of one or more analyzed or a multivalent binding agent.
[0039] [00039] In any of the systems, kits, cartridges, and methods of the invention for detecting any assayed in a whole blood sample, disruption of red blood cells can be performed using an erythrocyte lysis agent (ie, a lysis, or a non-ionic detergent). Erythrocyte lysis buffers that can be used in the methods of the invention include, without limitation, isotonic ammonium chloride solutions (optionally including carbonate and / or EDTA buffer), and hypotonic solutions. Alternatively, the erythrocyte lysis agent may be an aqueous solution of non-ionic detergents (for example, nonyl phenoxypolyethoxyethanol (NP-40), 4-octylphenol polyethoxylate (Triton-X100), Brij-58, or related non-ionic surfactants, and mixtures thereof). The erythrocyte lysis agent disrupts at least a little bit of the red blood cells, allowing a large fraction of certain whole blood components (eg, certain whole blood proteins) to be separated (eg, as a supernatant following centrifugation) from white blood cells , yeast cells, and / or bacterial cells present in the whole blood sample. Following the erythrocyte centrifugation and lysis, the resulting pellet is reconstituted to form an extract.
[0040] [00040] The methods, kits, cartridges and systems of the invention can be configured to detect a predetermined panel of analytes associated with the pathogen. For example, the panel may be a candida fungal panel configured to individually detect three or more among Candida guilliermondii, C. albicans, C. glabrata, C. krusei, C. Lusitaniae, C. parapsilosis, and C. tropicalis. In another embodiment, the panel may be a bacterial panel configured to individually detect three or more of coagulase negative Staphylococcus, Enterococcus faecalis, E. faecium, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli. In a particular embodiment, the panel can be a viral panel configured to individually detect three or more of Cytomegalovirus (CMV), Epstein Barr Virus, BK Virus, Hepatitis B Virus, Hepatitis C Virus, Herpes Simplex Virus (HSV), HSV1, HSV2, respiratory syncytial virus (RSV), Influenza; Influenza A, Influenza A H1 subtype, Influenza A H3 subtype, Influenza B, Human herpes virus 6, Human Herpes Virus 8, Human Metapneumovirus (hMPV), Rhinovirus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Adenovirus. The panel can be a bacterial panel configured to individually detect three or more d among E. coli, CoNS (coagulase negative staph), Pseudomonas aeruginosa, S. aureus, E. faecium, E. faecalis, and Klebsiella pneumonia. The panel may be a bacterial panel configured to individually detect three or more of A. fumigates, and A. flavum. The panel can be a bacterial panel configured to individually detect three or more of Acinetobacter baumannii, Enterobacter aeraogenes, Enterobacter cloacae, Klebsiella oxytoca, Proteus mirabilis, Serratia marcescens, Staphylococcus haemolyticus, Stenotro-phomonas maltophilia, Streptococcus agtreococcus and Streptococcus pyogenes. The panel can be a meningitis panel configured to individually detect three or more of Streptococcus pneumonia, H. influenza, Neisseria Meningitis, HSV1, HSV2, Enterovirus, Listeria, E. coli, Streptococcus Group B. The panel can be configured to individually detect three or more of N. gonnorrhoeae, S. aureus, S. pyogenes, CoNS, and Borrelia burgdorferi. The panel can be configured to individually detect three or more of C. Difficile, Toxin A, and Toxin B. The panel can be a pneumonia panel configured to individually detect three or more of Streptococcus pneumonia, MRSA, Legionella, C. pneumonia, and Mycoplasmic Pneumonia. The panel can be configured to individually detect three or more of the selected treatment-resistant mutations of mecA, vanA, vanB, NDM-1, KPC, and VIM. The panel can be configured to individually detect three or more of H. influenza, N. gonnorrhoeae, H. pylori, Campylobacter, Brucella, Legionella, and Stenotrophomonas maltophilia. The panel can be configured to detect the total viral load caused by CMV, EBV, BK, HIV, HBV, and HCV viruses. The panel can be configured to detect fungal load and / or bacterial load. Viral load determination can be using a standard curve and measuring the sample against this standard curve or some other method of quantifying the pathogen in a sample. The quantitative measurement method can include Real-Time PCR, competitive PCR (ratio of two competing signals) or other methods mentioned here. The panel can be configured to detect the immune response in an individual by monitoring PCT, MCP-1, CRP, GRO-alpha, High mobility group 1 protein (HMBG-1), IL-1 receptor, antagonist of IL-1, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18 receptor, macrophage inflammatory protein (MIP-1 ), macrophage migration inhibiting factor (MIF), osteopontin, RANTES (regulated on activation, expressed and secreted normal T cell; or CCL5), Th1, Th17, and / or TNF-α. The panel can be configured to individually detect three or more among Ehrlichea, Mycobacterium, Syphillis, Borrelia burgdorferi, Cryptococcus, Histoplasma, and Blastomyces. The panel can be an influenza panel configured to individually detect three or more among Influenza A, Influenza B, RSV, Parainfluenza, Meta-pneumovirus, Rhinovirus, and Adenovirus.
[0041] [00041] The methods, kits, cartridges, and systems of the invention can be configured to reduce the sample for sample variability by determining an MRI signal before and after hybridization. The addition of derived nanoparticles to the sample prior to methods for enhancing the cluster can provide a reference value, internal T2 signal that can be subtracted or used to modify the T2 signal after clustering and binding of the analyzed particle derived. This method can also be used to determine or control the cartridge for cartridge variability.
[0042] [00042] The terms "aggregation", "agglomeration", and "grouping" are used interchangeably in the context of the magnetic particles described here and mean the connection of two or more magnetic particles to another, for example, through a multivalent analyte, form of the analyzed, antibody, nucleic acid molecule, or other binding entity or molecule. In some cases, the magnetic particle agglomeration is reversible.
[0043] [00043] "Analyzed" means a substance or a constituent of a sample to be analyzed. Exemplary subjects analyzed include one or more species of the following: a protein, a peptide, a polypeptide, an amino acid, a nucleic acid, an oligonucleotide, RNA, DNA, an antibody, a carbohydrate, a polysaccharide, glucose, a lipid, a gas (e.g., oxygen or carbon dioxide), an electrolyte (e.g., sodium, potassium, chloride, bicarbonate, BUN, magnesium, phosphate, calcium, ammonia, lactate), a lipoprotein, cholesterol, a fatty acid, a glycoprotein, a proteoglycan, a lipopolysaccharide, a cell surface marker (for example, CD3, CD4, CD8, IL2R, or CD35), a cytoplasmic marker (for example, CD4 / CD8 or CD4 / viral load), a therapeutic agent, a metabolite of a therapeutic agent, a marker for the detection of a weapon (for example, a chemical or biological weapon), an organism, a pathogen, a pathogen by-product, a parasite (for example, a protozoan or helminth), a protista, a fungus (for example, yeast, i or mold), a bacterium age, an actinomycete, a cell (for example, a total cell, a tumor cell, a stem cell, a white blood cell, a T cell (for example, exhibiting CD3, CD4, CD8, IL2R, CD35, or other markers surface), or another cell identified with one or more specific markers), a virus, a prion, a plant component, a plant by-product, plant by-product, algae, an algae by-product, plant growth hormone, a insecticide, an artificial toxin, an environmental toxin, an oily component, and components derived therefrom. As used herein, the term "small molecule" refers to a drug, medication, medication, or other chemically synthesized compound that is contemplated for human therapeutic use. As used here, the term "biological" refers to a substance derived from a biological source, not synthesized and which is contemplated for human therapeutic use. A "biomarker" is a biological substance that can be used as an indicator of a particular disease state or a particular physiological state of an organism, usually a biomarker is a protein or other native compound measured in body fluid whose concentration reflects the presence or severity or gradation of a disease or dysfunction state, can be used to monitor the therapeutic progress of the treatment of a disease or disorder or dysfunction, or can be used as a surrogate measure of progression or clinical outcome. As used herein, the term "metabolic biomarker" refers to a substance, molecule, or compound that is synthesized or biologically derived which is used to determine a patient's condition or an individual's kidney and liver function. As used here, the term "genotyping" refers to the ability to determine genetic differences in specific genes that may or may not affect the phenotype of the specific gene. As used here, the term "phenotype" refers to a resulting biological expression, (metabolic or physiological) of the protein pool by the genotype. As used herein, the term "gene expression profile" refers to the ability to determine the rate or amount of production of a gene product or the activity of gene transcription in a specific tissue, in a spatial or temporal manner. As used here, the term "proteomic analysis" refers to a protein pattern or arrangement to identify major differences in proteins or peptides in normal and diseased tissues. The additional specimens analyzed are described here. The analyzed term also includes components of a sample that are a direct product of a biochemical means of amplifying the initial target analyzed, such as the product of a nucleic acid amplification reaction.
[0044] [00044] By an "isolated" nucleic acid molecule is meant a nucleic acid molecule that is removed from the environment in which it naturally occurs. For example, a naturally occurring nucleic acid molecule present in the cell genome or as part of a gene bank, is not isolated, however, the same molecule, separated from the rest of the genome, as a result of, for example, a cloning, amplification, or enrichment event is "isolated". Typically, an isolated nucleic acid molecule is free of nucleic acid regions (e.g., coding regions) with which it is immediately contiguous, at the 5 'or 3' ends, in the naturally occurring genome. Such isolated nucleic acid molecules can be part of a vector or a composition and still be isolated, since such a vector or composition is not part of its natural environment.
[0045] [00045] As used herein, "bonded" means attached or bonded by covalent bonds, non-covalent bonds, and / or bonded by Van der Waals forces, hydrogen bonds, and / or other intermolecular forces.
[0046] [00046] The term "magnetic particle" refers to particles including materials of high positive magnetic susceptibility, such as paramagnetic compounds, superparamagnetic compounds, and magnetite, ferric gamma oxide, or metallic iron.
[0047] [00047] As used herein, "non-specific reversibility" refers to the colloidal stability and robustness of magnetic particles against non-specific aggregation in a liquid sample and can be determined by subjecting the particles to the intended test conditions in the absence of a pooling portion specific (that is, an analysand or agglomerator). For example, non-specific reversibility can be determined by measuring the T2 values of a solution of magnetic particles before and after incubation in a uniform magnetic field (defined as <5000 ppm) at 0.45T for 3 minutes at 37 ° Ç. Magnetic particles are considered to have non-specific reversibility if the difference in T2 values before and after subjecting the magnetic particles to the intended test conditions varies by less than 10% (for example, by less than 9%, 8%, 6%, 4%, 3%, 2%, or 1%). If the difference is greater than 10%, then the particles exhibit irreversibility in the buffer, diluents, and matrix tested, and manipulation of the particle and matrix properties (for example, coating and buffer formulation) may be required to produce a system in which the particles have non-specific reversibility. In another example, the test can be applied by measuring the T2 values of a solution of magnetic particles before and after incubation in a gradient magnetic field of 1Gauss / mm-10000Gauss / mm.
[0048] [00048] As used here, the term "NMR relaxation rate" refers to a measurement of any of the following in a T1, T2, T1 / T2 hybrid, T1rho, T2rho, and T2 * sample. The systems and methods of the invention are designed to produce a characteristic NMR relaxation rate if an analyte is present in the liquid sample. In some cases, the NMR relaxation rate is characteristic of the amount of analyte present in the liquid sample.
[0049] [00049] As used here, the term "T1 / T2 hybrid" refers to any detection method that combines a T1 and T2 measurement. For example, the value of a T1 / T2 hybrid can be a composite signal obtained by combining, ratio, or difference between two or more different T1 and T2 measurements. The T1 / T2 hybrid can be obtained, for example, using a pulse sequence in which T1 and T2 are alternatively measured or acquired in an interval mode. In addition, the T1 / T2 hybrid signal can be acquired with a pulse sequence that measures a relaxation rate which is comprised of the mechanisms or both T1 and T2 relaxation rates.
[0050] [00050] A "pathogen" means an agent that causes disease or illness to its host, such as an infectious organism or particle, capable of producing a disease in another organism, and includes, however, is not limited to bacteria, or by-products of pathogen. "Pathogen by-products" are those biological substances that arise from the pathogen that can be harmful to the host or stimulate an excessive host immune response, for example, pathogen antigen (s), metabolic substances, enzymes, biological substances, or toxins.
[0051] [00051] By "analyte associated with the pathogen" is understood an analyte characteristic of the presence of a pathogen (for example, a bacterium, fungus, or virus) in a sample. The analyte associated with the pathogen can be a particular substance derived from a pathogen (for example, a protein, nucleic acid, lipid, polysaccharide, or any other material produced by a pathogen) or a mixture derived from a pathogen (for example, whole cells , or total viruses). In certain cases, the analyte associated with the pathogen is selected because it is characteristic of the genus, species, or specific strain of the pathogen being detected. Alternatively, the analyte associated with the pathogen is selected to verify a property of the pathogen, such as resistance to a particular therapy. For example, the analyte associated with the pathogen may be a gene, such as a Van A gene or Van B gene, characteristic of resistance to vancomycin in several different bacterial species.
[0052] [00052] By "pulse sequence" or "RF pulse sequence" is meant one or more radio frequency pulses to be applied to a sample and designed to measure, for example, certain NMR relaxation rates, such as spin echo sequences . A pulse sequence can also include the acquisition of a signal following one or more pulses to minimize noise and improve the accuracy of the resulting signal value.
[0053] [00053] As used here, the term "signal" refers to an NMR relaxation rate, frequency change, susceptibility measurement, diffusion measurement, or correlation measurements.
[0054] [00054] As used here, reference to the "size" of a magnetic particle refers to the average diameter for a mixture of the magnetic particles as determined by microscopy, light scattering, or other methods.
[0055] [00055] As used herein, the term "substantially monodispersed" refers to a mixture of magnetic particles having a polydispersity in the size distribution as determined by the shape of the particle size distribution curve in light scattering measurements. The FWHM (mean maximum total amplitude) of the particle distribution curve less than 25% of the peak position is considered to be substantially monodisperse. In addition, only one peak should be observed in light scattering experiments and the position of the peak should be within a standard deviation of a population of known monodisperse particles.
[0056] [00056] By "relaxation of T2 per particle" is meant the relaxation of average T2 per particle in a population of magnetic particles.
[0057] [00057] As used here, "unfractionated" refers to an assay in which none of the components of the sample being tested are removed following the addition of magnetic particles to the sample and prior to the NMR relaxation measurement.
[0058] [00058] It is contemplated that the units, systems, methods, and processes of the claimed invention cover the variations and adaptations developed using the information of the modalities described here. Throughout the description, where units and systems are described as having, including, or including specific components, or where processes and methods are described as having, including, or including specific steps, it is contemplated that, in addition, there are units and systems of the present invention that consist essentially of, consist of, and recited components, and that there are processes and methods according to the present invention that essentially consist of, or consist of the recited processing steps. It should be understood that the order of steps or order to perform certain actions is immaterial, unless otherwise specified, as long as the invention remains operable. In addition, in many cases two or more steps or actions can be carried out simultaneously.
[0059] [00059] Other features and advantages of the invention will be evident from the following detailed description, drawings, and claims. Brief Description of Drawings
[0060] [00060] Figure 1A is a schematic diagram of an NMR unit for detecting a response and signal from a sample to an RF pulse sequence, according to an illustrative embodiment of the invention.
[0061] [00061] Figure 1B describes a typical spiral configuration around a sample tube to measure a relaxation signal in a 20 µL sample.
[0062] [00062] Figures 2A-2E illustrate micro spiral geometries that can be used in NMR (for excitation and / or detection); drawings include, but are not limited to, a coiled solenoid spiral (Figure 2A), a planar spiral (Figure 2B), a MEMS solenoid spiral (Figure 2C), a MEMS Helmholz spiral (Figure 2D), and a spiral (Figure 2E), according to an illustrative embodiment of the invention. The manufacture of three-dimensional lithographic spiral of well characterized spirals used in the detection of MR is also established and can be used for these applications, Demas and others "Electronic characterization of lithographically patterned microcoils for high sensitivity NMR detection" J Magn Reson 200: 56 (2009 ).
[0063] [00063] Figure 3A is a drawing describing an aggregation assay of the invention. The magnetic particles (dots) are coated with a binding agent (i.e., antibody, oligo, etc.) such that in the presence of the analyte, or multivalent binding agent, aggregates are formed. The dotted circles represent the diffusion portion or sphere of the total fluid volume that a molecule of the solution can experience through its diffusion during a T2 measurement (the exact path taken by a water molecule is random, and this drawing is not for scale). The aggregation (right side) depletes the sample portions of microscopic magnetic non-uniformities that disrupt the T2 signal from the water, leading to an increase in T2 relaxation.
[0064] [00064] Figure 3B is a graph describing the polydispersed model and showing that T2 will transit between the two points on this curve when the particles form clusters of specific sizes. The response curve will be linear with respect to the addition of the analyzed, however, non-linear with respect to the volume fraction of the clusters, because the particles transit between state 1 and state 2. The slope of the response curve is directly proportional to the sensitivity of the test.
[0065] [00065] Figures 4A-4C are drawings describing different test formats for the tests of the invention. Figure 4A describes an agglomerative sandwich immunoassay in which two populations of magnetic particles are designed to bind to two different epitopes of an analyte. Figure 4B describes a competitive immunoassay in which the assay in a liquid sample binds to a multivalent binding agent (a multivalent antibody), whereby inhibiting aggregation. Figure 4C depicts a hybridization-mediated agglomerative assay in which two populations of particles are designed to bind the first and second portions of a nucleic acid target, respectively.
[0066] [00066] Figure 5 illustrates a modular cartridge concept in sections that can be packaged and stored separately. This is done, for example, so that the input module (shown elevated with the inverted Vacutainer tube attached) can be sterilized while the module holding the reagent in the middle is not. This allows the component containing the reagents to be the only refrigerated component.
[0067] [00067] Figures 6A-6F describe a Vacutainer input module. Figure 6A shows in the inverted position after the user has removed the closure of the Vacutainer tube and placed the cartridge over it. Figure 6B shows the mold in the pathway that the blood will follow outside the Vacutainer and inside the sample loading region once the cartridge is connected on the right side. The metallic seal can be the base of the channels, forming a low-cost molded part with closed channels. Figure 6C is a cross-sectional view showing the ventilation tube that allows air to enter the vial when blood leaves and fills the sample region. Figures 6D-6F describe an input module for sample aliquot designed to interface with uncapped vacutainer tubes, and to aliquot two of a sample volume that can be used to perform, for example, a Candida assay. The inlet module has two hard plastic parts, which are ultrasonically welded together and sealed to form a network of channels to allow a flow path to form within the first well that overflows to the second sample well. A soft part of the vacutainer seal is used for a seal with the vacutainer. It has a hole for the sample flow, and a ventilation hole to allow the flow to occur.
[0068] [00068] Figure 7A is a drawing describing the components of the competitive creatinine assay in Example 6. A magnetic particle decorated with creatinine is used in combination with a creatinine antibody to form an aggregation system. The creatinine present in a liquid sample competes with the magnetic particles for the antibody, leading to a reduction in aggregation with increased creatinine concentration. The change in aggregation is observed as a change in the relaxation rate of T2 of the hydrogen nuclei in the water molecules of the liquid sample. By comparing the T2 relaxation rate of the liquid sample with a standard curve, the creatinine concentration is determined.
[0069] [00069] Figure 7B is a design architecture describing the competitive tacrolimus assay in Example 9.
[0070] [00070] Figure 7C is a drawing describing the architecture of the Candida agglomerative sandwich test of Example 10.
[0071] [00071] Figures 8A-8C are a series of graphs showing the response curve for competitive creatinine assays. Figure 8A is a graph showing a standard curve for the competitive creatinine assay in Example 6 correlating with the T2 relaxation rate observed with the creatinine concentration in the liquid sample. Figure 8B shows the T2 response of a particle decorated with creatinine with 2 different antibody preparations. Preparation 1 is pre-production (with aggregated antibody) and Preparation 2 is purified production (no aggregated antibody is present). Figure 8C shows the T2 response of a particle decorated with non-aggregated antibody, biotinylated antibody and deliberately multimerized antibody, and confirms the increased ability to group multivalent agglomeration agents.
[0072] [00072] Figure 9 is a graph showing a standard curve for the competitive tacrolimus test of Example 9 correlating with the T2 relaxation rate observed for a liquid sample with the tacrolimus concentration in the liquid sample.
[0073] [00073] Figure 10 is a graph depicting a creatinine inhibition curve (see, Example 7) to use an antibody coated particle and a multivalent amino-dextran-creatinine binding agent to induce the cluster competing with any analyzed target ( creatinine) present in the sample to cause particle clustering. The binding agent used is a 40kDa dextran with ~ 10 creatinines per dextran molecule.
[0074] [00074] Figure 11 is a graph describing the evaluation of Tac-dextran conjugates for clustering capacity (see, Example 8) by performing a titration. As noted, such increased molecular weight of Tac-dextran results in the improved T2 signal.
[0075] [00075] Figure 12 is a graph describing evaluation of Tac-dextran conjugates for clustering capacity (see, Example 8) by performing a titration. As noted, the higher substitution improved the T2 signal.
[0076] [00076] Figure 13 is a graph describing the evaluation of Tac-BSA conjugates for pooling capacity (see, Example 8) by performing a titration similar to that used for Tac-dextran conjugates. As noted, cluster performance varies with tacrolimus replacement ratio.
[0077] [00077] Figure 14 is a graph describing a result of the T2 assays to detect antibiotin antibody using the magnetic particles prepared in the blood and PBS matrices as described in Example 1.
[0078] [00078] Figure 15 is a graph describing the results of T2 assays to detect the antibiotin antibody using magnetic particles prepared with (open circle) and without (filled circle) a protein block as described in Examples 8 and 9.
[0079] [00079] Figure 16 is a graph describing the results of T2 assays to detect the antibiotin antibody using magnetic particles prepared having a BSA block (triangle, square, dark filled diamond) or an FSG block (circle and dark gray X's) as described in Example 2.
[0080] [00080] Figures 17A and 17B are schematic of supplied particle coverings.
[0081] [00081] Figures 18A-18B describe the results of T2 assays to detect biotin in a competitive assay format described in Example 4. Figure 18A describes the experimental results in the buffer; at the same time as Figure 18B describes the experimental results in lysed blood.
[0082] [00082] Figure 19A is a table and 19B is a graph describing a repeatability of Candida measurements by methods of the invention over a period of eight days. To determine the repeatability of T2 measurement in human whole blood infected with C. albicans, we conducted a day eight study in which the same donor who reached the maximum and amplified the sample, was hybridized to the superparamagnetic particles (n = 3) every day and the resulting T2 values were recorded (see, Example 13). The precision in execution shown in Figure 19A and in general is according to CV’s of all measurements less than 12%. The repeatability observed during the eight-day course is shown in Figure 19B (mean T2 values +/- the 95% confidence intervals measured from the same donor who reached the maximum u amplified the samples during the eight-day course) with CVs less than 10% across the Candida concentration range and 6% for the negative control.
[0083] [00083] Figure 20 is a schematic that describes the workflow for detecting a bacterial or fungal pathogen in a whole blood sample (see, Examples 14 and 17).
[0084] [00084] Figures 21A and 21B are graphs describing the results of donor samples. Figure 21A is a graph describing the results obtained from 16 experiments designed to assess assay performance on 6 different donor blood samples that peaked with a range of C. albicans cells (see, Example 13). Each data point is the average +/- of the 95% confidence interval (n = 48). At the lowest test concentration (10 cells / mL), we failed to detect Candida albicans 37% of the time (6 out of 16 experiments); however, 100 cells / mL of Candida albicans was detected 100% of the time. This suggests that the assay can strongly detect at concentrations of C. albicans greater than or equal to 100 cells / mL with no major performance inhibition introduced through the donor's blood samples. Figure 21B is a graph depicting a result obtained from 7 experiments designed to assess the performance of the assay on 6 blood samples from different donors that peaked with a range of C. krusei cells (see, Example 13). Each data point is the average +/- of the 95% confidence interval (n = 21). We detected at 10 cells / mL in any of the experimental runs, however, we detected at 100 cells / mL for all experimental runs. This suggests the LOD between 10 and 100 cells / ml.
[0085] [00085] Figure 22 is a point diagram showing the T2 values measured for five clinical isolates of C. albicans that peaked at 400 pL of whole blood at concentrations ranging from 0 to 1E4 cells / mL. The plotted results are the average +/- 1SD. The data indicate, despite the dispersion of absolute T2 values obtained between the different isolates, at 50 cells / mL all values are above those of the control without Candida (3 replicate measurements from 20 independent tests, total of 60 different cluster reactions) .
[0086] [00086] Figures 23A and 23B are ROC plots of T2 results generated in 10 cells / mL (Figure 23A) and 50 cells / mL (Figure 23B). The area under the curve at 10 cells / mL is 0.72 (95CI = 0.56 to 0.88) while at 50 cells / mL the area under the curve is 0.98 (95CI = 0.95 to 1.001). The area under the curve is generally used to quantify the accuracy of the diagnosis; in this case our ability to discriminate between a Candidemic patient with an infection of 10 cells / mL or 50 cells / mL versus a patient without Candidemia. At 10 cells / mL the area under the curve is 0.72 which means that if the T2 assay was performed on a randomly chosen person with Candidemia at an infection level of 10 cells / mL, there is a 72% chance of its T2 value is greater than that of a person without Candidemia. The clinical accuracy of the test is much higher at 50 cells / mL with the area under the curve at 0.98. Again indicating that in a person with Candidemia at this level of infection, the T2 test would determine a higher value than a sample from a patient without Candidemia 98% of the time. See, Example 13.
[0087] [00087] Figure 24 is a graph describing the sensitivity of the assay using the standard thermocycle (~ 3 hours response time) and a process that combines the annealing / stretching steps (~ 2 hours, 13 minutes response time). The combination of the annealing and stretching steps in thermocycling reduces the total TAT test to 2.25 hours without compromising the test sensitivity.
[0088] [00088] Figure 25 is a graph depicting a change in the T2 signal with PCR cycling (see, Example 14). The results demonstrate that the methods and systems of the invention can be used to perform PCR in real time and provide quantitative information on the amount of target nucleic acid present in a sample.
[0089] [00089] Figure 26 is a series of photographs showing a simple magnetic separator / PCR block insert.
[0090] [00090] Figure 27 is an image showing the amount of DNA generated by amplification of (1) 100 copies of genomic C. albicans amplified in the presence of 3 'and 5' single probe nanoparticles of C. albicans; the particles were kept on the side wall during PCR through the magnetic field, (2) 100 copies of amplified genomic C. albicans without nanoparticles, and (3) 100 copies of genomic amplified C. albicans in the presence of 3 'and 5' nanoparticles of single probe of C.albicans; in the magnetic field.
[0091] [00091] Figures 28A-28E are schematic views of a sample tube containing an immobilized portion of magnetizable metal foam (shaded), magnetic particles (circles), and analyzed (triangles). A magnetizable metal foam, for example, made of nickel, can be inserted into a duct and immobilized by exposure to heat, which contracts the conduit around the metal foam, resulting in an impermeable seal. A sample containing magnetic particles and analyzed is then inserted at one end of the conduit (Figure 28A). Then, the conduit is exposed to a magnet (Figure 28B), and the magnetic particles are attracted by the metal foam and become magnetically captured in its pores, or crevices. The average pore diameter in the metal foam is, for example, between 100-1000 microns. The analyzed molecules can be charged to the metal foam by attaching to a magnetic particle, or the fluid can be forced through the metal foam to reach the captured magnetic particles. At the same time that they are captured in the metal foam, the magnetic particles have enhanced interactions, since they are now confined and are closer to the other magnetic particles, and the cluster formations are enhanced. The metal foam is then demagnetized (Figure 28C), that is, the magnetic field of the metal foam becomes insignificant. The magnetic particles and analyzed particle complexes largely remain in the metal foam, since the diffusion of magnetic particle agglomerates is relatively low, although some natural diffusion of the analyzed in and out of the metal foam occurs (Figure 28D). Alternatively, the magnetizable metal foam (hollow cylinder) is free floating in the sample tube with the magnetic particles (circles), and analyzed (stars). The magnetization and demagnetization of free-floating metal foam is used to increase the rate of aggregate formation.
[0092] [00092] Figure 29 is a table describing the T2MR results for 32 clinical specimens indicates that fourteen specimens are positive for Candida. The test identifies four specimens containing C. krusei or C. glabrata, seven specimens containing C. albicans or C. tropicalis, and three containing C. parapsilosis. A solid black line indicates the decision threshold (T2 = 128 msec) (see, Example 16). Detailed Description
[0093] [00093] The invention features systems, devices, and methods for the rapid detection of analyte or determination of analyte concentration in a sample. The systems and methods of the invention employ magnetic particles, an NMR unit, optionally one or more incubation stations at different temperatures, optionally one or more vortexes, optionally one or more centrifuges, optionally a fluid handling station, an optionally robotic system, and optionally one or more modular cartridges. The systems, devices and methods of the invention can be used to test a biological sample (for example, blood, sweat, tears, urine, saliva, semen, serum, plasma, cerebrospinal fluid (CSF), feces, vaginal fluid or tissue, sputum , nasopharyngeal aspirate or mop, tear fluid, mucus, or epithelial mop (oral mop), tissues, organs, bones, teeth, or tumors, among others). Alternatively, the systems, devices, and methods of the invention are used to monitor an environmental condition (for example, plant growth hormone, insecticides, artificial or environmental toxins, nucleic acid sequences that are important for insect susceptibility / resistance, algae and algae subgroups), as part of a bioremediation program, for use on plants or farm animals, or to identify environmental hazards. Similarly, the systems, devices, and methods of the invention are used to detect and monitor biological warfare or biological warfare agents, such as ricin, Salmonella typhimurium, botulinum toxin, aflatoxin, mycotoxins, Francisella tularesis, smallpox, anthrax, or others.
[0094] [00094] The magnetic particles can be coated with a binder portion (i.e., antibody, oligonucleotide, aptamer etc.) such that in the presence of the analyte, or multivalent binding agent, the aggregates are formed. The aggregation depletes the sample portions of the microscopic magnetic non-uniformities that disrupt the T2 signal from the solvent, leading to an increase in T2 relaxation (see, Figure 3).
[0095] [00095] The T2 measurement is a single measurement of all turns in the set, the measurements typically lasting 1-10 seconds, which allows the solvent to travel hundreds of microns, a long distance relative to microscopic non-uniformities in the liquid sample. Each solvent molecule samples a volume in the liquid sample and the T2 signal is an average (total net signal) of all (nuclear gyrations) in the solvent molecules in the sample; in other words, the T2 measurement is a net measurement of the total environment experienced by a solvent molecule, and is an average measurement of all microscopic non-uniformities in the sample.
[0096] [00096] The relaxation rate of T2 observed for the solvent molecules in the liquid sample is dominated by magnetic particles, which in the presence of a magnetic field form high magnetic dipole moments. In the absence of magnetic particles, the relaxation rates of T2 observed for a liquid sample are typically long (ie, T2 (water) = ~ 2000 ms, T2 (blood) = ~ 1500 ms). When the particle concentration increases, the microscopic non-uniformities in the sample increase and the diffusion of the solvent through these microscopic non-uniformities leads to an increase in the rotational decoherence and a decrease in the T2 value. The observed T2 value depends on the particle concentration in a non-linear way, and on the relaxation per particle parameter.
[0097] [00097] In the aggregation tests of the invention, the number of magnetic particles, and if the number of aggregated particles is present, remains constant during the test. The spatial distribution of the particles changes when the particles clump together. Aggregation changes the average "experiment" of a solvent molecule because the particle location in the aggregates is promoted, rather than more uniform particle distributions. At a high degree of aggregation, many solvent molecules do not experience microscopic non-uniformities created by magnetic particles and T2 is close to that of solvent. When the fraction of aggregated magnetic particles increases in a liquid sample, the observed T2 is the average of the non-uniform suspension of aggregated and unique (non-aggregated) magnetic particles. The assays of the invention are designed to maximize the change in T2 with aggregation to increase the sensitivity of the assay to the presence of analyzed, and differences in the concentration of the analyzed.
[0098] [00098] There are two regimes for particle grouping and T2 affects based on the particle size (see, Figure 3B, the threshold is typically approximately 100 nm of particle diameters). For any given assay of a liquid sample the particle count for magnetic particles 250 nm in size can be approximately 1 × 107 particles, whereas for magnetic particles 30 nm in size it can be approximately 1 × 1013. This is because the smaller particles have less relaxation per particle (for the same type of material), resulting in an inherent sensitivity disadvantage. In a typical assay of the invention, the magnetic particles are selected such that T2 increases with an increase in the fraction of aggregated particles.
[0099] [00099] The test of the invention can be designed to change the direction of T2 in the presence of the analyzed (see Figures 4A-4C). For example, the assay can be an agglomerative sandwich immunoassay in which two populations of magnetic particles bind to different epitopes of an analyte (see, Figure 4A); a competitive assay in which the subject is competing with a multivalent binding agent to inhibit the aggregation of magnetic particles (see Figure 4B); or a hybridization-mediated cluster in which two populations of magnetic particles attach to a first and second portion of an oligonucleotide (see Figure 4C). The additional competitive format may include when two particle agglutination portions bond without the agglomerator (for example, DNA oligonucleotides are designed so that two nanoparticles have two different oligos and they can ring together and when heated the analyzed or amplicon or target DNA competes or breaks the np ring).
[0100] [000100] Other formats to carry out the assays of the invention can be used, such as: (i) a target sample can be incubated in the presence of a magnetic particle that has been decorated with specific agglutinating portions for a target analyte and a screening agent. multivalent binding, in an inhibition assay the binding of the analyzed to the magnetic particles blocks the agglomeration of the magnetic particles with the multivalent binding agent; (ii) a target sample can be incubated in the presence of a magnet that has been decorated with the specific agglutinating portions for a target analyte and a multivalent binding agent, in a disintegration test the analyte is exposed to a preformed aggregate the multivalent binding agent and the magnetic particle and the analyzed one displaces the multivalent binding agent to reduce the aggregation in the liquid sample; or (iii) a target sample can be incubated in the presence of a magnetic particle that has been decorated with the specific agglutinating portions for a target analyte and the target analyte alone to form a unique self-assembling population of magnetic particles, in an assay of inhibition or disaggregation test the presence of the analyte's binding to the magnetic particles blocks the auto-agglomeration of the magnetic particles; or (iv) a target sample can be incubated in the presence of a soluble agglomeration agent and a magnetic particle decorated with the analyte or analogue of the analyte, in an inhibition test the presence of the analyte binds the soluble agglomeration agent blocking the agglomeration of the patches.
[0101] [000101] Where a multivalent binding agent (binder) is employed, multiple analyzed are bound to a vehicle (for example, a simple synthetic scaffold, or a larger vehicle or polysaccharide protein, such as BSA, transferrin, or dextran). Magnetic particles
[0102] [000102] The magnetic particles described here include those described, for example, in United States Patent No. 7,564,245 and United States Patent Application Publication No. 20030092029, each of which is incorporated herein by reference. The magnetic particles are generally in the form of conjugates, that is, a magnetic particle with one or more binding moieties (for example, an oligonucleotide, nucleic acid, polypeptide, or polysaccharide) attached thereto. The binding portion causes a specific interaction with a target analyte. The binding portion specifically binds to a selected target analyte, for example, a nucleic acid, polypeptide, or polysaccharide. In some cases, the bond causes the conjugates to aggregate, resulting in a change, for example, a decrease (for example, in the case of smaller magnetic particles) or an increase (for example, in the case of larger magnetic particles) in the time of gyro-gyro relaxation (T2) of adjacent water protons in an aqueous solution (or protons in a non-aqueous solvent). Alternatively, the analyte binds to a preformed aggregate in a competitive breakdown test (for example, an aggregate formed from a multivalent bonding agent and magnetic particles), or competes with a multivalent bonding agent for bonding portions in the particles in an inhibition test (that is, the formation of aggregates is inhibited in the presence of the analyte).
[0103] [000103] Conjugates have high relaxation due to the superparamagnetism of their iron, metal oxide, or other iron or ferrimagnetic materials. The compounds of iron, cobalt, and nickel and their alloys, rare earth elements such as gadolinium, and certain intermetallics, such as gold and vanadium are ferromagnets that can be used to produce superparamagnetic particles. The magnetic particles can be monodispersed (a single crystal of a magnetic material, for example, metal oxide, such as superparamagnetic iron oxide, by magnetic particle) or polydispersed (for example, a plurality of crystals per magnetic particle). Magnetic metal oxide can also include cobalt, magnesium, zinc, or mixtures of these metals with iron. The important elements and aspects of magnetic particles that are useful for producing the conjugates include: (i) a high relaxation, that is, a strong effect on relaxation of water (or other solvent), (ii) a functional group to which the portion of bond may be covalently attached, (iii) a low non-specific bond of interactive portions to the magnetic particle, and / or (iv) stability in the solution, that is, the magnetic particles remain suspended in the solution, have not precipitated and / or the nps maintain their ability to be employed in the method described (ie, nps have a shelf life).
[0104] [000104] Magnetic particles can be linked to the bonding portions through functional groups. In some embodiments, the magnetic particles can be associated with a polymer that includes functional groups selected, in part, to enhance the non-specific reversibility of magnetic particles. The polymer can be a synthetic polymer, such as, but not limited to, polyethylene glycol or silane, natural polymers, or derivatives of synthetic or natural polymers, or a combination thereof. The polymer can be hydrophilic. In some embodiments, the polymer "coating" is not a continuous film around the magnetic metal oxide, however, it is a "mesh" or "cloud" of extended polymer chains trapped and surrounding the metal oxide. The polymer can include polysaccharides and derivatives, including dextran, pullanan, carboxidextran, carboxymethyl dextran, and / or reduced carboxymethyl dextran. The metal oxide can be a collection of one or more crystals that contact each other, or that are individually captured or surrounded by the polymer.
[0105] [000105] Alternatively, the magnetic particles can be associated with non-polymeric functional group compositions. The methods for synthesizing stabilized magnetic particles, functionalized without associated polymers are described, for example, in Halbreich et al., Biochimie, 80: 379 (1998).
[0106] [000106] Magnetic particles typically include mono and polycrystals of metal oxide of about 1-25 nm, for example, about 3-10 nm, or about 5 nm in diameter per crystal. The magnetic particles can also include a polymer component in the form of a core and / or coating, for example, about 5 to 20 nm thick or more. The total size of the magnetic particles can be, for example, 20 to 50 nm, 50 to 200 nm, 100 to 300 nm, 250 to 500 nm, 400 to 600 nm, 500 to 750 nm, 700 at 1,200 nm, from 1,000 to 1,500 nm, or from 1,500 to 2,000 nm.
[0107] [000107] Magnetic particles can be prepared in a variety of ways. It is preferred that the magnetic particle has functional groups that connect the magnetic particle to the binding portion. Carboxy-functionalized magnetic particles can be made, for example, according to a Gorman method (see PCT Publication No. WO00 / 61191). In this method, reduced carboxymethyl (CM) dextran is synthesized from commercial dextran. CM-dextran and iron salts are mixed together and are then neutralized with ammonium hydroxide. The resulting carboxy-functionalized magnetic particles can be used to couple amino-functionalized oligonucleotides. Carboxy-functionalized magnetic particles can also be made of polysaccharide-coated magnetic particles by reacting with bromine or chloroacetic acid on a strong base to trap carboxyl groups. In addition, carboxy-functionalized particles can be made of amino-functionalized magnetic particles by converting amino to carboxy groups by using reagents such as succinic anhydride or maleic anhydride.
[0108] [000108] The magnetic particle size can be controlled by adjusting the reaction conditions, for example, using low temperature during the neutralization of iron salts with a base as described in United States Patent No. 5,262,176. Materials of uniform particle size can also be made by fractionating the particles using centrifugation, ultrafiltration, or gel filtration, as described, for example, in United States Patent No. 5,492,814.
[0109] [000109] Magnetic particles can also be synthesized according to a Molday method (Molday, RS and D. MacKenzie, "Imunospecific ferromagnetic iron-dextran reagents for the labeling and magnetic separation of cells," J. Imunol. Methods, 52 : 353 (1982)), and treated with periodate to form aldehyde groups. The magnetic particles containing aldehyde can then be reacted with a diamine (for example, ethylene diamine or hexanediamine), which will form a Schiff's base, followed by reduction with sodium borohydride or sodium cyanoborohydride.
[0110] [000110] Dextran-coated magnetic particles can be made and cross-linked with epichlorohydrin. The addition of ammonia reacts with epoxy groups to generate amine groups, see Hogemann, D., et al., Improvement of MRI probes to allow efficient detection of gene expression Bioconjug. Chem., 11: 941 (2000), and Josephson et al., "High-efficiency intracellular magnetic labeling with novel superparamagnetic-Tat peptide conjugates," Bioconjug. Chem., 10: 186 (1999). This material is known as cross-linked iron oxide or "CLIO" and when functionalized with amine it is referred to as amine-CLIO or NH2-CLIO. Carboxy-functionalized magnetic particles can be converted to amino-functionalized magnetic particles by using water-soluble carbodiimides and diamines such as ethylene diamine or hexane diamine.
[0111] [000111] Magnetic particles can be formed from an iron fluid (ie, a stable colloidal suspension of magnetic particles). For example, the magnetic particle can be a composite of including multiple metal oxide crystals of the order of a few tens of nanometers in size and dispersed in a fluid containing a surfactant, which adsorb on the particles and stabilize in them, or by precipitation, in a basic medium, a solution of metal ions. Suitable iron fluids are sold by Liquids Research Ltd. Under the references: WHKS1S9 (A, B or C), which is a water-based iron fluid including magnetite (Fe3O4), having particles of 10 nm in diameter; WHJS1 (A, B or C), which is an iron fluid based on isoparaffin including magnetite particles (Fe3O4) of 10 nm in diameter; and dextran BKS25, which is a water-based iron fluid stabilized with dextran, including 9 nm diameter magnetite (Fe3O4) particles. Other iron fluids suitable for use in the systems and methods of the invention are oleic acid stabilized iron fluids provided by Ademtech, which includes approximately 70% by weight of a-Fe2O3 particles (approximately 10 nm in diameter), 15% by weight octane, and 15% by weight of oleic acid.
[0112] [000112] Magnetic particles are typically a composite including multiple metal oxide crystals and an organic matrix, and having a surface decorated with functional groups (i.e., amine groups or carboxy groups) for the attachment of the bonding portions to the surface of the magnetic particle. For example, the magnetic particles useful in the methods of the invention are those commercially available from Dynal, Seradyn, Kisker, Miltenyi Biotec, Chemicell, Anvil, Biopal, Estapor, Genovis, Thermo Fisher Scientific, JSR micro, Invitrogen, and Ademtech, as well as those described United States Patent Nos. 4,101,435; 4,452,773; 5,204,457; 5,262,176; 5,424,419; 6,165,378; 6,866,838; 7,001,589; and 7,217,457, each of which is incorporated herein by reference.
[0113] [000113] Avidin or streptavidin can be attached to magnetic particles for use with a biotinylated binding moiety, such as an oligonucleotide or polypeptide (see, for example, Shen et al., "Magnetically labeled secretin retains receptor affinity to pancreas acinar cells," Bioconjug Chem., 7: 311 (1996)). Similarly, biotin can be attached to a magnetic particle for use with a binding portion labeled by avidin. Alternatively, the bonding portion is covalently attached to the surface of the magnetic particle; the particles can be decorated with IgG molecules; the particles can be decorated with anti-his antibodies; or the particles can be decorated with FAbs labeled by his.
[0114] [000114] Low molecular weight materials can be separated from magnetic particles by ultrafiltration, dialysis, magnetic separation, or other means before use. For example, binding agents and unreacted binding moieties can be separated from those of magnetic particle conjugates by magnetic separation or size exclusion chromatography. In certain cases the magnetic particles can be fractionated by size to produce mixtures of particles of a particular average size and diameter range.
[0115] [000115] For certain assays requiring high sensitivity, detection of the analyte using T2 relaxation assays may require the selection of an appropriate particle to allow sufficiently sensitive analyte-induced agglomeration. Higher sensitivities can be achieved using particles containing multiple superparamagnetic iron oxide cores (5-15 nm in diameter) within a single larger polymer matrix or iron fluid assembly (100 nm-1200 nm in total diameter, such as particles having an average diameter of 100 nm, 200 nm, 250 nm, 300 nm, 500 nm, 800 nm, or 1000 nm), or using particles or materials of higher magnetic moment with higher density, and / or particle with high iron content. Without being limited to theory, it is postulated that these types of particles provided a sensitivity gain of more than 100 × due to a much larger number of iron atoms per particle, which is believed to lead to an increase in sensitivity due to decreased number of particles present in the test solution and possibly a higher amount of superparamagnetic iron affected by each cluster event.
[0116] [000116] Particle relaxation and particle size is a useful term for selecting an ideal particle for high sensitivity assays. Ideally, this term will be as broad as possible. Particle relaxation is a measurement of the effect of each particle on the measured T2 value. The higher this number, the smaller the number of particles needed to produce a given T2 response. In addition, reducing the concentration of particles in the reactive solution can improve the analytical sensitivity of the assay. Particle relaxation may be a more useful parameter because the density of iron and relaxation can vary from magnetic particle to magnetic particle, depending on the components used to prepare the particles (see, Table 1). Particle relaxation is proportional to the saturation magnetization of a superparamagnetic material.
[0117] [000117] The base particle for use in the systems and methods of the invention can be any of the commercially available particles identified in Table 2.
[0118] [000118] The magnetic particles for use in the systems and methods of the invention can have a hydrodynamic diameter from 10 nm to 1200 nm, and containing on average 8x102 - 1x1010 metal atoms per particle, and having a relaxation per particle of 1 × 104 - 1x1013 mM-1s-1. The magnetic particles used in the systems and methods of the invention can be any of the drawings, composites, or sources described above, and can also be modified as described here for use as an MRI change.
[0119] [000119] In addition to particle relaxation, several other practical issues must be addressed in the selection and design of magnetic particles for tests of high analytical sensitivity.
[0120] [000120] For example, the use of large particles (i.e., 1000 nm or greater) may be desired to maximize the iron content and relaxation per particle. However, we observed that particles of this size tend to settle quickly out of solution. We observed that particle sedimentation does not typically interfere with the assay if the magnetic particle sizes are kept below 500 nm. When the use of a particle above 500 nm in the described tests or smaller particles with high density are employed, sedimentation is monitored and the effect on the T2 measurement is determined. It has been found that a magnetic particle size of about 100-300 nm is ideal for stability in terms of sedimentation, even after functionalization (increasing the hydrodynamic diameter to 300 nm by approximately 50 nm), and provides high sensitivity allowed by high particle relaxation. The particle density certainly plays a role in the fluctuation. As such, the relative density of the solution and particles plays an important role in the sedimentation of the particle. Consequently, a possible solution to this problem is the use of floating magnetic particles (i.e., a hollow particle, or particle containing both a low density matrix and high density metal oxide). Sedimentation can affect T2 detection, so solution additives can be used to change the particle's ratio to solution density. T2 detection can be impacted by sedimentation if there is a significant proportion of the superparamagnetic material in the measured volume of the liquid. Sedimentation can be assessed by diluting the particles to a concentration such that the UV-Vis absorbance at 410 nm is between 0.6-0.8 absorbance units and then the absorbance is monitored for 90 minutes. If sedimentation occurs, the difference between the initial and final absorbances divided by the initial absorbance will be greater than 5%. If the% of sedimentation is above 5% then the particles are typically not suitable for use in assays requiring high analytical sensitivity. The magnetic particles used in the tests of the invention may, however, not be limited to the magnetic particles of non-settling. High sedimentation represents handling difficulties and can lead to reproducibility problems.
[0121] [000121] For magnetic particles in the order of 100 nm or greater, the multiple superparamagnetic iron oxide crystals that typically include the particle core result in a liquid dipole moment when in the presence of external magnetic fields, ie the dipole moment is a enough strength to overcome the Brownian movement. Non-specific reversibility is a measure of colloidal stability and robustness against non-specific aggregation. Non-specific reversibility is assessed by measuring the T2 values of a solution of particles before and after incubation in a uniform magnetic field (defined as <5000 ppm). Initial T2 values are typically 200 ms for a particle with an iron concentration of 0.01 mM Fe. If the difference in T2 values before and after incubation in the uniform magnetic field is less than 20 ms, the samples are considered reversible. In addition, 10% is a threshold allowing initial T2 measurements to reflect the particle concentration in the assay. If the difference is greater than 10%, then the particles will exhibit irreversibility in the tested buffer, matrices and diluents. The non-specific reversibility of the magnetic particles can be changed as described here. For example, colloidal stability and robustness against nonspecific aggregation can be influenced by the characteristics of the particles, the binding portions, the test buffer, the matrix and the test processing conditions. The conservation of colloidal stability and resistance to non-specific binding can be altered by chemical conjugation, blocking methods, buffer modifications, and / or changes in the processing conditions of the assay.
[0122] [000122] It was observed that a very important attribute for robust and reproducible tests is the monodispersity in the size distribution of the used magnetic particles, a distinction observed in post-coating of polydispersed particles versus pre-coating of monodispersed particles. Polydispersed batches of magnetic particles may require reproducibility and compromise sensitivity. Polydispersed samples can also present problems in terms of obtaining uniform coatings. For certain highly sensitive assays it is desirable for the magnetic particles to be substantially monodispersed in the size distribution (i.e., having a polydispersity index less than about 0.8 - 0.9). Alternatively, the assays of the invention can be designed to accommodate the use of polydisperse magnetic particles.
[0123] [000123] It has been determined that the assays of the invention require monitoring of a change in the cluster states of the agglomeration assays and that the measurement of a change in the cluster is likely to require a significant fraction of clustered particles (for example, considered to be> 1 -10%), the total number of particles in an assay should be minimized to allow for higher sensitivity. However, a sufficient number of particles must be present to allow the use of the T2 dynamic detection range. It was found that superior sensitivity is observed when the number of magnetic particles (or molar equivalent) is approximately in the same order of magnitude as the number (or molar equivalent) of the analyte being detected, and the magnitude of the number (or molar equivalent) of multivalent binding agents employed (ie, in an inhibition assay).
[0124] [000124] For proteinaceous samples it may also be required to modify the surface of the magnetic particle to reduce the non-specific binding of base proteins to the magnetic particles. The non-specific binding of base proteins to the particles can induce or prevent particle clustering, resulting in false signals and / or absence of false signals. For example, in some cases the surface of the magnetic particle may include blocking agents covalently attached to the surface of the magnetic particle, which reduce non-specific binding of base proteins. There are a variety of agents that one can use to achieve the desired effect, and in some cases, it is an ideal combination of agents (see, Table 3; exemplary particles, and binding portions).
[0125] [000125] Thus, it was discovered that a block of protein may be required to obtain sensitivity and assay activity, particularly in proteinaceous samples (for example, plasma samples or whole blood samples), which is comparable with sample results. non-proteinaceous buffer. Some commonly used protein blockers that can be used in the preparations provided include, for example, bovine serum albumin (BSA), fish skin gelatin (FSG), bovine gamma globulin (BGG), lysozyme, casein, peptidase, or milk skimmed-milk powder. In certain embodiments, a magnetic particle coating includes BSA or FSG. In other embodiments, a combination of coatings are combinations of those exemplary coatings listed in Table 3.
[0126] [000126] In addition, non-specific binding may be due to lipids or other non-proteinaceous molecules in the biological sample. For non-specific binding mediated by non-proteinaceous, changes in pH and ionic resistance of the buffer can be selected to enhance the repulsive forces of the particle, however, not enough to limit the results of the intended interactions.
[0127] [000127] Test reagents
[0128] [000128] The assays of the invention can include reagents to reduce non-specific binding to magnetic particles. For example, the assay can include one or more proteins (for example, albumin (for example, human or bovine albumin), fish skin gelatin, lysozyme, or transferrin); low molecular weight amines (<500 Daltons) (for example, amino acids, glycine, ethylamine, or mercaptoethanol amine); and / or water-soluble non-ionic surfactants (for example, polyethylene glycol, Tween® 20, Tween® 80, Pluronic®, or Igepal®).
[0129] [000129] The surfactant can be selected from a wide variety of soluble non-ionic surfactants including surfactants that are generally commercially available under IGEPAL trade name GAF Company. IGEPAL's liquid non-ionic surfactants are composed of polyethylene glycol p-isooctylphenyl ether and are available in various molecular weight designations, for example, IGEPAL CA720, IGEPAL CA630, and IGEPAL CA890. Other suitable nonionic surfactants include those available under the trade name TETRONIC 909 from BASF Wyandotte Corporation. This material is a tetrafunctional block copolymer surfactant ending in primary hydroxyl groups. Suitable nonionic surfactants are also available under the trade name VISTA ALPHONIC from Vista Chemical Company and such materials are ethoxylates which are nonionic biodegradable products derived from linear primary alcohol mixtures of various molecular weights. The surfactant can also be selected from poloxamers, such as polyoxyethylene-polyoxypropylene block copolymers, such as those available under the trade names Synperonic PE series (ICI), Pluronic® series (BASF), Supronic, Monolan, Pluracare, and Plurodac, polysorbate surfactant, such as Tween® 20 (sorbitan monolaurate PEG-20), and glycols such as ethylene glycol and propylene glycol.
[0130] [000130] Such non-ionic surfactants can be selected to provide an appropriate amount of detergency for an assay without having a detrimental effect on assay reactions. In particular, surfactants can be included in a reaction mixture for the purpose of suppressing non-specific interactions between various ingredients in the aggregation assays of the invention. Nonionic surfactants are typically added to the liquid sample before an amount of 0.01% (weight / weight) to 5% (weight / weight).
[0131] [000131] Non-ionic surfactants can be used in combination with one or more proteins (eg albumin, fish skin gelatin, lysozyme, or transferrin) also added to the liquid sample before an amount and 0.01% (weight / weight) to 5% (weight / weight).
[0132] [000132] In addition, the assays, methods, and cartridge units of the invention may include additional suitable buffer components (e.g., Tris base, selected to provide a pH of about 7.8 to 8.2 in the reaction environment ); and chelating agents to recover cations (for example, EDTA disodium, ethylene diamine tetraacetic acid (EDTA), citric acid, tartaric acid, glucuronic acid, saccharic acid or suitable salts thereof). Connection Portions
[0133] [000133] In general, a binding moiety is a molecule, synthetic or natural, that specifically binds or otherwise attaches, for example, covalently or non-covalently to bind to or hybridize to, a target molecule, or another binding portion (or, in certain embodiments, with an aggregation-inducing molecule). For example, the binding portion may be an antibody directed to an antigen or any protein-protein interaction. Alternatively, the linker may be a polysaccharide that binds to a corresponding target or a synthetic oligonucleotide that hybridizes to a specific complementary nucleic acid target. In certain embodiments, the linking moieties can be designated or selected to serve, when linked to another linking moiety, as substrates for a target molecule such as enzyme in the solution.
[0134] [000134] Binding moieties include, for example, oligonucleotide binding moieties (DNA, RNA, or substituted or derived nucleotide substitutes), polypeptide binding moieties, antibody binding moieties, aptamers, and binding moieties polysaccharide. Oligonucleotide Binding Portions
[0135] [000135] In certain embodiments, the bonding portions are oligonucleotides, attached / attached to the magnetic particles using any of a variety of chemicals, by a single bond, for example, covalent, for example, at the 3 'or 5' end a functional group in the magnetic particle. Such binding portions can be used in the systems, devices, and methods of the invention to detect mutations (for example, SNPs, translocations, large deletions, small deletions, insertions, substitutions) or to monitor gene expression (for example, the presence expression, or changes in the level of gene expression, monitoring RNA transcription), or CHP analysis characteristic of the presence of a pathogen, sick state, or disease progression.
[0136] [000136] An oligonucleotide binding portion can be constructed using chemical synthesis. A double-stranded DNA binding moiety can be constructed by enzymatic binding reactions using procedures known in the art. For example, a nucleic acid (for example, an oligonucleotide) can be chemically synthesized using naturally occurring nucleotides or diversely modified nucleotides designed to increase the biological stability of molecules or to increase the physical stability of the duplex formed between complementary strains, for example , phosphorothioate derivatives and acridine-substituted nucleotides can be used. Nucleic acid can also be produced biologically using an expression vector in which a nucleic acid has been subcloned.
[0137] [000137] One method uses at least two populations of magnetic oligonucleotide particles, each with strong effects on relaxation with water (or another solvent). When the oligonucleotide-magnetic particle conjugates react with a target oligonucleotide, they form aggregates (for example, clusters of magnetic particles). At prolonged rest, for example, at night at room temperature, the aggregates form large clusters (micron-sized clusters). Using the methods of the invention, the formation of large clusters can be accomplished more quickly by employing multiple cycles of magnetic assisted agglomeration. Magnetic resonance imaging is used to determine the relaxation properties of the solvent, which are altered when the mixture of magnetic particles of magnetic oligonucleotide reacts with a target nucleic acid to form aggregates.
[0138] [000138] Certain embodiments employ a mixture of at least two types of magnetic metal oxide magnetic particles, each with a specific oligonucleotide sequence, and each with more than one copy of the oligonucleotide trapped, for example, covalently, by a magnetic particle . For example, the assay protocol may involve preparing a mixture of populations of oligonucleotide-magnetic particle conjugates and reacting the mixture with a target nucleic acid. Alternatively, the oligonucleotide-magnetic particle conjugates can be reacted with the target in a sequential manner. Certain modalities characterize the use of magnetic resonance to detect the reaction of the oligonucleotide-magnetic particle conjugates with the target nucleic acid. When a target is present, the dispersed conjugates come together to form small aggregates.
[0139] [000139] For example, the oligonucleotide bonding moieties can be bonded to the metal oxide via covalent bonding to a functionalized polymer or to the metal oxides functionalized by a non-polymeric surface. In the latter method, magnetic particles can be synthesized according to a method by Albrecht et al., Biochimie, 80: 379 (1998). Dimercapto-succinic acid is coupled is coupled to iron oxide and provides a carboxyl functional group.
[0140] [000140] In certain embodiments, the oligonucleotides are attached to the magnetic particles through a functionalized polymer associated with the metal oxide. In some embodiments, the polymer is hydrophilic. In certain embodiments, conjugates are made using oligonucleotides that have terminal amino, sulfhydryl, or phosphate groups, and superparamagnetic iron oxide magnetic particles containing amino or carboxy groups in a hydrophilic polymer. There are several methods for synthesizing magnetic particles derived from carboxy and amino.
[0141] [000141] In one embodiment, the oligonucleotides are attached to a particle through interaction of the ligand-protein bond, such as biotin-streptavidin, where the ligand is covalently attached to the oligonucleotide and the protein to the particle, or vice versa. This methodology can allow for faster reagent preparation.
[0142] [000142] Other forms of oligonucleotide can be used. For example, aptamers are single strand RNA or DNA oligonucleotides 15 to 60 bases in length that in solution form intramolecular interactions that fold the linear nucleic acid molecule into a three-dimensional complex that can then bind with high affinity to specific molecular targets ; generally with equilibrium constants in the range of 1 pM to 1 nM which is similar to some monoclonal antibody-antigen interactions. Aptamers can specifically bind to other nucleic acid molecules, proteins, small organic compounds, small molecules, and cells (organisms or pathogens). Polypeptide Binding Portions
[0143] [000143] In certain embodiments, the binding moiety is a polypeptide (i.e., a protein, polypeptide, or peptide), attached, using any of a variety of chemicals, to a single covalent bond in such a way as long as it does not affect the biological activity of the polypeptide. In one embodiment, the bond is made through the thiol group of a single reactive cysteine residue placed so that its modification does not affect the biological activity of the polypeptide. In this respect, the use of linear polypeptides, with cysteine at the C-terminus or N-terminus, provides a single thiol in a manner similar to that of alkanethiol which provides a thiol group at the 3 'or 5' end of an oligonucleotide. Similar bifunctional conjugation reagents, such as SPDP and reaction with the amino group of the magnetic particle and thiol group of the polypeptide, can be used with any thiol containing binding moiety. The types of polypeptides used as binding moieties can be natural and synthetic antibodies, antibody fragments, and polypeptide sequences. The peptide binding portions have a pair of bonds, that is, a molecule to which they selectively bind.
[0144] [000144] The use of peptides as binding moieties offers several advantages. For example, polypeptides can be constructed to have uniquely reactive residues, distal from the residues required for biological activity, for binding to the magnetic particle. The reactive residue can be a cysteine thiol, an N-terminal amino group, a C-terminal carboxyl group or a carboxyl group of aspartate and glutamate, etc. A single reactive residue on the peptide is used to guarantee a single binding site. These design principles can be followed with chemically synthesized peptides or biologically produced polypeptides.
[0145] [000145] The linking moieties may also contain amino acid sequences of naturally occurring (wild-type) polypeptides or proteins. For example, the natural polypeptide can be a hormone, (for example, a cytokine, a growth factor), a whey protein, a viral protein (for example, hemagglutinin), an extracellular matrix protein, a lecithin, or a ectodomain of a cell surface protein. Another example is a linker binding protein, such as streptavidin or avidin that binds biotin. In general, the resulting magnetic particle-binding portion is used to measure the presence of assayers in a test medium reacting with the binding portion.
[0146] [000146] Additionally, a polypeptide binding portion can be used in a universal reagent configuration, where the target of the binding portion (e.g., small molecule, linker, or pair of bonds) is pre-attached to the analyzed target for create a labeled analysand that, in the presence of particles decorated with polypeptide, induces the clustering.
[0147] [000147] Examples of protein hormones that can be used as binding moieties include, without limitation, platelet-derived growth factor (PDGF), which binds the PDGF receptor; insulin-like growth factor I and II (Igf), which binds the Igf receptor; nerve growth factor (NGF), which binds the NGF receptor; fibroblast growth factor (FGF), which binds the FGF receptor (for example, aFGF and bFGF); epidermal growth factor (EGF), which binds the EGF receptor; transformation growth factor (TGF, for example, TGFα and TGF-β), which binds the TGF receptor; erythropoietin, which binds the erythropoietin receptor; growth hormone (for example, human growth hormone), which binds the growth hormone receptor; and proinsulin, insulin, A-chain insulin, and B-chain insulin, all of which bind to the insulin receptor.
[0148] [000148] The receptor binding portions are useful for detecting and imaging the receptor cluster on the surface of a cell. Useful ectodomains include those of the Notch protein, Delta protein, integrins, cadherins, and other cell adhesion molecules. Antibody Binding Portions
[0149] [000149] Other polypeptide binding moieties include immunoglobulin binding moieties that include at least one immunoglobulin domain, and typically at least two such domains. An "immunoglobulin domain" refers to a domain of an antibody molecule, for example, a variable or constant domain. An "immunoglobulin superfamily domain" refers to a domain that has a three-dimensional structure related to an immunoglobulin domain, however, it is a non-immunoglobulin molecule. The immunoglobulin domains and immunoglobulin superfamily domains typically include two β-leaves of about seven β-filaments, and a conserved disulfide bond (see, for example, Williams and Barclay Ann. Rev Imunol., 6: 381 (1988 )). Proteins that include the domains of the Ig superfamily domains include the T cell receptors, CD4, platelet-derived growth factor receptor (PDGFR), and intercellular adhesion molecule (ICAM).
[0150] [000150] One type of immunoglobulin binding moiety is an antibody. The term "antibody", as used herein, refers to a naturally-sized, two-chain immunoglobulin molecule and an antigen-binding portion and fragments thereof, including synthetic variants. A typical antibody includes two heavy chain (H) variable regions (abbreviated here as VH), and two light chain (L) variable regions (abbreviated here as VL). The VH and VL regions can also be subdivided into regions of hypervariability, called "complementarity determination regions" (CDR), interspersed with the regions that are more conserved, called "structure regions" (FR). The extent of the structure region and CDR's has been precisely defined (see, Kabat, EA, et al. (1991) Sequences of Proteins of Immunological Interest, Fifth Edition, US Department of Health and Human Services, NIH Publication No. 91-3242, and Chothia et al., J. Mol. Biol., 196: 901 (1987)). Each VH and VL is composed of three CDR's and four FRs, arranged from the amino terminal to the carboxy terminal in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, and FR4.
[0151] [000151] An antibody can also include a constant region as part of a light and heavy chain. Light chains can include a kappa or lambda constant region gene at the COOH terminus (called CL). Heavy chains can include, for example, a gamma constant region (IgG1, IgG2, IgG3, IgG4; encoding about 330 amino acids). A gamma constant region can include, for example, CH1, CH2, and CH3. The term "life-size antibody" refers to a protein that includes a polypeptide that includes VL and CL, and a second polypeptide that includes VH, CH1, CH2, and CH3.
[0152] [000152] The term "antigen-binding fragment" of an antibody, as used herein, refers to one or more fragments of a life-sized antibody that maintains the ability to specifically bind to a target. Examples of antigen binding fragments include, however, are not limited to: (i) a Fab fragment, a monovalent fragment consisting of VL, VH, CL and CH1 domains; (ii) an F (ab ') 2 fragment, a divalent fragment including two Fab fragments linked by a disulfide bridge in the hinge region; (iii) and Fd fragment consists of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single branch of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544 (1989)), which consists of a VH domain; and (vi) an isolated complementarity determination region (CDR). In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined, using recombinant methods, by a synthetic linker that allows it to be made as a single protein chain in which the regions of VL and VH pair to form monovalent molecules (known as punk chain Fv (scFv); see, for example, Bird et al., Science 242: 423 (1988); and Huston et al., Proc. Natl. Acad. Sci. USA, 85: 5879 (1988)). Such single chain antibodies are also included in the term "antigen binding fragment".
[0153] [000153] A single domain antibody (sdAb, nanobody) is an antibody fragment consisting of a single variable antibody domain, and can also be used in the systems and methods of the invention. As a total antibody, sdAbs are able to selectively bind to a specific antigen. With a molecular weight of only 12-15 kDa, single domain antibodies are much smaller than ordinary antibodies (150-160 kDa) which are composed of two heavy protein chains and two light chains, and even smaller than Fab fragments (~ 50 kDa, a light chain and half a heavy chain) and single chain variable fragments (~ 25 kDa, two variable domains, one of a light chain and the other of a heavy chain). Polysaccharide Binding Portions
[0154] [000154] In certain embodiments, the bonding moiety is a polysaccharide, linked, for example, using any of a variety of chemicals, by a single bond, for example, a covalent bond, at one of the two ends, to a group functional in the magnetic particle. Polysaccharides can be synthetic or natural. Mono-, di-, tri- and polysaccharides can be used as the binding moiety. These include, for example, glycosides, N-glycosylamines, derivatives of O-acyl, derivatives of O-methyl, osazones, sugar alcohols, sugar phosphates when used with chemical substance appropriate to the magnetic particle.
[0155] [000155] One method for carrying out the bonding is to couple the avidin to a magnetic particle and to react the avidin-magnetic particle with commercially available biotinylated polysaccharides to produce polysaccharide-magnetic particle conjugates. For example, sially Lewis-based polysaccharides are commercially available as biotinylated reagents, and will react with avidin-CLIO (see, Syntesome, Gesellschaft fur medizinische Biochemie mbH.). The sialyl x tetrasaccharide Lewis (Slex) is recognized by the proteins known as Seletins, which are present on the leukocyte surfaces and function as part of the inflammatory cascade for leukocyte recruitment.
[0156] [000156] Still other targeting portions include a non-proteinaceous element, for example, a glycosyl modification (such as a Lewis antigen) or other non-proteinaceous organic molecule. Another method is the covalent coupling of the protein to the magnetic particle.
[0157] [000157] Another feature of the methods includes the identification of specific cell types, for hematological or histopathological investigations, for example, CD4 / CD3 cell counts and circulating tumor cells using any of the binding portions described above. Multivalent bonding agents
[0158] [000158] The assays of the invention may include a multivalent linker (i) containing multiple assays that are attached to a vehicle (for example, a simple synthetic scaffold, or a larger polysaccharide or carrier protein, such as BSA, transferrin, or dextran), or containing multiple epitopes for attachment, for example, to two or more populations of magnetic particles to form an aggregate.
[0159] [000159] Where a multivalent linker is employed, multiple analyzed can be linked to a vehicle (for example, a simple synthetic scaffold, or a larger carrier protein or polysaccharide, such as BSA, transferrin, or dextran). Alternatively, the multivalent linker can be a nucleic acid designed to bind to two or more populations of magnetic particles. Such multivalent binding agents act as binders and the test architecture is characterized by a competition between the analyte being detected and the multivalent binding agent (for example, in an inhibition test, competition test, or breakdown test).
[0160] [000160] The functional group present in the analyzed can be used to form a covalent bond with the vehicle. Alternatively, the analyte can be derived to provide a linker (i.e., a spacer separating the analyte from the vehicle in the conjugate) ending in a functional group (i.e., an alcohol, an amine, a carboxyl group, a sulfhydryl group, or a phosphate group), which is used to form the covalent bond with the vehicle.
[0161] [000161] The covalent connection of an analyte and a vehicle can be performed using a linker that contains reactive portions capable of reacting with such functional groups present in the analyte and in the vehicle. For example, a hydroxyl group of the analyzed can react with a carboxyl group on the linker, or an activated derivative thereof, resulting in the formation of an ester linking the two.
[0162] [000162] Examples of portions capable of reacting with sulfhydryl groups include α-haloacetyl compounds of the XCH2CO- type (where X = Br, Cl or I), which show particular reactivity for sulfhydryl groups, however, which can also be used to modify imidazolyl, thioether, phenol, and amino groups as described by Gurd, Methods Enzymol. 11: 532 (1967). N-maleimide derivatives are also considered selective for sulfhydryl groups, however, they may additionally be useful in coupling to amino groups under certain conditions. Reagents such as 2-iminothiolane (Traut et al., Biochemistry 12: 3266 (1973)), which introduce a thiol group through the conversion of an amino group, can be considered as sulfhydryl reagents if binding occurs through the formation of bridges disulfide.
[0163] [000163] Examples of reactive moieties capable of reacting with amino groups include, for example, alkylating and acylating agents. Representative alkylating agents include: (i) α-haloacetyl compounds, which show specificity for amino groups in the absence of reactive thiol groups and are of the XCH2CO- type (where X = Cl, Br or I), for example, as described by Wong, Biochemistry 24: 5337 (1979); (ii) N-maleimide derivatives, which can react with amino groups either through a Michael-type reaction or through acylation by addition to the ring carbonyl group, for example, as described by Smyth et al., J. Am. Chem. Soc. 82: 4600 (1960) and Biochem. J. 91: 589 (1964); (iii) aryl halides such as reactive nitroaloaromatic compounds; (iv) alkyl halides, as described, for example, by McKenzie et al., J. Proteína Chem. 7: 581 (1988); (v) aldehydes and ketones capable of forming Schiff's base with amino groups, the adductions formed generally being stabilized by reduction to produce a stable amine; (vi) epoxide derivatives such as epichlorohydrin and bisoxirans, which can react with phenolic amino, sulfhydryl, or hydroxyl groups; (vii) chlorine-containing derivatives of s-triazines, which are very reactive to nucleophiles such as amino, sulfhydryl, and hydroxyl groups; (viii) aziridines based on s-triazine compounds detailed above, for example, as described by Ross, J. Adv. Cancer Res. 2: 1 (1954), which reacts with nucleophiles such as ring-opening amino groups; (ix) scaryl acid diethyl esters as described by Tietze, Chem. Ber. 124: 1215 (1991); and (x) α-haloalkyl ethers, which are more reactive alkylating agents than normal alkyl halides because of activation caused by the ether oxygen atom, as described by Benneche et al., Eur. J. Med. Chem . 28: 463 (1993).
[0164] [000164] Representative amino-reactive acylating agents include: (i) isocyanates and isothiocyanates, particularly aromatic derivatives, which form stable urea and thiourea derivatives respectively; (ii) sulfonyl chlorides, which have been described by Herzig et al., Biopolymers 2: 349 (1964); (iii) acid halides; (iv) active esters such as nitrophenylester or N-hydroxysucinimidyl esters; (v) acidic anhydrides such as mixed, symmetric, or N-carboxyanhydrides; (vi) other reagents useful for amide bond formation, for example, as described by M. Bodansky, Principles of Peptide Synthesis, Springer-Verlag, 1984; (vii) acylazides, for example, where the azide group is generated from a preformed hydrazide derivative using sodium nitrite, as described by Wetz et al., Anal. Biochem. 58: 347 (1974); and (viii) imidoesters, which form stable amidines in reaction with amino groups, for example, as described by Hunter and Ludwig, J. Am. Chem. Soc. 84: 3491 (1962). Aldehydes and ketones can be reacted with amines to form Schiff's bases, which can advantageously be stabilized through reductive amination. The alkoxylamino moieties readily react with ketones and aldehydes to produce stable alkoxamines, for example, as described by Webb et al., Bioconjugate Chem. 1:96 (1990). Examples of reactive moieties capable of reacting with carboxyl groups include diazo compounds such as diazoacetate esters and diazoacetamides, which react with high specificity to generate groups of esters, for example, as described by Herriot, Adv. Proteína Chem. 3: 169 (1947). Carboxyl-modifying reagents such as carbodiimides, which react through formation of O-acylurea followed by formation of amide bond, can also be employed.
[0165] [000165] It will be appreciated that functional groups in the analyzed and / or in the vehicle can, if desired, be converted to other functional groups before the reaction, for example, to confer selectivity or additional reactivity. The so-called zero-length linkers, involving direct covalent bonding of a reactive chemical group of the analyzed one with a reactive chemical group of the vehicle without introducing additional binding material can, if desired, be used according to the invention. More generally, however, the linker will include two or more reactive portions, as described above, connected by a spacer element. The presence of such a spacer allows bifunctional linkers to react with specific functional groups in the analyzed and in the vehicle, resulting in a covalent bond between the two. The reactive moieties in a linker can be the same (homobifunctional linker) or different (heterobifunctional linker, or, where several dissimilar reactive moieties are present, heteromultifunctional linker), providing a variety of potential reagents that can perform the covalent bond between the analyzed and the vehicle. Spacer elements in the linker typically consist of straight or branched chains and can include a C1-10 alkyl, a heteroalkyl of 1 to 10 atoms, a C2-10 alkene, a C2-10 alkaline, C5-10 aryl, an acyclic system 3 to 10 atoms, or - (CH2CH2O) nCH2CH2-, where n is 1 to 4. Typically, a multivalent linker will include 2, 3, 4, 5, 6, 7, 8, 15, 50, or 100 ( for example, from 3 to 100, from 3 to 30, from 4 to 25, or from 6 to 20) conjugates of the analyzed. Multivalent linkers are typically 10 kD to 200 kD in size and can be prepared as described in the Examples. Analytes
[0166] [000166] The modalities of the invention include devices, systems, and / or methods for detecting and / or measuring the concentration of one or more analyzed in a sample (for example, a protein, a peptide, an enzyme, a polypeptide, a amino acid, a nucleic acid, an oligonucleotide, a therapeutic agent, a metabolite of a therapeutic agent, RNA, DNA, circulating DNA (for example, from a cell, tumor, pathogen, or fetus), an antibody, an organism, an viruses, bacteria, a carbohydrate, a polysaccharide, glucose, a lipid, a gas (for example, oxygen, and / or carbon dioxide), an electrolyte (for example, sodium, potassium, chloride, bicarbonate, BUN, magnesium, phosphate , calcium, ammonia, and / or lactate), general chemical molecules, calcium, ammonia, and / or lactate), general chemical molecules (creatinine, glucose), a lipoprotein, cholesterol, a fatty acid, a glycoprotein, a proteoglycan, and / or a lipopolysaccharide). Those analyzed may include identification of specific cells or cell types. The analyte (s) may include one or more biologically active substances and / or metabolite (s), markers (s), and / or other indicator of biologically active substances. A biologically active substance can be described as a single entity or a combination of entities. The term "biologically active substance" includes, without limitation, medications; vitamins; mineral supplements; substances used for the treatment, prevention, diagnosis, cure or mitigation of disease or illness; or substances that affect the structure or function of the body; or prodrugs, which become biologically active or more active after they have been placed in a predetermined physiological environment; or biologically toxic agents such as those used in biological warfare including organisms such as anthrax, ebola, Salmonella typhimurium, Marburg virus, plague, cholera, Francisella tulariesis (tularemia), brucellosis, Q fever, Bolivian hemorrhagic fever, Coccidioides mycosis, mormo, Melioidosis, Shigella, Rocky Mountain spotted fever, typhus, Psittacosis, yellow fever, Japanese B encephalitis, Rift Valley fever, and smallpox; naturally occurring toxins that can be used as weapons include ricin, aflatoxin, SEB, botulinum toxin, saxitoxin, and many mycotoxins. Those analyzed may also include organisms such as Candida albicans, Candida glabrata, Candida krusei, Candida parapsilosis, Candida tropicalis, Coagulase negative Staphalococcus, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, Ba , Bacteroides fragilis, Bacteroides fragilis, blaSHV, Burkholderia cepacia, Campylobacter jejuni / coli, Candida guilliermondii, Candida lusitaniae, Clostridium pefringens, Enterobacter aeraogenesl, Enterobacter cloacae, Enterobacteriaceae bacteria, King, hysteria, sp. containing Mec A gene (MRSA), Morganela morgana, Neisseria meningitides, Neisseria spp., non-meningitide, Prevotella buccae, Prevotella intermedia, Prevotella melaninogenica, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Salmonella enteric, Serratia marcescens ha, Staphylococcus lyticus; Analyzes may also include viral viral organisms such as dsDNA viruses (eg, adenovirus, herpes virus, poxvirosis); SsDNA virus (+) sense DNA (for example, parvovirus); dsRNA virus (for example, reovirus); (+) Sense RNA (for example, picornaviirus, togavirus); Virus (-) sense RNA (-) ssRNA (for example, orthomyxovirus, rabdovirus); (+) Sense RNA from ssRNA-RT virus with DNA intermediate in the life cycle (for example, retrovirus); and dsDNA-RT virus (for example, hepadnavirus).
[0167] [000167] Opportunistic infections that can be detected using the systems and methods of the invention include, without limitation, fungal, viral, bacterial, protozoal infections, such as: 1) fungal infections, such as those by Candida spp. (non-resistant and drug-resistant strains), C. albicans, C. krusei, C. glabrata, and Aspergillus fumigates; 2) gram negative infections, such as those by E. coli, Stenotrophomonas maltophilia, Klebsiella pneumonia / oxytoca, and Pseudomonas aeruginosa; and 3) gram positive infections, such as those caused by Staphylococcus spp., S. aureus, S. pneumonia, Enterococcus ssp. (And faecalis and E. faecium). The infection can be coagulase-negative staphylococcus, Corynebacterium spp., Fusobacterium spp., Morganella morganii, Pneumocystis jirovecii (previously known as Pneumocystis carinii), F. hominis, S. pyogenes, Pseudomonas aeruginosa, polyomavirus JC virus that causes polyomavirus progressive multifocal), Acinetobacter baumanni, Toxoplasma gondii, cytomegalovirus, Aspergillus spp., Kaposi's Sarcoma, Cryptosporidium spp., Cryptococcus neoformans, and Histoplasma capsulatum.
[0168] [000168] Non-limiting examples of broad categories of analytes that can be detected using the devices, systems, and methods of the invention include, without limitation, the following therapeutic categories: anabolic agents, antacids, antiasthmatic agents, anticholesterolemic agents and antilipids, anti -coagulants, anticonvulsants, antidiarrhea, antiemetics, antiinfectives, anti-inflammatory agents, anti-manic agents, anti-nausea, antineoplastic agents, anti-obesity agents, antipyretic and analgesic agents, antispasmodic agents, antithrombotic agents, anti-anesthetic agents, anti-anxiety agents, anti-anxiety agents, anti-anxiety agents appetite suppressants, biologicals, brain dilators, coronary dilators, decongestants, diuretics, diagnostic agents, erythropoiesis agents, expectorants, gastrointestinal sedatives, hyperglycemic agents, hypnotics, hypoglycemic agents, ion exchange resins, laxatives, mineral supplements, mu agents colitic, neuromuscular drugs, peripheral vasodilators, psychotropic, sedatives, stimulants, thyroid and antithyroid agents, uterine relaxants, vitamins and prodrugs.
[0169] [000169] Biologically active substances that can be detected using the devices, systems, and methods of the invention include, without limitation, medications for the digestive system or gastrointestinal tract, for example, antacids, reflux suppressants, antiflatulences, antidoopaminergics, inhibitors of proton pump, H2 receptor antagonists, cytoprotectors, prostaglandin analogs, laxatives, antispasmodics, antidiarrhea, bile acid scavengers, and opioids; medications for the cardiovascular system, for example, beta-receptor blockers, calcium channel blockers, diuretics, cardiac glycosides, antiarrhythmics, nitrate, anti-analgesics, vasoconstrictors, vasodilators, peripheral activators, ACE inhibitors, angiotensin receptor blockers, alpha blockers , anticoagulants, heparin, HSGAGs, antiplatelet drugs, fibrinolytics, antihemophilic factors, hemostatic drugs, hypolipemic agents, and statins; medications for the central nervous system, for example, hypnotics, anesthetics, antipsychotics, antidepressants, antiemetics, anticonvulsants, antiepileptics, anxiolytics, barbiturates, drugs for movement disorders, stimulants, benzodiazepine, cyclopyrrone, dopamine antagonists, antihistamines, antihistamines , emetics, cannabinoids, 5-HT antagonists; pain and / or awareness medications, for example, NSAIDs, opioids and orphans such as paracetamol, tricyclic antidepressants, and anticonvulsants; for musculoskeletal disorders, for example, NSAIDs, muscle relaxants, and neuromuscular drug anticolinersterase; eye medications, for example, adrenergic neuron blockers, astringents, eye lubricants, topical anesthetics, sympathomimetics, parasympatholytics, mydriatics, cycloplegics, antibiotics, topical antibiotics, sulfa drugs, aminoglycosides, fluoroquinolones, antivirals, antifungals, NIDs , corticosteroids, mast cell inhibitors, adrenergic agonists, beta-blockers, carbonic anhydrase / hyperosmotic inhibitors, cholinergics, myotics, parasympathomimetics, prostaglandins, prostaglandin, agonist / prostaglandin inhibitors, nitroglycerin; medications for the ear, nose and oropharynx, for example, sympathomimetics, antihistamines, anticholinergics, NSAIDs, steroids, antiseptics, local anesthetics, antifungals, cerumenolytics; medications for the respiratory system, for example, bronchodilators, NSAIDs, antiallergics, antitussives, mucolytics, decongestants, corticosteroids, beta-receptor antagonists, anticholinergics, steroids; medications for endocrine problems, for example, androgen, antiandrogen, gonadotropin, corticosteroids, growth hormone, insulin, antidiabetics, thyroid hormone, antithyroid drugs, calcitonin, diphosphonate, and vasopressin analogs; medications for the reproductive system or urinary system, for example, antifungals, alkalizing agents, quinolones, antibiotics, cholinergics, anticholinergics, anticholinesterase, antispasmodics, 5-alpha reductase inhibitor, selective alpha-1 blockers, and sildenafil; contraceptive medications, for example, oral contraceptives, spermicides, and depot contraceptives; medications for obstetrics and gynecology, for example, NSAIDs, anticholinergics, hemostatic drugs, antifibrinolytics, hormone replacement therapy, bone regulator, beta-receptor agonists, follicle stimulating hormone, luteinizing hormone, LHRH gamolinic acid, release inhibitor gonadotropin, progestogen, dopamine agonist, oestrogen, prostaglandin, gonadorrelin, clomiphene, tammoxifene, and dietilstilbestrol; skin medications, for example, emollients, antipruritic, antifungal, disinfectants, scabicide, pediculicide, tar products, vitamin A derivatives, vitamin D analog, keratolytics, abrasives, systemic antibiotics, topical antibiotics, hormones, peeling agents, exudates, fibrinolytics, proteolytics, sunscreens, antiperspirants, and corticosteroids; medications for infections and infestations, for example, antibiotics, antifungals, antileptic drugs, antituberculosis drugs, antimalarials, anthelmintics, amoebicidal antivirals, antiprotozoans, and antiserum; medications for the immune system, for example, vaccines, immunoglobulin, immunosuppressants, interferon, monoclonal antibodies; medications for allergic disorders, for example, antiallergic, antihistamines, and NSAIDs; nutrition medications, for example, tonics, iron preparations, electrolytes, vitamins, anti-obesity drugs, anabolic drugs, hematopoietic drugs, and food product drugs; medications for neoplastic disorders, for example, cytotoxic drugs, sex hormones, somatostatin inhibitors, recombinant interleukins, G-CSF, and erythropoietin; diagnostic medications, for example, contrast agents; and cancer medications (anticancer agents).
[0170] [000170] Biologically active substances that can be detected using the devices, systems and methods of the invention include, without limitation, hematological agents, such as anti-anemia agents, hematopoietic anti-anemia agents, coagulation agents, anticoagulants, hemostatic coagulation agents, coagulation agents platelet inhibitors, thrombolytic enzyme coagulation agents, and plasma volume dilators; anticoagulants, heparin, HSGAGs, antiplatelet drugs, fibrinolytics, anti-hemophilic factors, hemostatic drugs. Examples of antithrombotics (e.g., thrombolytics, anticoagulants, and antiplatelet drugs) that can be detected using the devices, systems, and methods of the invention include vitamin K antagonists such as acenocoumarol, chlorindione, dicumarol, diphenadione, ethyl bicarbonate, phenprocoumon, fenindione, thioclomarol, and warfarin; heparin group (platelet aggregation inhibitors) such as antithrombin III, bemiparin, dalteparin, danaparoid, enoxaparin, heparin, nadroparin, parnaparin, reviparin, sulodexide, and tinzaparin; other platelet aggregation inhibitors such as abciximab, acetylsalicylic acid (aspirin), aloxiprine, beraprost, ditazole, carbasalate calcium, chlorichromen, clopidogrel, dipyridamol, epoprostenol, eptifibatide, indobufen, iloprost, tyropyridine, tyropyridine, tyropyridine, tyropyridine, trichloridine, tyropyridine, trichloridine, tyridine, prophylamide and triflusal; enzymes such as alteplase, ancrod, anistreplase, brinase, drotrecogin alfa, fibrinolysin, procein C, reteplase, saruplase, streptocinase, tenecteplase, and urokinase; direct thrombin inhibitors such as argatroban, bivalirrudin, desirrudin, lepirudin, melagatran, and ximelagatran; other antithrombotics such as dabigatran, defibrotide, dermatan sulfate, fondaparinux, and rivaroxaban; and others such as citrate, EDTA, and oxalate.
[0171] [000171] Other biologically active substances that can be detected using the devices, systems and methods of the invention include those mentioned in Basic and Clinical Pharmacology (LANGE Basic Science), Katzung and Katzung, ISBN 0071410929, McGraw-Hill Medical, 9th edition (2003 ). Medical Conditions
[0172] [000172] The methods of the invention can be used to monitor one or more analyzed in the diagnosis, control, and / or treatment of any of a wide range of medical conditions. Various categories of medical conditions include, for example, pain disorders; changes in body temperature (for example, fever); nervous system dysfunction (eg, syncope, myalgia, movement disorders, numbness, sensory loss, delirium, dementia, memory loss, or sleep disorders); the eyes, ears, nose, and throat; circulatory and / or respiratory functions (for example, dyspnoea, pulmonary edema, cough, hemoptysis, hypertension, myocardial infarction, hypoxia, cyanosis, cardiovascular collapse, congestive heart failure, edema, or shock); gastrointestinal function (eg, dysphagia, diarrhea, constipation, GI bleeding, jaundice, ascites, indigestion, nausea, vomiting); urinary and renal tract function (eg, acidosis, alkalosis, electrolyte and fluid imbalances, azotemia, or urinary abnormalities); reproduction and sexual function (for example, erectile dysfunction, menstrual disorders, hirsutism, virilization, infertility, disorders associated with pregnancy, and standard measurements); skin (for example, eczema, psoriasis, acne, rosacea, skin infection, immune skin disorders, or photosensitivity); blood (for example, hematology); genes (for example, genetic disorders); drug response (for example, miscellaneous drug responses); and nutrition (eg, obesity, eating disorders, or nutritional assessment). Other medical fields with which the modalities of the invention find use include oncology (for example, neoplasms, malignancies, angiogenesis, paraneoplastic syndromes, or oncological emergencies); hematology (eg anemia, hemoglobinopathies, megalooblastic anemias, hemolytic anemias, aplastic anemia, myelodysplasia, bone marrow failure, polycythemia vera, miloproliferative diseases, acute myeloid leukemia, chronic myeloid leukemia, lymphoid malignancies, plasma cell disorders, transfusion biology , or transplants); hemostasis (for example, coagulation disorders and thrombosis, or disorders of the platelet and vessel wall); and infectious diseases (eg, sepsis, septic shock, fever of unknown origin, endocarditis, stings, burns, osteomyelitis, abscesses, food poisoning, pelvic, bacterial inflammatory disease (eg gram positive, gram negative, varied (nocardia, actimoyces , mixed), mycobacterial, spiroquetal, rickettsia, or mycoplasma); chlamydia; viral (DNA, RNA), fungal and algal infections; protozoal and helminth infections; endocrine diseases; nutritional diseases; and metabolic diseases.
[0173] [000173] Other medical conditions and / or fields with which the modalities of the invention find use include those mentioned in Harrison's Principles of Internal Medicine, Kasper et al., ISBN 0071402357, McGraw-Hill Professional, 16th edition (2004), as well as those mentioned in Robbins Basic Pathology, Kumar, Cotran, and Robbins, eds., ISBN 1416025340, Elsevier, 7th edition (2005).
[0174] [000174] Medical tests (for example, blood tests, urine tests, and / or other human or animal tissue tests) that can be performed using the various modalities of the invention described here include, for example, general chemistry tests (for example , analyzed include albumin, blood urea nitrogen, calcium, creatinine, magnesium, phosphorus, total protein, and / or uric acid); electrolyte tests (for example, analyzed include sodium, potassium, chloride, and / or carbon dioxide); diabetes tests (for example, analyzed include glucose, hemoglobin A1C, and / or microalbumin); lipid tests (for example, analyzed include apolipoprotein A1, apolipoprotein B, cholesterol, triglyceride, low density lipoprotein cholesterol, and / or high density lipoprotein cholesterol); nutritional assessment (eg, analyzed includes albumin, prealbumin, transferrin, retinol binding protein, alpha-1 acid glycoprotein, and / or ferritin); liver tests (for example, analyzed include alanine transaminase, albumin, alkaline phosphatase, aspartate transaminase, direct bilirubin, gamma glutamyl transaminase, lactate dehydrogenase lactate dehydrogenase, immunoglobulin A, immunoglobulin G, immunoglobulin M, prealbumin, total protein and / or bilirubin, total protein and / or bilirubin total0; cardiac tests (for example, analyzed include apolipoprotein A1, apolipoprotein B, cardiac troponin-1, creatine kinase, creatine kinase MB isoenzyme, high-sensitivity CRP, mass of creatine kinase MB isoenzyme myoglobin, and / or pro-brain natriuretic peptide N-terminal); tests for anemia (for example, analyzed include ferritin, folate, homocysteine, haptoglobin, iron, soluble transferrin receptor, total iron binding capacity, transferrin, and / or vitamin B12); pancreatic tests (for example , analyzed include amylase and / or lipase), kidney diseases (for example, analyzed include albumin, alfalfa-microglobulin, alpha2-macroglobuli na, beta2-microglobulin, cystatin C, retinol binding protein, and / or transferrin); bone tests (for example, analyzed include alkaline phosphatase, calcium, and / or phosphorus); monitoring of cancer marker (eg analyzed include total PSA); thyroid tests (for example, analyzed include free thyroxine, free triiodothyronine, thyroxine, thyroid stimulating hormone, and / or triiodothyronine); fertility tests (for example, analyzed include beta-human chorionic gonadotropin); therapeutic drug monitoring (for example, analyzed include carbamazepine, digoxin, digitoxin, gentamicin, lidocaine, lithium, N-acetyl procainamide, phenobarbital, phenytoin, procainamide, theophylline, tobramycin, valproic acid, and / or vancomycin); immunosuppressive drugs (for example, analyzed include cyclosporin A, sirolimus, and / or tacrolimus); tests for complementary activity and / or autoimmune disease (eg, analyzed include C3 complement, C4 complement, C1 inhibitor, C-reactive protein, and / or rheumatoid factor); polyclonal / monoclonal gammopathies (for example, analyzed include immunoglobulin A, immunoglobulin G, immunoglobulin M, 1 g of kappa and / or lambda light chains, immunoglobulin G subclasses 1, 2, 3, and / or 4); tests for infectious disease (for example, analyzed include anti-streptolysin O); tests for inflammatory disorders (for example, analyzed include alpha1 acid glycoprotein, alpha1-antitrypsin, ceruloplasmin, C-reactive protein, and / or haptoglobin); allergy test (for example, analyzed include immunoglobulin E); urine protein tests (for example, analyzed include alpha1-microglobulin, immunoglobulin G, 1 g kappa and / or lambda light chains, microalbumin, and / or urine / cerebrospinal fluid protein); tests for protein - CSF (for example, analyzed include immunoglobulin G and / or urine / cerebrospinal fluid protein); toxicology tests (for example, analyzed include serum acetaminophen, serum barbiturates, serum benzodiazepines, serum salicylate, serum tricyclic antidepressants, and / or urine ethyl alcohol); and / or tests for drugs of abuse (for example, analyzed include amphetamine, cocaine, barbiturates, benzodiazepines, ecstasy, methadone, opiate, phencyclidine, tetrahydrocannabinoids, propoxyphene, and / or metaqualone). Specific cancer markers that can be detected using the methods, devices, cartridges, and kits of the invention include, without limitation, 17-beta-hydroxysteroid dehydrogenase type 1, Abl 2 interacting, actin-related 2/3 protein complex subunit , Albumin, Aldolase A, placental-type alkaline phosphatase, Alpha 1 antitrypsin, Alpha-1 acid glycoprotein 1, Alpha-2-HS glycoprotein, Alpha lactalbumin, Alpha-2-macroglobulin, Alpha-fetoprotein (AFP), family 5 of Angiogenin ribonuclease RNase A, Angiopoietin 1, Angiopoietin 2, Antigen identified by monoclonal antibody Ki-67, Antileukoproteinase 1 (SLPI), Apolipoprotein A1, ATP7B, β2-microglobulin, B-ce11 CLL / lymphoma 2, BR protein associated with BCL2 , BRCA2, BrMS1, butyrate-induced transcription 1, CA15.3 / CA27-29, cancer antigen 125, cancer antigen 15.3, cancer antigen 19.9, cancer antigen 602, cancer antigen 72-4 / TAG-72 , galactotransferase antigen associated with cancer, cancer-associated serum antigen (CASA), carcinoembryonic antigen (CEA), Catenin beta 1, Cathepsin D, Cathepsin member 8, chemokine CC 4 (HCC-4), CCL21 (small inducible cytokine A21), CCL5, CD15, CD24, CD34, CD44, cell division protein kinase 5, ceruloplasmin, cervical cancer protooncogene protein p40 1, c-Ets1, TCP1 containing caparonin, subunit 3, chemokine (cc motif) inducible small cytokine A4 by ligand 4 (CCL4, MIP-1-beta), chemokine ligand 5, protein 1 as chitinase-3 (YKL-40), intracellular chloride channel 4 (CLIC4), choriogonadotropin beta chain, Claudina-3, Claudina-4, clusterin, factor II coagulation (prothrombin), coagulation factor III, a coagulation factor XIII chain, coagulation factor XIII chain, collagen C-terminal peptide I, colony-stimulating factor 2, colony-stimulating factor 3 3 complement, C-reactive protein, creatinine kinase brain (CKB), similar to phosphorus small atase CTD, Cyclin D1, cyclin-dependent kinase 6 (CDK 6), cyclin-dependent kinase inhibitor 1 (p21), Cyclooxygenase-1, Cytochrome c oxidase Va, Cytochrome c-1, Desmin, Distroglycan 1, Endogline, Endothelin 1, epidermal growth factor (EGFR) receptor, epidermal growth factor (EGF), erythropoietin, E-selectin, EST translocation variant 4 (EST 4), extracellular matrix metalloproteinase inductor (EMMPRIN), Ferritin H, Ferritin L, fibroblast growth factor 2, fibronectin, Fit-3 ligand, Fluorodeoxyglucose-PET (FDG-PET) with CA125, Fms-related tyrosine kinase 1 (VEGFR-1), GADD45A, Geminine, N-acetyltransferase glyphosate, precursor granulin-epithelin (GEP), growth differentiation factor 15, Haptoglobin 1, Haptoglobulin-a-subunit, HE4 (human epidymis protein), Her2, HER2-neu, hK10, hK11, hK13, hk6, hk7, hK8, HLA class II Doβ, hLMH1, hLMH2, HNF-1β, hu chorionic interleukin 2-gonadotropin-β subunit mana, human chorionic gonadotropin (hCG), IGFBP-2, IL-2R alpha (soluble interleukin 2 alpha receptor), immunoglobulins, immunosuppressive acidic protein (lAP), indoleamine 2,3-dioxigenase, growth factor-binding protein 1 insulin-like, insulin-like growth factor-binding protein 2, insulin-like growth factor-binding protein 3, Integrin α-V, Integrin αvβ6, intercellular adhesion molecule, Interferon alpha 1, Interleucin 1 alpha, Interleukin 1 beta, Interleukin 10, Interleukin 12A, Interleukin 16, fragment of Inter-α-trypsin inhibitor, Kallikrein 8, Keratin, Keratin 18, Keratin, cytoskeletal 19 type I (cytokeratin 19), Kit ligand, KRAS, Lactotransferrin, β3 laminate, Leptil-selectin, luteinizing hormone releasing hormone receptor, 90k Mac-2 binding protein, macrophage colony stimulation factor, Macrophage migration inhibitor factor, mammary serum antigen, Mamoglobin B, M-CAM, MIR21 , Mesot elina, MMP3, mucin-like glycoprotein antigen, Myosin X, beta nerve growth factor, Netrin-1, neuroendocrine secretory protein 55, neutrophil defensin 1, neutrophil defensin 3, Nm23-H1, non-metallic cell protein 2 , non-metastatic cell protein (NM23A), O-acyltransferase domain containing 2, OVX1, OX40, P53, Paraoxonase 2, Pcaf, p-glycoprotein, phosphibosylaminoimidazole carboxylase, platelet-derived growth factor receptor, beta receptor platelet-derived growth factor, platelet endothelial cell adhesion molecule (PECAM-1), platelet factor 4, pregnancy-associated plasma protein A, pregnancy zone protein, Procol-lys 1,2 oxoglute 5-digixyg 3, Procol-lys 1,2 oxoglute 5-digoxyg 1, progesterone receptor (PR), Prolactin, prostate secretory protein PSP94, prostate specific antigen (PSA), Prostatin, protein kinase C-binding protein 1, p- selectin, Pyrroline-5-carboxylate re dutase 1, Protein G signaling Regulator 12, Reticulocalbine, S-100 alpha chain, s-adenosylomocysteine hydrolase, serum proteinamyloid A, transmembrane domain protein seven, sex-determining factor Y-box-4, Sialila SSEA-1 , small inducible cytokine A18 (CCL18, MlP-4), small inducible cytokine A2 (CCL2), small inducible cytokine (CCL3) (macrophage inflammatory protein 1-alpha, small inducible cytokine B5 (CXCL5), Somatostatin, growth factor of somatotropin, growth factor, squamous cell carcinoma antigen 1, squamous cell carcinoma antigen 2, steroid hormone receptors, Survivin, Syndecan-1, Gamma synuclein, Tetranectin, Tetraspanin 9, TGF-α, Thymidine phosphorylase (TP ), Thyroglobulin (Tg), metalloproteinase 2 tissue inhibitor, tissue specific transplant P35B antigen, tissue type plasminogen activator (tPA), Topoisomerase II, transfer receptor p90 CD71, alpha growth factor transfo rant, transforming growth beta 1 factor, outer mitochondrial membrane translocase, Transtiretin, Transtiretin fragment (realbumin), trophoblast glycoprotein, Tropomyosin 1 alpha chain (alpha-tropomyosin), Trypsin, β2 tubulin, β3 tubulin, member 5 of tumor necrosis factor (ligand) superfamily (CD154), member 6 of the tumor necrosis factor (ligand) superfamily (Fas ligand), tumor necrosis factor alpha, tumor necrosis factor p75 / p55, tumor necrosis factor super family member 6 receptor (fas), protein 1 associated with tumor necrosis factor receptor, tumor protein p53, ubiquitin conjugating enzyme E2C (Ubiquitin cong enz), urinary angiostatin (uAS), vascular endothelial growth factor ( VEGF), vascular smooth muscle growth promoting factor (VSGPIF-Spondin), VEGF (165) b, V-erb-b2, vitamin D-binding protein, vitamin K-dependent protein C, Vitronectin, Von Willebrand factor, tumor Wilms 1 (WT-1), WW domain binding protein 11, X-box binding protein 1, and YKL-40. See Polanski et al., Biomarker Insights, 1: 1 (2006); Cherneva et al., Biotechnol. & Biotechnol. EQ. 21/2007/2: 145 (2007); Alaoui-Jamali et al., J. Zhejiang Science B 7: 411 (2006); Basil et al., Cancer Res. 66: 2953 (2006); Suh et al., Expert Rev. Mol. Diagn. 10: 1069 (2010); and Diamandis, E. P., Molecular and Cellular Proteomics 3: 367 (2004).
[0175] [000175] Other analyzed that can be detected using the devices, systems and methods of the invention include those mentioned in the Tietz Textbook of Clinical Chemistry and Molecular Diagnostics, Burtis, Ashwood, and Bruns, ISBN 0721601898, Elsevier, 4th edition (2006).
[0176] [000176] The methods, kits, cartridges, and systems of the invention can be configured to detect a predetermined combination panel of analyzers that can be used to understand the individual's medical condition. For example, a combination panel may include the detection of pathogens, therapeutic agents used to treat the suspected pathogen (s), and a potential biomarker to monitor therapeutic pharmacological progress (efficacy or pharmacokinetics), or monitor the presence of the pathogen or pathogen by-products. In addition, one might consider a disease treatment panel configured for use to detect a disease or a disease biomarker, the level or concentration of a therapeutic drug for use in treating suspected disease, a potential biomarker for monitoring therapeutic pharmacological progress ( efficacy or pharmacokinetics), and general chemistry biomarker or other physiological marker of disease or treatment effect. In this way, analyte detection panels can be used to inform and induce to make appropriate medical decision.
[0177] [000177] For example, the systems and methods of the invention can be used to monitor immunocompromised individuals following allogeneic transplantation. In transplant recipients who receive a solid organ, bone marrow, hematopoietic stem cell, or other allogeneic donations, there is a need to monitor imne status, ortion function, and if necessary, quickly and precisely identify opportunistic infections. For example, there is a need to monitor the levels of creatinine and tacrolimus from the same blood sample as an individual when monitoring drug concentration and kidney function can assist and guide the physician for optimal post-transplant therapy. The optimization of therapy is a firm balance of prevention of rejection, but also to guarantee the immune function to fight opportunistic infections and in general results in individual acceptance enhanced with immunosuppressive therapy. In large part, transplant recipients succumb to transplant rejection, graft versus host disease, or opportunistic infections. In the first two, immunosuppressive agents can remove or inhibit the reactions. However, if the individual has an underlying infection, then clinical control is challenging. For a specific example, a heart, a lung transplant individual presenting with a fever of unknown origin enters a health care facility. The individual is started on broad-spectrum antibiotics until the culture results are known. If the condition worsens, and the culture reveals a specific infection, for example, candida, a specific antifungal, fluconazole, can be administered to the known individual. However, this antifungal can alter the levels of the immunosuppressive agent given to almost all allogeneic transplant recipients, tacrolimus. In testing for both tacrolimus and creatinine levels, the doctor stops tacrolimus, believing that fluconazole will beat the infection, and quickly. Under this regimen, the individual may get worse if the candida species is resistant to fluconazole, and the individual is then started on an appropriate antifungal agent. However, since tacrolimus can be stopped, immunosuppressive therapy is out of control and the individual may become unresponsive to any additional therapy and death can result. Thus, if there is a test to simultaneously monitor creatinine (kidney function), blood tacrolimus levels, and the accurate identification of opportunistic infections, the individual above may have been saved.
[0178] [000178] The systems and methods of the invention can include a multiplex, no sample preparation, simple detection method, automated system to determine the drug level, toxicity or adverse effect determinant, and the identification of the pathogen playing a critical role in the scenario of the immunocompromised individual. For example, a cartridge having portals or cavities containing 1) magnetic particles having specific creatinine antibodies decorated on its surface, 2) magnetic particles having specific tacrolimus antibodies on its surface, and 3) magnetic particles having specific nucleic acid probes to identify pathogen species can be employed to quickly determine and provide clinical control values for a given transplant individual. Opportunistic infections that can be monitored in such individuals, and any other patient populations at risk of infection, include, without limitation, fungal infections; candida (resistant and non-resistant strains); gram negative bacterial infections (for example, E. coli, stenotrophomonas maltophilia, Klebsiella pneumonia / oxytoca, or Pseudomonas aeruginosa); and gram positive bacterial infections (for example, Staphylococcus species: S. aureus, S. pneumonia, E. faecalis, and E. faecium). Other opportunistic infections that can be monitored include coagulase negative staphylococcus, Corynebacterium spp., Fusobacterium spp., And Morganella morganii, and viral organisms, such as CMV, BKV, EBC, HHV-6, HIV, HCV, HBV, and HAV.
[0179] [000179] The systems and methods of the invention can also be used to monitor and diagnose cancer patients as part of a multiplexed diagnostic test. One specific form of cancer, colorectal cancer, has shown positive promise for personalized medical treatment for a specific solid tumor. Pharmacogenetic markers can be used to optimize the treatment of colorectal cancer and others. There is significant individual genetic variation in drug metabolism of 5FU, capecitabine, irinotecan, and oxaliplatin that influence, however, the toxicity and efficacy of these agents. Examples of genetic markers that include UGT1A1 * 28 lead to reduced conjugation of SN-38, the active metabolite of irinotecan, resulting in an increased rate of adverse effects, especially neutropenia. To a lesser extent, increased 5-FU toxicity is predicted by DPYD * 2A. A variable number of tandem repeat polymorphisms in the thymidylate synthase enhancing region, in combination with a single C> G nucleotide polymorphism, can predict a weaker response to 5-FU. The effectiveness of oxaliplatin is influenced by polymorphisms in components of DNA repair systems, such as ERCC1 and XRCC1. Polymorphic changes in the endothelial growth factor receptor are likely to predict the efficacy of cetuximab. In addition, the cell-mediated cytotoxic effect dependent on cetuximab antibody can be reduced by polymorphisms at the immunoglobin G fragment C receptors. Polymorphic changes in the VEGF gene and the hypoxia-inducible factor 1 alpha gene are also believed to play a role in variability of therapy outcome. Thus, the identification of such polymorphisms in individuals can be used to assist physicians with treatment decisions. For example, PCR-based genetic tests have been developed to assist physicians with therapeutic treatment decisions for individuals with non-small cell lung cancer (NSCLC), colorectal cancer (CRC) and gastric cancer. Expression of ERCC1, TS, EGFR, RRM1, VEGFR2, HER2, and mutation detection in KRAS, EGFR, and BRAF are available for doctors to order to identify the ideal therapeutic option. However, these PCR tests are not available on site, so the sample must be released to the outside laboratory. These solid tumors are often biopsied and FFPE (formalin fixed samples, embedded in paraffin (tissues) are prepared. The systems and methods of the invention can be used without the 5 to 7 days of return to obtain data and information and use of samples The required systems and methods of the invention can provide a simple platform for analyzing samples, without sample preparation, for multiple types of analyte, such as cancer for chemotherapeutic drugs, genotyping, toxicity and efficacy markers can revolutionize practice personalized medicine and provide accurate, rapid diagnostic testing.
[0180] [000180] The systems and methods of the invention can also be used to monitor and diagnose neurological disease, such as dementia (a loss of cognitive ability in a previously unimpaired person) and other forms of cognitive impairment. Dementia can be broadly categorized into two groups: cortical dementia and subcortical dementia. Cortical dementias include: Alzheimer's disease, vascular dementia (also known as multiple infarct dementia), including Binswanger's disease, Lewy body dementia (DLB), alcohol-induced persistent dementia, Korsakoff syndrome, Wernicke encephalopathy, degenerations frontotemporal longarms (FTLD), including Pick's disease, frontotemporal dementia (or frontal FTLD variant), semantic dementia (or FTLD temporal variant), progressive non-fluent aphasia, Creutzfeldt-Jakob disease, pugilistic dementia, Moyamoya disease, tebestia (often confuses with cancer), posterior cortical atrophy or Benson syndrome. Subcortical dementias include dementia due to Huntington's disease, dementia due to hypothyroidism, dementia due to Parkinson's disease, dementia due to vitamin B1 deficiency, dementia due to vitamin B12 deficiency, dementia due to folate deficiency, dementia due to syphilis , dementia due to subdural hematoma, dementia due to hypercalcemia, dementia due to hypoglycemia, AIDS dementia complex, pseudo-dementia (a major major depressive episode with prominent cognitive symptoms), persistent substance-induced dementia (related to psychoactive use and previously absintheism) , dementia due to multiple etiologies, dementia due to other general medical conditions (ie, end-stage renal failure, cardiovascular disease, etc.), or dementia not otherwise specified (used in cases where no specific criteria are met). Alzheimer's disease is a common form of dementia. Since dementia is fundamentally associated with many neurodegenerative diseases, the ability to test for these proteins, as disease biomarkers, along with the drug or drug metabolite levels on a single platform will help a doctor adjust the dosage, change a regimen, or generally monitor disease progression. These tests are currently conducted externally in locations away from the individual and caregiver. Thus, in order to have the ability to monitor drug and biomarker levels in the same detection system, on-site will provide a huge advantage for this debilitating and devastating disease. The method, according to the claims of the invention, can be a multiplex, no sample preparation, single detection method, automated system to determine the drug level, toxicity or adverse effect determinant, and the potential biomarker of the progression of disease. For example, a cartridge having ports or cavities containing 1) magnetic particles having specific protein dementia biomarker antibodies decorated on its surface, 2) magnetic particles having specific antibodies on its surface, and 3) magnetic particles having nucleic acid probes for identifying protein expression levels can be employed to quickly determine and provide clinical control values for a particular dementia patient.
[0181] [000181] The systems and methods of the invention can also be used to monitor and diagnose infectious disease in an automated, multiplexed sample preparation system. Examples of pathogens that can be detected using the devices, systems and methods of the invention include, for example, Candida (resistant and non-resistant strains), for example, C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis; A. fumigatus; E. coli, Stenotrophomonas maltophilia, Klebsiella pneumonia / oxytoca, P. aeruginosa; Staphylococcus spp. (for example, S. aureus or S. pneumonia); E. faecalis, E. faecium, Coaglulase negative staphylococcus spp., Corynebacterium spp., Fusobacterium spp., Morganella morganii, Pneumocystis jirovecii, formerly known as pneumocystis carinii, F. hominis, streptococcus pyogenes, Pseudomonas aeruginosa, causing progressive multifocal leukoencephalopathy), Acinetobacter baumanni, Toxoplasma gondii, Cytomegalovirus, Aspergillus spp., Kaposi's sarcoma, cryptosporidium, Cryptococcus neoformans, and Histoplasma capsulatum, among other bacteria, yeast, fungi, proton, actin, proti, acti, pri parasitic, protist and helminthic infectious organisms.
[0182] [000182] The systems and methods of the invention can be used to identify and monitor the pathogenesis of the disease in an individual, to select therapeutic interventions, and to monitor the effectiveness of the selected treatment. For example, for a patient having or at risk for a viral infection, the systems and methods of the invention can be used to identify the infectious virus, viral load, and monitor the leukocyte count and / or biomarkers indicative of the status of the infection. The virus identity can be used to select an appropriate therapy. Therapeutic intervention (for example, a particular antiviral agent) can also be monitored to correlate the treatment regimen with the circulating concentration of antiviral agent and viral load to ensure that the patient is responding to treatment.
[0183] [000183] The systems and methods of the invention can be used to monitor a viral infection in an individual, for example, with a viral panel configured to detect Cytomegalovirus (CMV), Epstein Barr Virus, BK Virus, Hepatitis B virus, Hepatitis C, Herpes simplex virus (HSV), HSV1, HSV2, respiratory syncytial virus (RSV), Influenza; Influenza A, Influenza A subtype H1, Influenza A subtype H3, Influenza B, Human Herpes Virus 6, Human Herpes Virus 8, Human Metapneumovirus (hMPV), Rhinovirus, Parainfluenza 1, Parainfluenza 2, Parainfluenza 3, and Adenovirus. The methods of the invention can be used to monitor an appropriate therapy for the individual with a viral infection (e.g., Abacavir, Acyclovir, Acyclovir, Adefovir, Amantadine, Amprenavir, Ampligen, Arbidol, Atazanavir, Atripla, Boceprevir, Cidofovir, Combivir, Darunavir , Delavirdine, Didanosine, Docosanol, Edoxudine, Efavirenz, Emtricitabine, Enfuvirtide, Entecavir, Famciclovir, Fomivirsen, Fosamprenavir, Foscarnet, Fosfonet, Ganciclovir, Ibacitabine, Immunovir, Inhibitor, Type Idoxuridine, Idoxurin II, Interferon type I, Interferon α, Interferon β, Lamivudine, Lopinavir, Lovirida, Maraviroc, Moroxidine, Methisazone, Nelfinavir, Nevirapine, Nexavir, nucleoside analogs, Oseltamivir (Tamiflu), Peginterferon alfa-2a, Penciclirir, Penciclovir, Penciclovir, Podophyllotoxin, Raltegravir, reverse transcriptase inhibitor, Ribavirin, Rimantadine, Ritonavir, Piramidine, Saquinavir, Stavudine, Tea tree oil, Tenofovi r, Tenofovir disoproxil, Tipranavir, Trifluridine, Trizivir, Tromantadine, Truvada, Valacyclovir (Valtrex), Valganciclovir, Vicriviroc, Vidarabine, Viramidine, Zalcitabine, Zanamivir (Relenza), or the product to be used for the administration of the drug and zidovudine patient.
[0184] [000184] The systems and methods of the invention can also be used to monitor HIV / AIDS patients. When doctors suspect an acute infection (for example, in a patient with a report of recent high-risk composting in association with symptoms and signs of acute retroviral syndrome), an HIV RNA test is usually performed. High levels of HIV RNA detected in plasma through the use of sensitive amplification assays (PCR, bDNA, or NASBA), in combination with an undetermined or negative HIV antibody test, support the diagnosis of acute HIV infection. Monitoring individuals with HIV / AIDS for viral load levels, drug, CD4 cell counts, and toxicity patterns in a single platform diagnostic method would provide distinct advantages to an individual. The systems and methods of the invention can be used in a simple detection method, no sample preparation, multiplexed, automated system to determine the drug level, the determinants of toxicity or adverse effect, and the potential biomarker of disease progression. For example, a cartridge having ports or cavities containing 1) magnetic particles having specific CD4 cell antibodies decorated on their surface, 2) magnetic particles having specific toxicity biomarker antibodies on their surface, and 3) magnetic particles having specific acid probes nucleic acid to identify viral load levels could be employed to quickly determine and provide clinical control values for a given HIV / AIDS patient.
[0185] [000185] The systems and methods of the invention can also be used to monitor and diagnose immune disease in an individual (e.g., Crohn's disease, ileitis, enteritis, inflammatory bowel disease, irritable bowel syndrome, ulcerative colitis, as well as disease non-gastrointestinal immune system). The relatively recent development of genetically engineered agents has the potential to alter the treatment of immune disease radically, and Remicade (also known as Infliximab, an anti-TNF antibody) has been introduced as a new therapeutic class with high efficacy, rapid onset, prolonged effect, and improved tolerance. However, these agents are expensive and at least a third of eligible patients do not provide a useful answer. Finding a way to predict those who will respond, and for early release, is therefore of obvious importance. TNF polymorphisms also seem to influence the nature and frequency of extraintestinal manifestations of inflammatory bowel disease (IBD). Several large controlled experiments indicate that remicade has a role in the treatment of patients with moderate to severely active Crohn's disease and in Crohn's disease fistulation. Small studies have shown possible associations between poor response to remicade and increasing mucosal levels of activated NF-kappaB, homozygosity for TNFR2 exon 6 polymorphism (Arg196Arg genotype), positivity for perinuclear antineutrophil cytoplasmic antibodies (ANCA), and with the presence of increased numbers of activated lamina provide mononuclear cells producing interferon-gamma and TNF-alpha. Thus, monitoring Crohn's disease patients for TNF-alpha patterns and toxicity in a simple platform diagnostic method would have distinct advantages. The method according to the claims of the invention can be a simple detection method, no sample preparation, multiplexed, automated system to determine the drug level, toxicity or adverse effect determinants, and the potential biomarker of disease progression. For example, a cartridge having ports or cavities containing 1) magnetic particles having specific anti-TNF-alpha antibodies decorated on its surface, 2) magnetic particles having specific toxicity biomarker antibodies, and 3) magnetic particles having specific probes to identify markers of progression disease could be employed to quickly determine and provide clinical control values for a given Crohn's disease or IBD patient.
[0186] [000186] The systems and methods of the invention can also be used to monitor and diagnose infectious disease and inflammation in a no-sample, automated, multiplexed system. Such systems and methods can be used to monitor, for example, bacteremia, sepsis, and / or Systemic Inflammatory Response Syndrome (SIRS). Early diagnosis is clinically important when this type of infection, if left untreated, can lead to organ dysfunction, hypoperfusion, hypotension, refractory (septic) shock / SIRS shock, and / or Multiple Organ Dysfunction Syndrome (MODS). For a typical patient, many bacterial or fungal infections are the result of incubation upon admission to a healthcare setting and are referred to as healthcare associated infections (HAI), also known as nosocomial, hospital-acquired infections. or beginning in hospital. Among the best-known categories of bacteria to infect immunocompromised patients are MRSA (Methicillin-resistant Staphylococcus aureus), gram-positive bacteria and Helicobacter, which is gram-negative. While there are antibiotic drugs that can treat diseases caused by Gram-positive MRSA, there are currently few effective drugs for Acinetobacter. Common pathogens in bloodstream infections are coagulase-negative staphylococci, Enterococcus, and Staphylococcus aureus. In addition, Candida albicans and pathogens for pneumonia, such as Pseudomonas aeruginosa, Staphylococcus aureus, Klebsiella pneumoniae, and Haemophilus influenza are responsible for many infections. Pathogens for urinary tract infections include Escherichia coli, Candida albicans, and Pseudomonnas aeruginosa. Gram-negative enteric organisms are additionally common in urinary tract infections. Surgical site infections include Staphylococcus aureus, Pseudomonas aeruginosa, and coagulase-negative staphylococci. The infectious agent can be selected from, without limitation, pathogens associated with sepsis, such as Acinetobacter baumannii, Aspergillus fumigatis, Bacteroides fragilis, B. fragilis, blaSHV, Burkholderia cepacia, Campylobacter jejuni / coli, Candida guilliermondii, C. albicans, C. glabrata, C. krusei, C. Lusitaniae, C. parapsilosis, C. tropicalis, Clostridium pefringens, Coagulase negative Staph, Enterobacter aeraogenes, E. cloacae, Enterobacteriaceae, Enterococcus faecalis, E. faecium, Escherichia coli, Haemophilus influenzae, King Kingae, Klebsiella oxytoca, K. pneumoniae, Listeria monocytogenes, Mec A gene (MRSA), Morganella morgana, Neisseria meningitidis, Neisseria spp. non-meningitidis, Prevotella buccae, P. intermedia, P. melaninogenica, Propionibacterium acnes, Proteus mirabilis, P. vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Serratia marcescens, Staphylococcus aureus, S. haemolyticus, S. maltophomophys, S. saprophytis, S. maltophilia, S. maltophilia, Streptococcus agalactie, S. bovis, S. dysgalactie, S. mitis, S. mutans, S. pneumoniae, S. pyogenes, and S. sanguinis; or any other infectious agent described here. In certain cases, the method and system will be designed to verify that the infectious agent carries a Van A gene or Van B gene characteristic of vancomycin resistance; mecA for resistance to methicillin, NDM-1 and ESBLfor more generally resistance to beta-lactams.
[0187] [000187] Sepsis and septic shock are serious medical conditions that are characterized by an inflammatory state or septic shock are serious medical conditions that are characterized by an inflammatory state of the whole body (systemic inflammatory response syndrome or SIRS) and the presence of an infection known and suspected. Sepsis is defined as SIRS in the presence of an infection, septic shock is defined as sepsis with refractory arterial hypotension or abnormalities of hypoperfusion in spite of adequate fluid resuscitation, and severe sepsis is defined as sepsis with organ dysfunction, hypoperfusion, or hypotension. Several studies have examined the value of combining currently available markers, and thus, the platform as described provides the single or simultaneous determination of the levels of factors such as GRO-alpha, High mobility group 1 protein (HMBG-1), receptor IL-1, IL-1 receptor antagonist, IL-1b, IL-2, IL-4, IL-6, IL-8, IL-10, IL-12, IL-13, IL-18, protein inflammatory macrophage (MIP-1), macrophage migration inhibiting factor (MIF), osteopontin, RANTES (regulated on activation, expressed and secreted normal T cell; or CCL5), TNF-α, C-reactive protein (CRP), CD64 , and monocyte chemotactic protein 1 (MCP-1). In addition, systems and methods can be designed to monitor certain sepsis-specific proteins, such as adenosine deaminase binding protein (ABP-26), inducible nitric oxide synthase (iNOS), lipopolysaccharide binding protein (LBP), and procalcitonin (PCT). Thus, this platform reduces the empirical protocols and / or use of non-specific / general antimicrobials that may or may not be targeting the specific pathogen and / or the underlying system dysfunction for a given patient. This platform provides rapid and accurate diagnoses, which can be indicative of effective therapy, providing a key component to provide an essential component for structuring the physician's decision and reducing morbidity and mortality.
[0188] [000188] To determine whether a patient has sepsis, it is necessary to identify the presence of a pathogen. To most effectively treat a patient, early initiation of appropriate therapy is critical. Antimicrobial and other sepsis treatments rely on the classification of pathogens at multiple levels, including the identification of an agent as 1) bacterial, viral, fungal, parasitic or otherwise; 2) gram-positive, gram-negative, yeast, or mold, 3) species, and 4) susceptibility.
[0189] [000189] Each of these levels of specificity improves the time of initiation of appropriate therapy, and each step in addition to lowering the mat will induce a restriction of therapeutic agents for the most specific group. Without absolute susceptibility data, empirical methods of care rely on available information on the pathogen (at whatever level) and the pattern of pathogen frequency and susceptibility tilt in the hospital from another care location. Thus, certain categories of pathogens are often assumed to be causative until more data exists to refine pathogen and therapy matching. Specifically, these targets fall into the ESKAPE category (which is a series of resistant pathogens) and the SPACE category, which is a group of highly virulent pathogens that require isolation from patients.
[0190] [000190] In addition, to identify these pagogens in multiple types of samples (blood, tissues, urine, etc.), another method for distinguishing symptomatic patients, for example, patients with systemic inflammatory syndrome, or SIRS, from septic patients, must use biomarkers that correlate either individually or using an index to identify patients with infection. In cases where infections are not detected due to interference from antimicrobial therapy with diagnoses, immune system control of the therapy, or otherwise, these biomarkers, which can be multiple types of analyzed (cytokines, metabolites, DNA, RNA expression / gene, etc.) will indicate infection and thus sepsis.
[0191] [000191] To generate the diagnostic information required for both, the presence of an infection and some level of species identification, a panel can be: (i) gram-positive groups (for example, S. aureus, and CoNS (negative staph coagulase))); (ii) gram-positive chains / pairs (for example, Strep spp., mitis, pneumonia spp., agalactiae spp., pyogenes spp., Enterococcus spp. (E. faecium, E. fecalis); (iii) gram- negative (for example, E. coli, Proteus spp., Klebsiella spp., Serratia spp., Acinetobacter spp., Stenotrophomonas spp.); (iv) SPACE (for example, Serratia spp., Pseudomonas spp., Acinetobacter spp., Citrobacter spp., Enterobacter spp.); (V) Pseudomonas (for example, Pseudomonas spp.); (Vi) ESKAPE (E. faecium, Staphylococcus aureas, Klebsiella spp., Acinetobacter spp., Pseudomonas spp., Enterobacter spp.) and (vii) Pan-Bacterial (all bacterial species).
[0192] [000192] This panel should be used in conjunction with a fungal test for full coverage. The categories represent the information required for an effective intervention with appropriate therapy, since each caregiver site will have an empirically derived method based on a positive response to gram +, gram-, etc. The species identified in each category represent those that fit under each topic, but are not inclusive. In addition, a pan-bacterial marker is included to cover all species that are not covered by the diagnostic method employed for each category. In addition, the combination of results will also provide an indication of the species, although not completely, if included as described above. Positive and negative cross-references by category allow a process of elimination method to identify some of the species, probabilistically.
[0193] [000193] The systems and methods of the invention can likewise be used to monitor and diagnose heart disease in an individual, such as a myocardial infarction. Cardiac markers or cardiac enzymes are proteins that leak from injured myocardial cells and are used to assess cardiac injury. Cardiac markers include, without limitation, the SGOT enzymes, LDH, the MB subtype of the creatine kinase enzyme, and cardiac troponins (T and I). Thus, in the acute adjustment, monitoring of Troponin I and T, as well as other potential biomarkers of cardiac ischemia, in addition to drug therapy and toxicity patterns in a single-platform diagnostic method would have distinct advantages. The systems and methods of the invention can be used to provide a multiplexed sample-free preparation, single detection method, automated system to determine the drug level, the determinants of adverse effect or toxicity, and the potential biomarker of disease progression. For example, a cartridge having portals or cavities contains 1) magnetic particles having specific antitroponin I or troponin T antibodies decorated on its surface, 2) magnetic particles having specific toxicity biomarker antibodies on its surface, and 3) magnetic particles having probes Specific measures to identify progression disease markers can be used to quickly determine and provide clinical control values for a given patient with myocardial infarction.
[0194] [000194] One or more multi-cavity cartridges may be configured for use in the systems and methods of the invention and prepared (s) with at least one patient's whole blood sample; magnetic particles to detect each of the analyzed to be detected (one or more smaller molecules; one or more metabolites of one or more smaller molecules; metabolic biomarker as described for the liver function panel); and dilution and wash buffers. Liver function tests are done on a plasma or serum sample from a patient or plasma sample and blood analysis in a clinical biochemical laboratory provides crucial data regarding the condition of the patient's liver. A "liver function panel" is a blood test in which low or high levels of one or more enzymes can point to liver damage or disease. For example, the liver function panel may include one or more of the following assays for detecting analyte: one or more smaller molecules; one or more metabolites of one or more smaller molecules; a biological, metabolic biomarker; genotyping, gene expression profile; and proteomic analysis.
[0195] [000195] A liver function panel can include analysis of one or more of the following proteins in a biological sample from a patient or individual: 1) albumin (the main constituent of total protein in the liver; while the remainder is called globulin; albumin; must be present as 3.9 to 5.0 g / dL, hypoalbuminemia indicates inferior nutrition, inferior protein catabolism, cirrhosis or nephrotic syndrome); 2) aspartate transaminase (AST) (also known as serum glutamic oxaloacetic transaminase or aspartate aminotransferase, is an enzyme in liver parenchymal cells and is usually 10 to 34 IU / L; high levels are indicative of acute liver damage) ; 3) alanine transaminase (ALT) (also known as glutamic pyruvic serum transaminase or alanine aminotransferase, is an enzyme present in hepatocytes at levels between 8 to 37 IU / L; high levels are indicative of acute liver damage in viral hepatitis or paracetamol overdose; the ratio of AST to ALT is used to differentiate between the reasons for liver damage); 4) alkaline phosphatase (ALP) (an enzyme that is present in the cells lining the bile ducts of the liver; the normal range is 44 to 147 IU / L and the level increases in the case of infiltrative diseases of the liver, intrahepatic cholestasis or obstruction of the large bile duct); 5) Range glutamyl transpeptidase (GGT) (a more sensitive marker for cholestatic lesion than ALP, is very specific to the liver; the standard range is 0 to 51 IU / L; both acute and chronic alcoholic toxicity GGT; the ratio of an isolated elevation in ALP can be detected by GGT); 6) total bilirubin (TBIL) (an increase in total bilirubin can lead to jaundice and can be attributed to cirrhosis, viral hepatitis, hemolytic anemias or internal bleeding); 7) direct bilirubin; 8) prothrombin time (PTT) (liver cell damage and obstruction of bile flow can cause changes in blood clotting time); 9) alpha-fetoprotein test (high levels indicate hepatitis or cancer); 10) lactate dehydrogenase; and 11) mitochondrial antibodies (if present may indicate chronic active hepatitis, primary biliary cirrhosis, or other autoimmune disorders). The proteins described above would be analyzed on the liver function panel using the systems and methods of the invention.
[0196] (i) Metabolizadores inferiores. Certos fármacos são metabolizados mais lentamente do que o normal e o medicamento terá uma meia vida mais longa e possivelmente aumentam a probabilidade que causará efeitos colaterais. (ii) Metabolizadores normais. Os fármacos serão metabolizados em uma taxa média e desse modo são indicativos que há um benefício de tratamento e pontos para menos efeitos colaterais do que em outros indivíduos que não metabolizam esses medicamentos particulares também. (iii) Metabolizadores intermediários. Os fármacos podem ou não ser metabolizados em uma taxa média. Pelo menos um gene envolvido no metabolismo de fármaco é suspeito de funcionar anormalmente. Em seguida, há uma predisposição para metabolizar certos fármacos diferentemente. (iv) Metabolizadores ultrarrápidos. Os fármacos são metabo-lizadas mais rapidamente e mais eficazmente do que a média. Considerando que a taxa metabólica é mais alta do que média, alguns medicamentos são inativados mais cedo ou são excretados mais cedo do que o normal e o medicamento pode não ter a eficácia desejada. [000196] An additional liver function panel may include the genotyping of cytochrome P450 enzymes. Cytochrome P450 genotyping tests are used to determine how a patient or healthy individual metabolizes a drug. The results of cytochrome P450 tests can be used to divide individuals into four main types: (i) Lower metabolizers. Certain drugs are metabolized more slowly than normal and the drug will have a longer half-life and possibly increase the likelihood that it will cause side effects. (ii) Normal metabolizers. Drugs will be metabolized at an average rate and thus are indicative that there is a treatment benefit and points to fewer side effects than in other individuals who do not metabolize these particular drugs as well. (iii) Intermediate metabolizers. Drugs may or may not be metabolized at an average rate. At least one gene involved in drug metabolism is suspected of functioning abnormally. Then there is a predisposition to metabolize certain drugs differently. (iv) Ultra-fast metabolizers. Drugs are metabolised more quickly and more effectively than the average. Considering that the metabolic rate is higher than average, some medications are inactivated earlier or are excreted earlier than normal and the medication may not have the desired effectiveness.
[0197] [000197] Currently, the genotyping of the genes responsible for these enzymes by a population has been shown, in which polymorphic differences in these enzymes can lead to variation in the efficacy and toxicity of some drugs. Genotyping requires a cell sample representative of the patient or individual's genome and the analysis is aimed at determining genetic differences in these clinically important genes.
[0198] [000198] Possible hepatic metabolic enzymes that may be part of a liver function panel include, however, are not limited to CYP2C19, CYP2D6, CYP2C9, CYP2C19, CYP1A2, NAT2, DPD, UGT1A1, 5HTT.
[0199] [000199] The invention features a multiplexed analysis of a single blood sample (e.g., a single blood draw or any other type of patient sample described here) from a patient to determine a) enzymatic state of the liver, as well as b ) the genotype of major metabolic enzymes to then be able to design pharmacotherapy regimens for optimal therapeutic care using the systems and methods of the invention.
[0200] [000200] The systems and methods of the invention can include one or more multilayer cartridges prepared with at least one patient's whole blood sample; magnetic particles to detect each of the analyzed to be detected; analyzed antibodies; multivalent binding agents; and / or dilution and wash buffers for use in a multiplexed assay as described above.
[0201] [000201] Nephrotoxicity
[0202] [000202] Renal toxicity is a common side effect of using xenobiotics and early, rapid detection of early stages of nephrotoxicity can help in understanding the medical decision. Early reports of detection of renal toxicity suggest that the increased mRNA expression of certain genes can be monitored. However, others have suggested that markers of renal toxicity can be detected in urine. These markers include: kim-1, lipocalin-2, neutrophil gelatinase-associated lipocalin (NGAL), timp-1, clusterin, osteopontin, vimentin, and heme oxygenase 1 (HO-1). More broadly, the detection of DNA, heavy metal ions or BUN levels in the urine can be useful clinical information. Accordingly, the methods and utility of the present invention also include the ability to detect these markers of renal toxicity. Optionally, a liver function panel may likewise include one or two official nephrotoxicity biomarkers, or vice versa. Nucleic Acid Amplification and Detection of Complex Samples
[0203] [000203] Systems and methods of the invention may include nucleic acid detection assays based on amplification conducted starting with complex samples (for example, for diagnostic, forensic, and environmental analyzes).
[0204] [000204] Sample preparation must likewise remove or provide resistance to common PCR inhibitors found in complex samples (for example, body fluids, soil, or other complex medium). Common inhibitors include those identified in Wilson, Appl. Environ. Microbiol., 63: 3741 (1997), among others. Inhibitors typically act by preventing cell lysis, degradation or sequestration of a target nucleic acid, and / or inhibiting polymerase activity. The most commonly used polymerase, Taq, is inhibited by the presence of 0.1% blood in a reaction. Very recently, mutant Taq polymerase was created, which is resistant to common inhibitors (eg, hemoglobin and / or humic acid) found in blood and soil (Kermekchiev et al., Nucl. Acid. Res., 37 (5): e40 , (2009)). Manufacturer's recommendations indicate that these mutations allow direct amplification of up to 20% of blood. Despite the resistance provided by the mutations, the detection of accurate real-time PCR is complicated due to the fluorescence extinction observed in the presence of a blood sample (Kermekchiev et al., Nucl. Acid. Res., 37: e40 (2009)).
[0205] [000205] The amplification of the DNA polymerase chain reaction or cDNA is a proven and trusted methodology; however, as discussed above, polymerases are inhibited by agents contained in crude samples, including but not limited to anticoagulants and hemoglobin commonly used. Recently, Taq mutant polymerases were created to harbor resistance to common inhibitors found in blood and soil. Currently available polymerases, for example, HemoKlenTaqTM (New England BioLabs, Inc., Ipswich, MA) as well as OmniTaqTM and OmniKlenTaqTM (DNA Polymerase Technology, Inc., St. Louis, MO) are mutant Taq polymerase (for example, N- terminal and / or point mutations) that make them capable of amplifying DNA in the presence of up to 10%, 20% or 25% whole blood, depending on the product and reaction conditions (See, for example, Kermekchiev et al., Nucl. Acids Res 31: 6139 (2003); and Kermekchiev et al., Nucl. Acids. Res., 37: e40 (2009); and see US Patent No. 7,462,475). In addition, Phusion® Blood Direct PCR Kits (Finnzymes Oy, Espoo, Finland), include a single-fusion DNA polymerase enzyme to incorporate a double-stranded DNA binding domain, which allows amplification under conditions that are typically inhibitory in conventional polymerases such as Taq or Pfu and allows DNA amplification in the presence of up to about 40% whole blood under certain reaction conditions. See Wang and another, Nuc. Acids Res. 32: 1197 (2004); and see U.S. Patent Nos. 5,352,778 and 5,500,363. In addition, Kapa Blood PCR Mixes (Kapa Biosystems, Woburn, MA), provides a genetically engineered DNA polymerase enzyme that allows direct amplification of whole blood to up to about 20% of the reaction volume under certain reaction conditions. Despite these innovations, direct optical detection of generated amplicons is not possible with existing methods since fluorescence, absorbance, and other methods linked to light produce signals that are extinguished by the presence of blood. See Kermekchiev et al., Nucl. Acid. Res., 37: e40 (2009).
[0206] [000206] We found that complex samples such as whole blood can be directly amplified using about 5%, about 10%, about 20%, about 25%, about 30%, about 25%, about 40 %, and about 45% or more whole blood in amplification reactions, and that the resulting amplicons can be directly detected from the amplification reaction using magnetic resonance (MR) relaxation measures in the addition of conjugated magnetic particles attached to the complementary oligonucleotides target nucleic acid sequence. Alternatively, the magnetic particles can be added to the sample before amplification. Thus, methods are provided for using nucleic acid amplification in a complex dirty sample, hybridization of the resulting amplicon to paramagnetic particles, followed by direct detection of hydridized magnetic particle conjugate and target amplicons using the particle-based detection systems magnetic. In particular embodiments, the direct detection of conjugates of hydridized magnetic particle and amplicons is by relaxation measures by MR (for example, T2, T1, T1 / T2 hybrid, T2 *, etc.). Also provided are methods that are kinetic for quantifying the original nucleic acid copy number within the sample (for example, sampling and detection of nucleic acid in predefined cycle numbers, comparison of endogenous internal control nucleic acid, use of nucleic acid from endogenous reinforced homologous competitive control).
[0207] [000207] The terms "amplification" or "amplify" or derivatives thereof as used herein mean one or more methods known in the art to copy a target or standard nucleic acid, thereby increasing the number of copies of a selected nucleic acid sequence . Amplification can be exponential or linear. A target or standard nucleic acid can be DNA or RNA. Sequences amplified in this way form an "amplified region" or "amplicon". Primer probes can be designed easily by those skilled in the art to target a specific standard nucleic acid sequence. In certain preferred embodiments, the resulting amplicons are short to allow rapid cycling and copy generation. The size of the amplicon may vary when necessary to provide the ability to separate target nucleic acids from targetless nucleic acids. For example, amplicons can be less than about 1,000 nucleotides in length. Desirably, amplicons are 100 to 500 nucleotides in length (for example, 100 to 200, 150 to 250, 300 to 400, 350 to 450, or 400 to 500 nucleotides in length).
[0208] [000208] While the exemplary methods described below refer to amplification using polymerase chain reaction ("PCR"), numerous other methods are known in the art for nucleic acid amplification (for example, isothermal methods, rotation, etc.). Those skilled in the art will understand that these other methods can be used in place of, or in conjunction with, PCR methods. See, for example, Saiki, "Amplification of Genomic DNA" in PCR Protocols, Innis et al., Eds., Academic Press, San Diego, Calif., Pp 13-20 (1990); Wharam and gold, Nucleic Acids Res. 29: E54 (2001); Hafner and another, Biotechniques, 30: 852 (2001). Other amplification methods suitable for use with the present methods include, for example, polymerase chain reaction (PCR) method, reverse transcription PCR (RT-PCR), ligase chain reaction (LCR), amplification system with transcription-based (TAS), transcription-mediated amplification (TMA), nucleic acid sequence-based amplification method (NASBA), the filament displacement amplification method (SDA), the loop-mediated isothermal amplification method ( LAMP), the amplification initiated by isothermal and chimeric nucleic acid initiator (ICAN), and the method of the intelligent amplification system (SMAP). These methods, as well as others, are well known in the art and can be adapted for use in conjunction with provided methods of detecting amplified nucleic acid.
[0209] [000209] The PCR method is a technique for preparing many copies of a specific standard DNA sequence. The PCR process is described in U.S. Patent Nos. 4,683,195; 4,683,202; and 4,965,188, each of which is incorporated herein by reference. A group of primers complementary to a standard DNA is designed, and a region flanked by the primers is amplified by DNA polymerase in a reaction including multiple amplification cycles. Each amplification cycle includes an initial denaturation, and up to 50 annealing cycles, filament elongation (or extension) and filament separation (denaturation). In each reaction cycle, the DNA sequence between the primers is copied. Primers can bind to the copied DNA as well as the original standard sequence, so the total number of copies increases exponentially over time. PCR can be performed according to Whelan, and another, Journal of Clinical Microbiology, 33: 556 (1995). Various modified PCR methods are available and well known in the art. Various modifications such as the "RT-PCR" method, in which DNA is synthesized from RNA using a reverse transcriptase before performing PCR; and the "TaqMan PCR" method, in which only a specific allele is amplified and detected using a fluorescently labeled TaqMan probe, and Taq DNA polymerase, are known to those skilled in the art. RT-PCR and variations thereof have been described, for example, in U.S. Patent Nos. 5,804,383; 5,407,800; 5,322,770; and 5,310,652, and references described herein, which are hereby incorporated by reference; and TaqMan PCR and related reagents for use in the method have been described, for example, in U.S. Patent Nos. 5,210,015; 5,876,930; 5,538,848; 6,030,787; and 6,258,569, which are hereby incorporated by reference.
[0210] [000210] LCR is a DNA amplification method similar to PCR, unless you use four primers instead of two and use the ligase enzyme to bind or join two segments of DNA. Amplification can be performed on a thermal cycler (for example, LCx of Abbott Labs, North Chicago, IL). CSF can be carried out, for example, as according to Moore and another, Journal of Clinical Microbiology 36: 1028 (1998). LCR methods and variations have been described, for example, in European Patent Application Publication No. EP0320308, and U.S. Patent No. 5,427,930 each of which are incorporated herein by reference.
[0211] [000211] The TAS method is a method to specifically amplify a target RNA in which a transcription is obtained from a standard RNA by a cDNA synthesis step and an RNA transcription step. In the cDNA synthesis step, a sequence recognized by a DNA-dependent RNA polymerase (i.e., a polymerase binding sequence or PBS) is inserted into the cDNA copy downstream of the target or marker sequence to be amplified using a primer. two-domain oligonucleotide. In the second step, an RNA polymerase is used to synthesize the multiple RNA copies of the cDNA standard. Amplification using TAS requires only a few cycles because DNA-dependent RNA transcription can result in 10-1000 copies for each copy of the cDNA standard. TAS can be performed according to Kwoh and another, PNAS 86: 1173 (1989). The TAS method has been described, for example, in International Patent Application Publication No. WO1988 / 010315 which is incorporated herein by reference.
[0212] [000212] Transcription-mediated amplification (TMA) is a transcription-based isothermal amplification reaction that uses RNA transcription by RNA polymerase and DNA transcription by reverse transcriptase to produce a target nucleic acid RNA amplicon. TMA methods are advantageous in that they can produce 100 to 1000 copies of amplicon per cycle of amplification, instead of PCR or LCR methods that produce only 2 copies per cycle. TMA has been described, for example, in U.S. Patent No. 5,399,491 which is incorporated herein by reference. NASBA is a transcription-based method to specifically amplify a target RNA from an RNA or DNA pattern. NASBA is a method used for the continuous amplification of nucleic acids in a single mixture at a temperature. A transcript is obtained from a standard RNA by a DNA-dependent RNA polymerase using a sense primer having a sequence identical to a target RNA and an antisense primer having a sequence complementary to the target RNA on the 3 'side and a promoter sequence that recognizes T7 RNA polymerase on the 5 'side. A transcript is also synthesized using the obtained transcript as a standard. This method can be performed as according to Heim, et al., Nucleic Aciss Res., 26: 2250 (1998). The NASBA method has been described in U.S. Patent No. 5,130,238 which is incorporated herein by reference.
[0213] [000213] The SDA method is an isothermal amplification method of nucleic acid in which the target DNA is amplified using a DNA strand replaced with a filament synthesized by a filament replacement DNA polymerase requiring 5 '-> exonuclease activity 3 'by a single strand cut by a restriction enzyme as a pattern for the next replication. A primer containing a restriction site is annealed to the standard, and then the amplification primers are annealed to the adjacent 5 'sequences (forming a cut). Amplification is started at a fixed temperature. Newly synthesized DNA strands are cut by a restriction enzyme and polymerase amplification begins again, displacing the newly synthesized strands. SDA can be performed according to Walker and another, PNAS, 89: 392 (1992). SDA fram methods described in U.S. Patent Nos. 5,455,166 and 5,457,027 each of which are incorporated by reference.
[0214] [000214] The LAMP method is an isothermal amplification method in which a loop is always formed at the 3 'end of a synthesized DNA, the primers are annealed within the loop, and specific amplification of the target DNA is performed isothermally. LAMP can be performed according to Nagamine and another, Clinical Chemistry. 47: 1742 (2001). LAMP methods have been described in U.S. Patent Nos. 6,410,278; 6,974,670; and 7,175,985 each of which are incorporated by reference.
[0215] [000215] The ICAN method is anisothermal amplification method in which the specific amplification of a target DNA is performed isothermally by a filament replacement reaction, a standard exchange reaction, and a cut-in reaction, using a chimeric initiator including RNA-DNA and DNA polymerase having a filament replacement activity and RNase H. ICAN can be performed according to Mukai and another, J. Biochem. 142: 273 (2007). The ICAN method has been described in U.S. Patent No. 6,951,722, which is incorporated herein by reference.
[0216] [000216] The SMAP method (MITANI) is a method in which a target nucleic acid is synthesized continuously under isothermal conditions using a primer group including two types of primers and DNA or RNA as a standard. The first primer included in the primer set includes, in the 3 'end region thereof, a sequence (Ac') hybridizable to a sequence (A) in the 3 'end region of a target nucleic acid sequence as well as, on the side 5 'of the aforementioned sequence (Ac'), a sequence (B ') hybridizable to a sequence (Bc) complementary to a sequence (B) existing on the 5' side of the aforementioned sequence (A) in the aforementioned target nucleic acid sequence. The second primer includes, in the 3 'end region thereof, a sequence of a (Cc') hybridizable to a sequence (C) in the 3 'end region of a sequence complementary to the aforementioned target nucleic acid sequence as well as a sequence of return (D-Dc ') including two nucleic acid sequences hybridizable to each other in an identical strand on the 5' side of the aforementioned (Cc ') sequence. SMAP can be performed according to Mitani and another, Nat. Methods, 4 (3): 257 (2007). SMAP Methods in Publication of U.S. Patent Application Nos. 2006/0160084, 2007/0190531 and 2009/0042197, each of which is incorporated herein by reference.
[0217] [000217] The amplification reaction can be designed to produce a specific type of amplified product, such as nucleic acids that are double-stranded; single filament; double filament with 3 'or 5' projections; or double-stranded with chemical binders at the 5 'and 3' ends. The amplified PCR product can be detected by: (i) hybridizing the amplified product to complementary oligonucleotides attached to the magnetic particles, where two different oligonucleotides are used which hybridize to the amplified product such that nucleic acid serves as a particle agglomeration of promotion of interparticle mooring; (ii) hybridization-mediated detection where the amplified product's DNA must first be denatured; (iii) detection mediated by hybridization where the particles for 5 'and 3' projections of the amplified product; (iv) binding of particles from chemical or biochemical binders to the terminals of the amplified product, such as streptavidin functionalized particles that bind to the biotin functionalized amplified product.
[0218] [000218] The systems and methods of the invention can be used to perform PCR in real time and provide quantitative information about the amount of target nucleic acid present in a sample (see Figure 25 and Example 14). Methods for conducting quantitative real-time PCR are provided in the literature (see for example: RT-PCR Protocols. Methods in Molecular Biology, Vol. 193. Joe O'Connell, ed. Totowa, NJ: Humana Press, 2002, 378 pp. ISBN 089603-875-0.). Example 14 describes use of the methods of the invention for real-time PCR analysis of a whole blood sample.
[0219] [000219] The systems and methods of the invention can be used to perform real-time PCR directly on opaque samples, such as whole blood, using modified magnetic nanoparticles with capture and magnetic separation probes. The use of real-time PCR allows the quantification of a target nucleic acid without opening the reaction tube after the PCR reaction begins.
[0220] [000220] Previous work has shown that in some cases the presence of particles in the PCR reaction could inhibit PCR. For these inhibitory particles, it is considered that the particles can be pulled alongside the tube (or another location inside the container) to keep them out of solution during the PCR reaction. The methods can be used to release the particles back into the suspension to allow them to hybridize to the PCR product and then remove them out of solution again.
[0221] [000221] In certain embodiments, the invention features the use of enzymes compatible with whole blood, for example, NEB Hemoklentaq whole blood enzyme, DNAP Omniklentaq, Kapa Biosystems, Thermo-Fisher Finnzymes Phusion enzyme.
[0222] [000222] The invention also features quantitative asymmetric PCR. In any of the real-time PCR methods of the invention, the method may involve the following steps: (i) aliquoting the whole blood into a prepared PCR master mixture containing superparamagnetic particles; (ii) before the first PCR cycle, close the tube until the PCR cycle is completed; (iii) load the tube on the thermal cycler; (iv) conducting "n" cycles of standard PCR thermal cycling; (v) conducting a T2 detection (the exact length of time and steps for this vary, depending on the particle and biochemical design method described below); and (vi) repeat steps (iv) and (v) until sufficient T2 readings are made for an accurate quantification of the initial target concentration.
[0223] [000223] The previous methods can be used with any of the following categories of aggregation or disaggregation detection described here, including:
[0224] [000224] A variety of impurities and whole blood components can be inhibitory to polymerase and hybridization of the primer. These inhibitors can lead to the generation of false positives and low sensitivity. To reduce the generation of false positives and low sensitivity when amplifying and detecting nucleic acids in complex samples, it is desirable to use a thermal stable polymerase not inhibited by the whole blood sample (see, for example, United States Patent No. 7,462,475 ) and include one or more internal PCR assay controls (see Rosenstraus et al. J. Clin Microbiol. 36: 191 (1998) and Hoofar et al., J. Clin. Microbiol. 42: 1863 (2004)). For example, to ensure that clinical specimens are successfully amplified and detected, the assay can include an internal control nucleic acid that contains primer binding regions identical to those of the target sequence. As shown in the Examples, the target nucleic acid and internal control were selected in such a way that each has a unique probe binding region that differentiates the internal control from the target nucleic acid. Reaction Kinetics
[0225] [000225] The reaction of magnetic and specific particles analyzed to form aggregates can be used to produce a diagnostic signal in the tests of the invention. In many instances, incubating the reaction mixture for a period of time is sufficient to form the aggregates. The methods, kits, cartridges, and devices of the invention can be configured to shorten the amount of time required to capture a particular analyte, or to produce aggregates of magnetic particles. While altering the total concentration of magnetic particles would appear to be a simple and straightforward approach to increase aggregation rates, this approach is complicated by (i) non-specific aggregation that can arise with high concentrations of magnetic particles, and (ii) the need to produce an observable signal change (ie, change in the relaxation signal) in response to aggregation in the presence of a low analyte concentration. Reaction kinetics can be improved, for example, by mechanically induced aggregation, by acoustically induced aggregation, by ultrasonically induced aggregation, by electrostatically induced aggregation, or by capture (for example, by exposing nanoparticles to a magnetic field, using a porous membrane , employing a magnetizable metal foam, or centrifugation) the magnetic particles in a portion of the liquid sample. NMR units
[0226] [000226] Systems for preparing the methods of the invention can include one or more NMR units. Figure 1A is a schematic diagram 100 of an NMR system for detecting a signal response from the liquid sample to an appropriate RF pulse sequence. A bias magnet 102 stabilizes a Bb 104 polarizing magnetic field through a sample 106. The magnetic particles are either liquid or lyophilized in the cartridge prior to its introduction into a sample well (the term "well" as used here includes any indentation, vessel, container, or support) 108 until the introduction of liquid sample 106 into well 108, or magnetic particles can be added to sample 106 before introduction of the liquid sample into well 108. An RF coil 110 and RF oscillator 112 provide an RF excitation at the Larmor frequency which is a linear function of the Bb polarizing magnetic field. In one embodiment, the spiral RF 110 is wrapped around the sample cavity 108. The RF excitation creates an unbalanced distribution in the rotation of the water protons (or free protons in a non-aqueous solvent). When the RF excitation is turned off, the protons "relax" to their original state and emit an RF signal that can be used to extract information about the presence and concentration of the analyzed. Coil 110 acts as an RF antenna and detects a signal, which based on the applied RF pulse sequence, the probes have different material properties, for example a T2 relaxation. The signal of interest for some cases of the technology is the gyro-gyro relaxation (usually 10 to 2000 milliseconds) and is called the T2 relaxation. The RF signal from coil 110 is amplified 114 and processed to determine the response of T2 (decay time) to excitation in the Bb polarization field. Cavity 108 can be a small capillary or other tube with nanoliters to microliters of the sample, including the analyzed and a properly sized coil wound around it (see Figure 1B). The coil is typically wrapped around the sample and sized according to the sample volume. For example (and without limitation), for a sample having a volume of about 10 ml, a solenoid coil about 50 mm long and 10 to 20 mm in diameter can be used; for a sample having a volume of about 40 μl, a solenoid coil about 6 to 7 mm long and 3.5 to 4 mm in diameter can be used; and for a sample having a volume of about 0.1 nl, a solenoid coil about 20 pm long and about 10 pm in diameter can be used. Alternatively, the coil can be configured as shown in any of Figures 2A-2E around or in proximity to the cavity. An NMR system can also contain multiple RF coils for detecting multiplexing purposes. In certain embodiments, the RF spiral has a conical shape with dimensions 6 mm × 6 mm × 2 mm.
[0227] [000227] Figures 2A-2E illustrate exemplary micro NMR coil (RF coil) designs. Figure 2A shows a 200 coil solenoid coil of about 100 pm in length, however one can consider the coil to be 200μm, 500 pm or up to 1000 pm in length. Figure 2B shows a "planar" coil 202 (the coil is not really planar, as long as the coil is finite in thickness) of about 1000 pm in diameter. Figure 2C shows a MEMS 204 solenoid coil defining a volume of about 0.02 μL. Figure 2D shows a schematic of a MEMS Helmholz 206 coil configuration, and Figure 2E shows a schematic of a 220 saddle coil configuration.
[0228] [000228] A 200 wrapped solenoid microbobin used for traditional NMR detection is described in Seeber et al., "Design and testing of high sensitivity micro-receiver coil apparatus for nuclear magnetic resonance and imaging," Ohio State University, Columbus, Ohio. A planar microbobin 202 used for traditional NMR detection is described in Massin et al., "High Q factor planar RF microcoil for micro-scale NMR spectroscopy," Sensors and Actuators A 97-98, 280-288 (2002). A Helmholtz 206 coil configuration features a cavity 208 for holding the sample, a top layer Si 210, a round base Si layer 212, and deposited metal coils 214. An Example of a Helmholtz 206 coil configuration used for NMR detection traditional is described in Syms and another, "MEMS Helmholz Coils for Magnetic Resonance Spectroscopy," Journal of Micromechanics and Micromachining 15 (2005) S1-S9.
[0229] [000229] An NMR unit includes a magnet (ie, a superconducting magnet, an electromagnet, or a permanent magnet). The magnet design can be open or partially closed, ranging from U- or C-shaped magnets, to magnets with three and four columns, to fully closed magnets with small openings for sample location. The disadvantage is the accessibility to the "sweet spot" of the magnet and mechanical stability (mechanical stability may be an issue where high field homogeneity is desired). For example, the NMR unit may include one or more permanent magnets, cylindrically shaped and made of SmCo, NdFeB, or other low-field permanent magnets that provide a magnetic field in the range of about 0.5 to about 1, 5 T (ie, suitable SmCo and NdFeB permanent magnets are available from Neomax, Osaka, Japan). For illustration and not limitation purposes, such permanent magnets may be a permanent box / dipole (PM) magnet assembly, or a hallbach design (see Demas et al., Concepts Magn Reson Part A 34A: 48 (2009)). NMR units may include, without limitation, a permanent magnet of about 0.5T of strength with a field homogeneity of about 20-30 ppm and a 40 μL sweet spot, centered. This field homogeneity allows a less expensive magnet to be used (fine tuning less acute of the assembly / chatter), in a system less inclined to fluctuations (eg temperature deviation, mechanical stability over a long period of time virtually any impact is too small to be observed ), tolerating motion ferromagnetic objects or conductors in the dispersion field (they have less of an impact, so less shielding is required), without compromising the test measurements (relaxation measurements and correlation measurements do not require a highly homogeneous field).
[0230] [000230] The coil configuration can be chosen or adapted for specific implementation of micro-NMR-MRS technology, provided that different coil configurations offer different performance characteristics. For example, each of these coil geometries has a different field alignment and performance. Planar coil 202 has an RF field perpendicular to the coil plane. The solenoid coil 200 has an RF field on the coil axis, and the Helmholtz 206 coil has an RF field across the two rectangular coils 214. The Helmholtz 206 and saddle spirals 220 have cross fields that would allow the permanent magnet bias field to be located above and below the cavity. Helmholtz 206 and saddle spirals 220 can be more effective for chip design, while solenoid coil 200 can be more effective when the magnetic particles of MRS and sample are kept in a micro tube
[0231] [000231] Micro-NMR devices can be manufactured by winding or printing the coils or by semiconductor manufacturing techniques of microelectromechanical system (MEMS). For example, a rolled or printed coil / sample cavity module can be about 100 μm in diameter, or as large as one centimeter or more. A MEMS unit or chip (hence named since it is manufactured in a semiconductor process like a matrix in a wafer) can have a coil that is about 10 μm to about 1000 pm in characteristic size, for example. The wound or printed coil / sample cavity configuration is referenced here as a module and the MEMS version is referenced here as a chip. For example, liquid sample 108 can be kept in a tube (for example, a capillary tube, pipette, or micro) with the coil wrapped around it, or it can be kept in cavities on the chip with the RF spiral surrounding the cavity. Alternatively, the sample is positioned for flow through a tube, capillary, or cavity in the vicinity of the RF spiral.
[0232] [000232] The basic components of an NMR unit include electrical components, such as an RF circuit tuned to a magnetic field, including an MR sensor, electronic receiver and transmitter that can be by including preamplifiers, amplifiers and circuitry. protection, data acquisition components, pulse programmer and pulse generator.
[0233] [000233] Systems containing NMR units with RF coils and micro cavities containing magnetic particle sensors described here can be designated for measurement of detection and / or concentration of specific (s) analyzed of interest by developing a model for phenomenon particle aggregation and development of an RF-NMR signal chain model. For example, experiments can be conducted for magnetic particle / analyte systems of interest by characterizing particle aggregation physicists, including, for example, the effects of affinities, relevant dimensions, and concentrations. Also, experiments can be conducted to characterize the NMR signal (s) (T2, T1, T2 *, T2rho, T 1rho and / or other signal characteristics, such as hybrid T1 / T2 signals and may also include however, they are not limited to diffusion, susceptibility, frequency) as characteristic particle aggregation or depletion and magnetic particle functions. Signal characteristics specific to the MRS (magnetic resonance shift) phenomenon in a given system can be used to enhance detection sensitivity and / or otherwise improve performance.
[0234] [000234] The NMR system may include a chip with RF coil (s) and micromachined electronics on it. For example, the chip can be micro-machined surface, such that the structures are built on top of a substrate. Where structures are built on top of the substrate and not inside it, the properties of the substrate are not as important as in volume micromachining, and expensive silicon wafers in volume micromachining can be replaced with less expensive materials such as glass or plastic. Alternative modalities, however, may include chips that are micromachined in volume. Surface micromachining usually starts with a wafer or other substrate and develops layers at the top. These layers are selectively recorded by photolithography and a wet recording involving an acid or a dry recording involving an ionized gas, or plasma. Dry recording can combine chemical recording with physical recording, or ion bombardment of the material. Surface micromachining can involve as many layers as necessary.
[0235] [000235] In some cases, a cheap RF coil can be integrated into a disposable cartridge and be a disposable component. The coil can be placed in a way that allows electrical contact with the circuit in the fixed NMR installation, or the coupling can be done by inducing a circuit.
[0236] [000236] Where the relaxation measurement is T2, the precision and repeatability (accuracy) will be a function of the sample's temperature stability as relevant to the calibration, the test stability, the signal-to-noise ratio (Y / N) , the pulse sequence for reorientation (for example, CPMG, BIRD, Tango, and the like), as well as signal processing factors such as signal conditioning (for example, amplification, rectification, and / or digitization of echo signals ), time / frequency domain transformation, and signal processing algorithms used. Signal-to-noise ratio is a function of the magnetic polarization field (Bb), sample volume, loading factor, coil geometry, coil Q-factor, electronic bandwidth, amplifier noise, and temperature.
[0237] [000237] In order to understand the required accuracy of T2 measurement, one should look at a hand-held test response curve and correlate the desired accuracy to determine the analyte concentration and the measurable accuracy, for example, T2 for some cases. Then an appropriate error budget can be formed.
[0238] [000238] For example, to obtain a 10-fold improvement in the detection limit of 0.02 ng / mL for Troponin (10-fold increase in sensitivity), it would be necessary to discern a delta-T2 less than about 5.6 milliseconds of a traditional T2 (not measured by MRS) of about 100 milliseconds. The minimum signal-to-noise ratio (Y / N) would need to be around 20 to detect this difference.
[0239] [000239] NMR units for use in the systems and methods of the invention can be those described in United States Patent No. 7,564,245, incorporated herein by reference.
[0240] [000240] The NMR units of the invention may include a small probehead for use in a portable magnetic resonance relaxometer as described in PCT Application No. WO09 / 061481, incorporated herein by reference.
[0241] [000241] The systems of the invention can be implantable or partially implantable in an individual. For example, the NMR units of the invention can include implantable radio frequency coils and optionally implantable magnets as described in PCT Application Nos. WO09 / 085214 and WO08 / 057578, each of which is incorporated herein by reference.
[0242] [000242] The systems of the invention may include a polymeric sample container to reduce, partially or completely, the contribution of the NMR signal associated with a sample container to the nuclear magnetic resonance parameter of the liquid sample as described in PCT Order No. WO09 / 045354, incorporated herein by reference.
[0243] [000243] The systems of the invention may include a disposable sample holder for use with the MR reader that is configured to allow a predetermined number of measurements (i.e., it is designed for a limited number of uses). The disposable sample holder can include none, part, or all, of the elements of the RF detection coil (i.e., such that the MR reader needs a detection coil). For example, the disposable sample holder may include a "reader" coil for RF detection which is by induction coupled to a "capture" coil present in the MR reader. When the sample container is inside the MR reader it is in close proximity to the pickup coil and can be used to measure the NMR signal. Alternatively, the disposable sample holder includes an RF coil for RF detection that is electrically connected to the MR reader after inserting the sample container. Thus, when the sample container is inserted into the MR reader, the appropriate electrical connection is stabilized to allow for detection. The number of uses available for each disposable sample holder can be controlled by disabling a fusible connection included in the electrical circuit with the disposable sample holder, or between the disposable sample holder and the MR reader. After the disposable sample holder is used to detect NMR relaxation in the sample, the instrument can be configured to apply excess current to the melt connection, causing the connection to break and rendering the coil inoperable. Optionally, multiple fused connections can be used, working in parallel, each connecting to a pickup in the system, and each broken individually for each use until all are broken and the disposable sample holder rendered inoperable. Cartridge units
[0244] [000244] Systems for carrying out the methods of the invention may include one or more cartridge units to provide a convenient method for placing all test reagents and consumables in the system. For example, the system can be customized to perform a specific function, or adapted to perform more than one function, for example, by means of changeable cartridge units containing microcavity arrangements with customized magnetic particles contained therein. The system can include a replaceable and / or interchangeable cartridge containing an array of cavities preloaded with magnetic particles, and designed for measuring the detection and / or concentration of a particular analyzed. Alternatively, the system can be used with different cartridges, each designated for detection and / or concentration measurements of different analyzed, or configured with separate cartridge modules for reagent and detection for a given assay. The cartridge can be sized to facilitate insertion and ejection of a housing for the preparation of the liquid sample which is transferred to other units in the system (ie, a magnetic assisted agglomeration unit, or an NMR unit). The cartridge unit itself can potentially interface directly with handling stations as well as the MR reader (s). The cartridge unit can be a modular cartridge having an input module that can be sterilized independent of the reagent module.
[0245] [000245] To manipulate biological samples, such as blood samples, there are numerous competing requirements for cartridge design, including what is necessary to sterilize for the input module to prevent cross-contamination and false positive test results, and what is necessary to include reagents in the package that cannot be easily sterilized using irradiation similar to standard terminal sterilization techniques. A sample aliquot entry module can be designed to interface with non-level vacutainer tubes, and for two aliquots a sample volume that can be used to perform, for example, a candida assay (see Figures 7D-7F). The vacutainer allows partial or full loading. The inlet module has two hard plastic parts, which become ultrasonically fused together and the spiral sealed to form a network of channels to allow a flow path to form within the first overflow well into the second sample well. A soft vacutainer seal part is used to form a seal with the vacutainer, and includes a sample flow port and a ventilation port. To overcome the flow resistance once the vacutainer is loaded and inverted, some hydrostatic pressure is required. Each time the sample is removed from the sample well, the well will be replenished by flow from the vacutainer.
[0246] [000246] A modular cartridge can provide a simple means of controlling cross-contamination during certain tests, including, but not limited to, the distribution of PCR products in multiple detection aliquots. In addition, a modular cartridge can be compatible with automated fluid dispensing, and provides a means to keep reagents in very small volumes for long periods of time (in excess of one year). Finally, pre-dispensing of these reagents allows concentration and volumetric precision to be established by the manufacturing process and provides an instrument point of care use that is more convenient because it may require much less accurate pipetting.
[0247] [000247] The modular cartridge of the invention is a cartridge that is separated into modules that can be packaged and if necessary sterilized separately. They can also be handled and stored separately, if, for example, the reagent module requires refrigeration but the detection module will not. Figure 5 shows a representative cartridge with an input module, a reagent module and a detection module that are attached to each other. In this mode, the input module would be packaged separately in a sterile package and the reagent and detection modules would be pre-assembled and packaged together.
[0248] [000248] During storage, the reagent module can be stored in a refrigerator at the same time as the input module can be stored in dry storage. This provides the added advantage that only a very small amount of cooler or cooler space is required to store many tests. At the time of use, the operator would retrieve a detection module and open the package, potentially using a sterile technique to prevent contamination with skin flora if required by the test. The Vacutainer tube is then uncapped and the inverted inlet module is placed in the tube as shown in Figure 6A. This module has been designed to be easily moldable using a single extraction tool as shown in Figures 6B and 6C and the top and bottom of the cartridge are sealed with a thin metal blade to prevent contamination and also to close the channels. Once the tube has been resealed using the inlet module, the inlet is connected on the right side and fitted over the rest of the cartridge. The inlet section includes a cavity with an overflow that allows sample tubes with between 2 and 6ml of blood to be used and still provides a constant depth interface for system automation. This is done by means of extravasation, where the blood that overflows the sampling cavity is simply included in the cartridge body, preventing contamination.
[0249] [000249] Alternatively, the modular cartridge is designed for a multiplexed assay. The challenge in multiplexed assays is to combine multiple assays that have incompatible assay requirements (ie, different incubation times and / or temperatures) in one cartridge. The cartridge can feature two main components: (i) a reagent module that contains all the individual reagents required for the complete assay panel, and (ii) the detection module. The detection modules contain only the parts of the cartridge that carry out the incubation, and can carry single tests or several tests, as needed. The detection module can include two detection chambers for a single test, the first detection chamber as the control and the second detection chamber for the sample. This cartridge format can be expandable in those additional tests that can be added including reagents and an additional detection module.
[0250] [000250] The operation of the module begins when the user inserts all or a portion of the cartridge into the instrument. The instruments trigger the test, aliquoting the tests in separate detection chambers. These individual detection chambers are then disconnected from the reagent strip and from each other, and progress through the system separately. Because the reagent module is separated and discarded, the smallest possible sample unit travels through the instrument, conserving internal instrument space. By dividing each assay into its own unit, different incubation times and temperatures are possible as each multiplexed assay is physically removed from the others and each sample is individually handled.
[0251] [000251] The cartridge units of the invention may include one or more populations of magnetic particles, such as a liquid suspension or dry magnetic particles that are reconstituted before use. For example, the cartridge units of the invention may include a compartment including from 1 × 106 to 1 × 1013 magnetic particles (for example, from 1 × 106 to 1 × 108, 1 × 107 to 1 × 109, 1 × 108 to 1 × 1010, 1 × 109 to 1 × 1011, 1 × 1010 to 1 × 1012, 1 × 1011 to 1 × 1013, or 1 × 107 to 5 × 108 magnetic particles) to test a single liquid sample. Systems
[0252] [000252] Systems for carrying out the methods of the invention may include one or more NMR units, cartridge units, and stirring units (for example, to break up non-specific magnetic particle interactions and redistribute the magnetic particles back into the liquid sample, or to simply shake the sample tube to completely mix the test reagents, such as sonication, vortexing, shaking, or ultrasound station to mix one or more liquid samples). Such systems may also include other components for performing an automated assay of the invention, such as a PCR unit for detecting oligonucleotides; a centrifuge, a robotic arm to release a liquid sample from unit to unit within the system; one or more incubation units; a fluid transfer unit (i.e., a pipetting device) for combining test reagents and a biological sample to form the liquid sample; a computer with a programmable processor for storage data, processing data, and for controlling the activation and deactivation of the various units according to one or more protocols present; and a cartridge insertion system for releasing pre-loaded cartridges into the system, optionally with instructions for the computer identifying the reagents and protocol to be used in conjunction with the cartridge.
[0253] [000253] The systems of the invention can provide an effective means for high productivity and real-time detection of analytes present in an individual's body fluid. Detection methods can be used in a wide variety of circumstances including, without limitation, identification and / or quantification of analytes that are associated with specific biological processes, physiological conditions, disturbances or disturbance stages. As such, the systems have a wide spectrum of utility in, for example, drug screening, disease diagnostics, phylogenetic classification, parental and forensic identification, disease return and onset, individual response to treatment versus population bases, and monitoring of therapy. Object devices and systems are also particularly useful for advancing clinical and preclinical therapeutics development stages, improving patient compliance, monitoring ADRs associated with a prescribed drug, developing individualized medicine, outsourcing blood testing from the central laboratory to the home or on a prescription basis, and monitoring therapeutic agents following regulatory approval. The devices and systems can provide a flexible system for personalized medicine. The system of the invention can be changed or altered together with a protocol or instructions for a programmable processor of the system to perform a wide variety of tests as described here. The systems of the invention offer many advantages of a laboratory environment contained in a desk-top or automated instrument of smaller size.
[0254] [000254] The systems of the invention can be used for simultaneously analyzed assays that are present in the same liquid sample over a wide concentration range, and can be used to monitor the rate of change of an analyzed concentration and / or concentration of PD markers or PK over a period of time in a single individual, or used to perform trend analysis on concentration, or PD markers, or PK, if they are concentrations of drugs or their metabolites. For example, if glucose is the subject of interest, the concentration of glucose in the sample at a given time as well as the rate of change in glucose concentration over a given period of time can be highly useful in preventing and preventing, for example, events hypoglycemic. In this way, the data generated with the use of an individual's fluidic systems and devices can be used to perform a trend analysis on the concentration of one analyzed in an individual. For example, a patient may be provided with a plurality of cartridge units to be used to detect a variety of assays at predetermined times. An individual can, for example, use different cartridge units on different days of the week. In some embodiments, the system software is designed to recognize a cartridge identifier instructing the computer system to execute a particular protocol to perform the test and / or process the data. The system protocols can be updated via an external interface, such as a USB drive or an internet connection, or in some modalities the complete protocol can be recorded on the barcode connected to the cartridge. The protocol can be optimized as necessary by alerting the user to various inputs (ie, to change the sample dilution, the amount of reagent supplied to the liquid sample, by changing an incubation time or magnetic assisted agglomeration time, or by changing the parameters of NMR relaxation collection). Where the multiplexed array is configured to detect a target nucleic acid, the assay can include a multiplexed PCR to generate different amplicons and then serially detect the different reactions. The multiplexed test optionally includes a logical arrangement in which targets are established by binary search to reduce the number of tests required (for example, positive or negative gram leads to tests based on different species that would only be conducted for one group or the other ).
[0255] [000255] The systems of the invention can perform a variety of tests, regardless of the analyzed being detected from a sample of body fluid. A protocol dependent on the identity of a cartridge unit being used can be stored on the computer system. In some embodiments, the cartridge unit has an identifier (ID) that is detected or read by the computer system, or a barcode (1D or 2D) on a card that then supplies specific assays or specific patient or individual information needed to be tracked or accessed with analysis information (for example, calibration curves, protocols, levels or concentrations of previous analyte). Where desired, a cartridge unit identifier is used to select a protocol stored in the computer system, or to identify the location of several assay reagents on a cartridge unit. The protocol to be performed on the system may include instructions to the system controller to perform the protocol, including, but not limited to, the particular test to be performed and a detection method to be performed. Once the test is performed by the system, indicative of data from an analyte in the biological sample is generated and communicated to a communications assembly, where it can be transmitted to the external device for processing, including without limitation, calculation of the analyzed concentration in the sample, or processed by the computer system and the result shown on a reading display.
[0256] [000256] For example, the identifier can be a barcode identifier with a series of white and black lines, which can be read by a barcode reader (or other type of detector) after inserting a cartridge unit . Other identifiers can be used, such as a series of alphanumeric values, colors, protrusions, RFID, or any other identifier that can be located on a cartridge unit and be detected or read by the computer system. The detector can also be an LED that emits light that can interact with an identifier that reflects light and is measured by the computer system to determine the identity of a particular cartridge unit. In some embodiments, the system includes a memory or storage device with a cartridge unit or detector to transmit the information to the computer system.
[0257] [000257] Thus, the systems of the invention can include an operational program to perform different tests, and encoded cartridges to: (i) report to the operational program whose pre-programmed test was being used; (ii) report the configuration of the cartridges to the operational program; (iii) inform the operating system the order of steps to perform the test; (iv) inform the system which pre-programmed routine to employ; (v) alert the user's input with respect to certain test variables; (vi) registering a patient identification number (the patient identification number can also be included in the Vacutainer that holds the blood sample); (vii) record certain cartridge information (ie, lot #, calibration data, cartridge tests, analytical data range, expiration date, storage requirements, acceptable sample specifics); or (viii) reporting test revisions or updates to the operational program (that is, so that newer versions of the assay would occur on cartridge updates only and not on the larger, more expensive system).
[0258] [000258] The systems of the invention may include one or more fluid transfer units configured to adhere to a robotic arm. The fluid transfer unit can be a pipette, such as an air displacement, reflux liquid, or syringe pipette. For example, the fluid transfer unit may also include a motor in communication with a programmable processor of the computer system and the motor may move a plurality of heads based on a programmable processor protocol. In this way, a system's programmable processor can include instructions or commands and can operate the fluid transfer unit according to the instructions for transferring liquid samples by withdrawing (to extract liquid in) or extending (to expel liquid) a piston in a closed air space. Both the volume of air moved and the speed of movement can be precisely controlled, for example, by the programmable processor. Mixing samples (or reagents) with diluents (or other reagents) can be activated by aspirating the components to be mixed in a standard tube and then repeatedly aspirating a significant fraction of the combined liquid volume up and down at one end. Dissolving dry reagents in a tube can be done in a similar way.
[0259] [000259] A system may include one or more incubation units for heating the liquid sample and / or for controlling the test temperature. Heating can be used in the incubation step of a test reaction to promote the reaction and shorten the duration required for the incubation step. A system may include a heating block configured to receive the liquid sample for a predetermined time at a predetermined temperature. The heating block can be configured to receive a plurality of samples.
[0260] [000260] The temperature of the system can be carefully regulated. For example, the system includes an enclosure maintained at a predetermined temperature (i.e., 37 ° C) using agitated temperature controlled air. Heat waste from each of the units will exceed what can be passively dissipated by simple inclusion by conduction and air convection. To eliminate waste heat, the system can include two compartments separated by an insulated floor. The upper compartment includes those portions of the components necessary for handling and measuring liquid samples, while the lower compartment includes the heat generating elements of the individual units (for example, the motor for the centrifuge, the motors for the stirring units, electronics for each of the separate units, and heating blockers for the incubation units). The lower floor is then ventilated and forced air cooling is used to take heat out of the system.
[0261] [000261] The MR unit may require more closely controlled temperature (for example, ± 0.1 ° C), and then may optionally include a separate enclosure into which air heated to a predetermined temperature is blown. The housing may include an opening through which the liquid sample is inserted and removed, and through which heated air is allowed to escape.
[0262] [000262] The following examples are mentioned in order to provide those skilled in the art with a full disclosure and description of how the devices, systems and methods described here are performed, made, and evaluated, and are intended to be examples of the invention and they are not intended to limit the scope of what inventors consider to be their invention. Example 1. Preparation of coated particles.
[0263] [000263] Soon, 1 mg of substantially monodispersed carboxylated magnetic particles was washed and resuspended in 100 μl of activation buffer, 10 mM MES. 30 μl of 10 mg / ml of 10kDa amino-dextran (Invitrogen) were added to the activation buffer and incubated on a rotator for 5 minutes at room temperature. For coupling the carboxyl groups to amines in dextran, 30 μl of 10mg / ml of 1-ethyl-3- [3-dimethylaminopropyl] carbodiimide hydrochloride (EDC) were added and incubated on a rotator for 2 hours at room temperature. The particles were removed by washing 3 × free dextran in 1 ml of PBS using magnetic separation, then resuspended in 1 ml of PBS. 100 μl of a 100 mM solution of Sulfo-NHS-biotin (Invitrogen) was used to decorate the amino groups on the dextran surface with biotin. After 30 minutes of incubation, the particles were washed 3 × in 1 ml of activation buffer. Then, a 100 μl protein block of 0.5 mg / ml bovine serum albumin (BSA) (Sigma) and 30 μl 10 mg / ml EDC were introduced and incubated overnight (Sigma). The prepared particles were washed 3 × in 1 ml of PBS and resuspended to the desired concentration.
[0264] [000264] The prepared particles synthesized with this protocol have been shown to provide similar results in T2 assays for detecting analyte, whether the samples include buffer or 20% lysed blood (see Figure 14). Variations of preparations where pre-biotinylated amino dextran was conjugated directly to particles in one step also resulted in similar performance in T2 assays in both buffer and blood samples. Example 2. Evaluation of particles prepared with and without a protein block.
[0265] [000265] Briefly, magnetic biotin-decorated amino-dextran particles prepared according to a method described in Example 1 were assayed in PBS and in 20% of blood lysed samples in a T2 antibiotin titration assay.
[0266] [000266] The test was carried out with the following procedure. 50 μL of matrix, PBS or 20% lysed blood sample, 50 μL of varying concentrations of the antibiotin antibody, and 50 μL of 1.0 μg / ml of secondary antibody were added to a 5 mm NMR tube. 150 μL of 0.02 mM Fe particles were then added to each tube (i.e., 2.7 × 108 particles per tube). The samples were then vortexed for 4 seconds and incubated in a heating block at 37 ° C for 2 minutes. Each sample was then vortexed again for 4 seconds, and incubated for another minute in the heating block at 37 ° C. Following the incubation, each sample was placed in a Bruker Minispec for 10 minutes, under a magnetic field. After 10 minutes, the sample was removed from the magnet, vortexed for 4 seconds, and incubated in a heating block at 37 ° C for 5 minutes. After 5 minutes, each sample was again vortexed and incubated in a heating block at 37 ° C for an additional 1 minute. T2 values were obtained using the Bruker Minispec program with the following parameters: Scans: 1; Gain: 75; Tau: 0.25; Echo Train: 3500; Total Echo Train: 4500; and Dummy Echos: 2. The values of Δ T2 were calculated: T2 - (T2) 0, and results are represented in Figure 14.
[0267] [000267] The particles synthesized with a protein block, AXN4, provided almost equal performance in blood and buffer (Figure 14). The graph represented in Figure 15 compares particles prepared with (open circle) and without (filled circle) a protein step block. Thus it was discovered that blocking protein may be necessary to activate similar functionality in blood matrices.
[0268] [000268] Additional protein blocking including, but not limited to, fish skin gelatin was also successful. The particles were prepared according to a method described above, with the exception that instead of using BSA as the protein block, fish skin gelatin (FSG) was replaced. The graph depicted in Figure 16 shows results of a T2 assay (as described above) using antibody titration for particles blocked with BSA and compared to FSG. The data indicate that there is little or no difference between the two protein blocking methods (see Figure 16). However, BSA proved to be a more reliable block. Example 3. Determination of the amount of dextran coating.
[0269] [000269] Attempts to increase the dextran coating density on the particles have been discovered to reduce the functionality of particles prepared in the blood. The particle preparation described in Example 1 above that demonstrated nearly equivalent buffer / blood performance used an excess of 10 × dextran base after a space filling model determined the amount of dextran to include in coating experiments. In an attempt to functionalize the particles with greater fidelity, increasing the dextran coating to an excess of 100010000 × dextran in particles generated by coating experiments having a thick dextran coating that produced a reduced blood response when compared to the buffer. We concluded that a moderate dextran density with a protein block may be desirable to produce a particle coating that works well in T2 assays in the presence of a blood sample (see Figures 17A and 17B). Example 4. Detection of a small molecule assay in whole blood samples.
[0270] [000270] Buffer / Analyze Preparation: 0.1% BSA, 0.1% Tween® in 1 × PBS: A 10% 20% Tween® solution by weight was prepared. Briefly, Tween® in 1X PBS was prepared. 500 ml of 0.2% Tween® solution was prepared by adding 10 ml of 10% Tween® to 490 ml of 1X PBS. A 2% BSA solution was prepared in a solution of 1 * PBS by weight. A 0.2% solution of BSA solution was prepared by adding 50 ml of 2% BSA in PBS to 450 ml of 1 × PBS. The dilutions were combined to prepare a final volume of 1 L and a final buffer concentration of 0.1% BSA, 0.1% Tween® in 1 × PBS.
[0271] [000271] PEG-FITC-Biotin assay: 100 μl of a 0.5 mM solution was prepared from 1 mM Tris HCl. 40 μl of PEG FITC biotin was mixed with 40 μl of 0.5 mM Tris HCl, and incubated for 15 minutes at room temperature. After 15 minutes, 70 μl of PEG-FITC-Biotin in 0.5 mM Tris HCl was added to 630 μl of 0.1% Tween® to prepare a 100 μM raw material solution. The raw material solution was mixed vigorously by vortexing. 200 μl of 100 pM solution was added to 900 μl of 0.1% Tween® to prepare analyzed at 20,000 nM. 10-fold dilutions were prepared at 0.02 nM Procedure:
[0272] [000272] 25 μΙ of appropriate analyte and 50 μΙ of lysed blood matrix 1: 5 were directly pipetted into a 5 mm NMR tube. The samples were vortexed for 4 seconds. 25 μΙ of primary antibiotin antibody (0.18 μg / ml diluted in 0.1% Tween 20, 0.1% BSA, 1 * PBS) was added, followed by an incubation at 37 ° C for 15 minutes. After 15 minutes, 50 μΙ of 3.0 μg / ml of secondary anti-mouse antibody (diluted in 0.1% Tween, 0.1% BSA, 1 * PBS) and 150 μl of 0.02 Fe particles mM (2.7 × 108 particles per tube) were added to the NMR Tube. The sample was then vortexed for 4 seconds and incubated for 5 minutes at 37 ° C. The sample was placed in a Bruker Minispec for 10 minutes, under a magnetic field. After 10 minutes, the sample was removed from the magnet and incubated for an additional 5 minutes. The sample was vortexed again for 4 seconds and incubated for an additional 1 minute. T2 values were taken using the Bruker Minispec program with the following parameters: Scans: 1; Gain: 75; Tau: 0.25; Echo Train: 3500; Total Echo Train: 4500; and Dummy Echos: 2. Example 5: Synthesis of antibody decorated particles.
[0273] [000273] Amine dextran-coated magnetic particles prepared as described in Example 1, can also be functionalized with antibodies by means of an SMCC-SATA bond (SMCC = succinimidyl 4- [N-maleimidomethyl] cyclohexane-1-carboxylate; SATA = N-succinimidyl-S-acetylthioacetate). The carboxylated magnetic particles are first conjugated to 10kDa of amino dextran by means of EDC chemistry as described above. The dextran-coated particles are also modified with an excess of sulfo-SMCC to provide a maleimide functional group. The antibodies are modified with a SATA linker, which first binds to the amines in the antibody. The binding of SATA is controlled to minimize the superfunctionalization of the antibody, which can lead to particle cross-linking or reduced antibody affinity. After deacetylation, the SATA linker exposes a thiol functional group, which can be used to bind directly to functionalized malemide particles that form a thioether bond. The number of antibodies conjugated to each particle can be measured using a BCA assay protein (Pierce). The connectors that provide SATA-like functionality have been used successfully, such as SPDP (N-Succinimidyl 3- [2-pyridyldithium] -propionate).
[0274] [000274] The antibody coated magnetic particles can be similarly prepared using the chemicals described above, but with direct covalent bonding to the base carboxylated particle. In some examples, it may be necessary to add additional coating to the particle surface, such as dextran, or a blocking agent. Similar chemicals can be used with alternate coatings for amino dextran, such as PEG or BSA. Example 6. Creatinine assay.
[0275] [000275] Briefly, the test includes the following: a target sample is incubated in the presence of a magnetic particle that has been decorated with creatinine, which is attached to the surface of the magnetic particles. The magnetic particles decorated with creatinine are designed to aggregate in the presence of the creatinine antibody. Each of the magnetic particles decorated with creatinine and creatinine antibody are added to the liquid sample containing creatinine, which competes with the magnetic particles for the creatinine antibody. Thus, the binding of creatinine to the antibody blocks the agglomeration of magnetic particles, and low levels of creatinine are marked by the formation of clusters. These clusters alter the longitudinal relaxation rates of the sample when exposed to a magnetic field, and the change in relaxation times T2 (measuring a change in the magnetic resonance signal of the surrounding water molecules) may be directly correlated to the presence and / or concentration of the analyzed in the target sample. Creatinine antibody
[0276] [000276] Establishing an antibody generation program for creatinine, a modified creatinine molecule was planned (COOH-creatinine) and conjugated to transferrin for immunization in BALB-C and AJ mice.
[0277] [000277] Thirty four stable antibody-producing clones were generated. These clones emerged from BALB-C (spleen cells) (n = 17) or AJ (n = 17) mice. The two genetically different mouse strains were selected for the known genetic differences in their immune systems. Criteria and a selection process have been developed to assess and identify an ideal monoclonal antibody for use in the assay. The antibody selection process included evaluation for BSA-creatinine binding by ELISA, antibody affinity / sensitivity / specificity by competitive ELISA assays using free creatinine and potential interferents, determining the ability of the antibody to be conjugated to the magnetic particle and functionality in a T2 magnetic relaxation change assay.
[0278] [000278] Using the established antibody selection criteria outlined above, seven ideal monoclonal antibodies were identified and selected as potential candidates in the assay. Magnetic particles coated with creatinine
[0279] [000279] Substantially monodisperse carboxylated magnetic particles were washed and resuspended in 100 μl of coupling buffer (50 mM MES, pH = 4.75). Sulfo-NHS (55 μmol in 200 μl of MES buffer) was added, and the mixture vortexed. To the mixture was added EDC (33.5 μmol in 200 μl of MES buffer). The solution was briefly vortexed and placed in an end over end mixer for 1 hour at room temperature, allowed to settle, and the supernatant removed. To the resulting solids, 1 ml of 1% BSA was added in PBS, and again the mixture was vortexed and placed in an end over end mixer for 15-18 hours at room temperature. The particles were allowed to settle, and the supernatant removed.
[0280] [000280] The BSA-coated particles were suspended in 0.5 ml of 0.01% PBS-T20 (10 mM phosphate buffer, pH = 7.4, 150 mM NaCl, with Tween® 20 to 0, 01%). Unreacted carboxyl groups were subjected to Methyl-PEG4-amine (20 μl of 10% v / v in DMSO) as a blocking agent. The mixture was vortexed and placed in an end over end mixer for 8 hours at room temperature. The resulting BSA-coated particles were washed repeatedly with 0.5 ml of 0.01% PBS-T20.
[0281] [000281] COOH-creatinine (66 μmol), EDC (140 μmol) and NHS (260 μmol) were combined with 300 μl of dry DMSO to form a suspension, which cleared when the reaction reached completion. BSA-coated particles were suspended in 0.5 mL of 0.01% PBS-T20 (pH = 8), followed by the addition of the activated COOH-creatinine solution. The resulting mixture was vortexed and placed in an end over end mixer for 4 hours at room temperature. The resulting particles were washed 3 ×, each with sonication using 1:15 and 1:30 DMSO: 0.01% (vol / vol) PBS-T20. The particles were washed 3 ×, then each with sonication using 0.01% PBS-T20. The particles were resuspended in 0.1% PBS-T20 (pH = 8), and 2 mg of NHS-PEG 2K in 200 μΙ of 0.01% PBS-T20 was added. The mixture was placed in an end over end mixer for 12-20 hours at room temperature. The particles were washed 3 ×, then each with sonication using 0.01% PBS-T20 to produce creatinine conjugated magnetic particles with sequential BSA, creatinine coating, block and PEG buffer.
[0282] [000282] The particles coated with creatinine were resuspended in assay buffer (100 mM glycine (pH = 9.0), 150 mM NaCl, 1% BSA, 0.05% ProClin® and Tween® 0, 05%).
[0283] [000283] The creatinine assay protocol was performed using particles conjugated by creatinine, and the soluble creatinine antibody with detection using the T2 signal was generated / completed. The competitive creatinine assay architecture is described in Figure 7A.
[0284] [000284] Solutions of magnetic particles, antibody and liquid sample were, where indicated, diluted with an assay buffer that included 100 mM Tris pH 7.0, 800 mM NaCl, 1% BSA, Tween® a 0.1% and 0.05% ProClin®.
[0285] [000285] The magnetic particles coated with creatinine were diluted to 0.4 mM Fe (5.48 × 109 particles / ml) in assay buffer, vortexed completely and allowed to equilibrate for 24 hours at 4-8 ° C.
[0286] [000286] The mouse anti-creatinine monoclonal antibody (described above) has been used as a multivalent binding agent for creatinine-conjugated magnetic particles. The antibody was diluted to a concentration of 0.8 μg / ml in assay buffer and vortexed completely.
[0287] [000287] Samples and calibrators were diluted 1 part of sample to 3 parts of assay buffer. The upper test range is approximately 4 mg / dL of creatinine. For samples with expected creatinine levels> 4 mg / dL, an additional sample dilution was performed using 1 part of the initial diluted sample to 4 parts of assay buffer.
[0288] [000288] The pre-diluted sample, assay buffer, magnetic particle and antibody solutions were each vortexed. 10 μL of each solution was added to a tube, and the tube was vortexed for 5 seconds.
[0289] [000289] The tube was then subjected to 12 minutes of magnetic assisted agglomeration in a gradient field, incubated for 5 minutes at 37 ° C, placed in the MR reader (T2 MR, Reader with 2200 Fluke Temperature Controller, with software NDxlient 0.9.14.1/hardware Version 0.4.13 Build 2, Firmware Version 0.4.13 Build 0) to measure the relaxation rate of T2 in the sample, and the relaxation rate of T2 in the sample was compared to a standard curve (see Figure 8A) to determine the creatinine concentration in the liquid sample. Performance of modified creatinine antibodies
[0290] [000290] Different creatinine antibodies were tested in the assay to ascertain the effect of the antibody on the agglomeration. We found that the performance of creatinine antibodies varied in their performance characteristics, when combined with magnetic particles coated with creatinine (see Figure 8B). The SDS-PAGE gel analysis of the two preparations revealed significantly enhanced aggregation in preparation 1, believed to arise from an increase in the creatinine binding valence for this antibody, which is added due to its purification process. For comparison, we multimerized another creatinine monoclonal antibody (14HO3) by biotinylating the antibody and multimerizing the antibody in the presence of streptavidin. The monomeric, biotinylated and multimerized monomeric forms were then tested with magnetic particles coated with creatinine to evaluate the effect of increased valence on T2 time. The results are described in Figure 8C, showing groups of forms of multimerized antibody at much lower concentrations than non-multimerized antibodies. This valence enhancement for the particle cluster was also observed using IgM antibodies. Example 7. Magnetic particle coated with creatinine antibody.
[0291] [000291] Using an alternative assay architecture, the assay includes the following: a target sample is incubated in the presence of (i) a magnetic particle that has been decorated with creatinine antibody; and (ii) a multivalent linker including multiple creatinine conjugates. The magnetic particles are designed to aggregate in the presence of the multivalent binding agent, however aggregation is inhibited by competition with creatinine in the liquid sample. Thus, the binding of creatinine to the antibody-coated particle blocks the agglomeration of magnetic particles in the presence of the multivalent binding agent, and low levels of creatinine are marked by the formation of clusters. These clusters alter the rates of longitudinal relaxation of the sample when exposed to a magnetic field and the change in the relaxation times of T2 (measuring a change in the magnetic resonance signal of the surrounding water molecules) can be directly correlated to the presence and / or concentration analyzed in the target sample.
[0292] [000292] Substantially monodispersed carboxylated magnetic particles were washed and resuspended in 300 μl of coupling buffer (50 mM MES, pH = 4.75), and sulfo-NHS (46 μmol) EDC (25 μmol) were added to the particles. The solution was briefly vortexed and placed in an end over end mixer for 1 hour at room temperature. The activated particles were washed with mL of 0.01% PBS-T20, and resuspended in 1 mL of 10% w / v amine-PEG-amine solution in 0.01% PBS-T20. The mixture was vortexed and placed in an end over end mixer for 2 hours at room temperature, and then washed 3 × with 0.01% PBS-T20.
[0293] [000293] BSA can be substituted for amine-PEG-amine as an alternating chemical. The magnetic particles coated with BSA were prepared as described in example 6, in the section describing magnetic particles coated with creatinine.
[0294] [000294] The particles were resuspended in 260 μl of 0.01% PBS-T20 and reacted with 198 μl of SMCC sulfo (5 mg / ml in 0.01% PBS-T20). The solution was briefly vortexed and placed in an end over end mixer for 1 hour at room temperature, and then washed 3 × with 0.01% PBS-T20 with 10 mM EDTA to produce SMCC coated particles.
[0295] [000295] SATA labeled antibody was prepared by combining SATA (30 nmol in DMSO) with antibody (2 nmol in PBS, pH = 7.4). The solution was placed in an end over end mixer for 1 hour at room temperature. Sulfhydryl groups blocked in SATA-labeled antibody were deprotected by treatment with deacetylation buffer (0.5M hydroxylamine hydrochloride at pH 7.4, 10 mM phosphate, 150 mM sodium chloride, 10 mM EDTA) for 1 hour and purified by a desalination column using PBS containing 10 mM EDTA before use.
[0296] [000296] As an alternative to SATA, SPDP-labeled antibody can be used. SPDP-labeled antibody was prepared by adding SPDP (10mmol in DMSO) with antibody (2nmol in PBS, pH 7.4). The solution was incubated for 1 hour at room temperature and purified by a desalination column. The SPDP disulfide bond in the SPDP labeled antibody was cleaved in a reaction with 5mM mercaptoethiamine and incubated for 10 minutes at room temperature. The SPDP labeled antibody cleaved by disulfide bond was purified by a desalination column prior to use.
[0297] [000297] The SMCC functionalized particles with deacetylated SATA modified antibody and PEG or BSA coating were combined and placed in an end over end mixer overnight at room temperature, washed 3 × with PBS-Tween® at 0 ° C, 05% 80, and resuspended in 0.01% PBS-T20 with 10 mM EDTA. A blocking agent (m-PEG-SH 2K) was added, the solution was placed in an end over end mixer for 2 hours, washed 2 × with 0.05% PBS-Tween® 80, and resuspended in PBS-Tween ® 80a 0.05%, BSA 1%, and ProClin® 0.05% to produce magnetic particles coated by antibody.
[0298] [000298] The BSA-coated particles functionalized from SMCC and disulfide cleaved SPDP-labeled antibody were combined and placed in an end over end mixer for 2 hours at room temperature, washed 2 times with PBS-Tween® 20 to 0, 01%, 10 mM EDTA, and resuspended in PBS, 0.01% T20, and 10 mM EDTA. A blocking agent, m-PEG-SH 2K (1 μmol), was added, and the solution was placed in an end over end mixer for 2 hours. A second blocking agent, n-ethyl maleimide (5 μmol), was added. The particles were mixed for 15 minutes, washed twice with 0.01% PBS-Tween® 20, and resuspended in pH 9, 100 mM Tris, 0.05% Tween® 80, 1% BSA, and ProClin ® at 0.05% to produce the antibody coated magnetic particles.
[0299] [000299] The procedure mentioned above can be used with creatinine antibodies, or creatinine antibodies can be coupled directly to the surface of the carboxylated magnetic particles by EDC coupling. Multivalent creatinine binding agents
[0300] [000300] COOH-creatinine was conjugated into 3 amino-dextran compounds (Invitrogen; MW 10k, 40k, and 70k with 6.5, 12, and 24 amino groups per dextran molecule respectively) and BSA by EDC coupling. The resulting multivalent amino-dextran-creatinine and BSA-creatinine binding agents can be used in the competitive inhibition assay described above. Degrees of substitution between 2-30 creatinines per serving of dextran have been achieved. An example of a creatinine inhibition curve is shown in Figure 10. The binding agent used is a 40kDa dextran with ~ 10creatinins per dextran molecule. Example 8. Preparation of multivalent tacrolimus linkers.
[0301] [000301] Tacrolimus conjugates were prepared using dextran and BSA. FK-506 was subjected to the olefin metathesis reaction using the second generation Grubbs catalyst in the presence of 4-vinylbenzoic acid as described below in Scheme 1. The crude product mixture was purified by normal phase silica gel chromatography.
[0302] [000302] Dextran-tacrolimus conjugates were prepared using three amino-dextrans of different molecular weight, each with a different amino group substitution.
[0303] [000303] 2.78 ml of EDC solution (40 mg / ml of EDC hydrochloride) and 2.78 ml of sulfo-NHS solution (64 mg / ml of sulfo-NHS) were combined with stirring. To this mixture was added 0.96 ml of solution of tacrolimus acid derivative (C21) (28.8 mg / ml of in DMSO) and the contents stirred for 30 minutes at room temperature to form the activated tacrolimus acid derivative (4.6 mM activated Tac solution). The activated tacrolimus was used immediately.
[0304] [000304] Various amino-dextran polymers were dissolved in 100 mM sodium phosphate buffer (pH 8.0) to prepare a 9.5 mg / mL raw material solution.
[0305] [000305] The activated Tac solution was added dropwise with stirring at room temperature to the amino-dextran raw material solution in the ratios classified below. Each reaction was stirred vigorously for at least 2 hours.
[0306] [000306] The resulting Tac-dextran conjugate was purified using a 5-step serial dialysis of each reaction product (1 ° -15% (v / v) aqueous DMSO; 2 ° - 10% (v / v) methanol aqueous; 3 ° to 5 ° high-purity water; at least 2 hours for each step; using 3,500 MWCO dialysis membrane for the 10K MW amino-dextran and a 7K MWCO dialysis membrane for the amino-dextran of 40K and 70K).
[0307] [000307] Following the purification, each sample was lyophilized and the dry weight determined. Multivalent binding agents were reconstituted prior to use.
[0308] [000308] After reconstitution, tacrolimus replacement ratios were calculated based on absorbance at 254nm.
[0309] [000309] Experiments were performed to determine at what size the dextran provided the most ideal agglomerative performance. Briefly, 10 μL of 10% MeOH, 1% BSA in PBS buffer pH 6.3, 20 μL of Dextran Tac agglomerator, 10K, 40K, 70K, in varying concentrations, and 10 μL of magnetic particles coated with Anti- 0.2 mM Tacrolimus in Fe were added to a 200 μL PCR Tube (2.7 × 109 particles per tube). The sample was vortexed using a plate mixer at 2000 rpm for 2 minutes, preheated for 15 minutes at 37 ° C in an incubation station, exposed to a magnet at the base and side for 1 minute each, repeated for 6 cycles, vortexed again for 2 minutes at 2000 rpm, incubated for 5 minutes in an incubator at 37 ° C containing a heat block projected onto the PCR tube, and the T2 was read in the MR Reader. The data indicates that the varying substitution / mlecular weight ratios of dextran Tac can result in the improved T2 signal (see Figure 11). In addition, the higher substitution also resulted in an improved response (see Figure 12). Conjugates of BSA
[0310] [000310] BSA-tacrolimus conjugates were prepared with varying degrees of tacrolimus substitution.
[0311] [000311] 34.5 μL of NHS solution (66.664 mg / mL in acetonitrile) and 552 μL of EDC (6.481 mg / mL in 50 mM MES pH 4.7) were combined with stirring. 515.2 μL of this mixture of EDC NHS was added dropwise in 220.8 μL of solution of tacrolimus acid derivative (C21) (33.33 mg / mL in acetonitrile) and the contents stirred for 1 hour at room temperature to form the activated tacrolimus acid derivative. The activated tacrolimus was used immediately.
[0312] [000312] BSA was dissolved in phosphate-buffered saline and acetonitrile to form a solution containing 5mg / mL of BSA in 40% acetonitrile.
[0313] [000313] Activated Tac solution was added dropwise with stirring at room temperature to the BSA solution in the ratios classified below. Each reaction was stirred vigorously for at least 2 hours.
[0314] [000314] The resulting Tac-BSA conjugates were purified using a PD10 size exclusion column pre-equilibrated with 40% acetonitrile. The eluent was collected in fractions of 1 mL and monitored for absorbance at 280nm to identify fractions containing BSA.
[0315] [000315] The fractions containing BSA were combined and the acetonitrile removed under vacuum.
[0316] [000316] Tac-BSA conjugates were evaluated for clustering capacity by performing a titration similar to that used for dextran-tacrolimus conjugates. As noted, cluster performance differs with the Tac substitution ratio (see Figure 13). Example 9. Tacrolimus competitive test protocol (antibody on particle architecture).
[0317] [000317] A tacrolimus assay using conjugated anti-tacrolimus antibody particles and multivalent BSA-tacrolimus binding agent with detection using an MR Reader was developed (see Example 6). This assay was designed to test whole blood samples that were extracted to release tacrolimus from erythrocytes and binding proteins (extraction of hydrophobic analyte from a sample can be achieved, for example, using the methodology described in US Patent No. 5,135,875 ). The architecture of the tacrolimus competitive assay is described in Figure 7B.
[0318] [000318] Solutions of magnetic particles and multivalent binding agent were, where indicated, diluted with an assay buffer that included 100 mM Glycine pH 9, 0.05% Tween® 80, 1% BSA, NaCl a 150 mM, 0.1% CHAPS.
[0319] [000319] A basic particle with COOH functionality was modified by sequential amino coating (PEG or BSA), covalent antibody binding, PEG buffer and PEG / protein block (as described in the examples above). The antibody-coated magnetic particles were diluted in 0.4 mM Fe (5.48x109 particles / ml) in assay buffer, and vortexed completely.
[0320] [000320] The multivalent linker was COOH-modified tacrolimus form covalently conjugated to BSA (as described in Example 8). The multivalent binding agent was diluted to 0.02 μg / ml in assay buffer, and vortexed completely.
[0321] [000321] The sample solution (10 μL) and the magnetic particle solution (10 μL) were combined and vortexed for five seconds and incubated at 37 ° C for 15 minutes. To this mixture, 20 μL of the multivalent binding agent were added and the resulting mixture was vortexed for five seconds and incubated at 37 ° C for 5 minutes.
[0322] [000322] Several samples were prepared as described above. All samples were subjected to magnetic assisted agglomeration in a gradient field for 1 minute. All samples were then placed on a tray removed from the magnetic field. Each sample was vortexed for at least five seconds and returned to the tray. All samples were again subjected to magnetic assisted agglomeration for 1 minute, followed by vortexing. This process was repeated twelve times for each sample.
[0323] [000323] The sample was incubated for 5 minutes at 37 ° C, placed in the MR Reader (see Example 6) to measure the sample's T2 relaxation rate, and the sample's T2 relaxation rate was compared to a curve (see Figure 9) to determine the concentration of tracrolimus in the liquid sample. Example 10. Candida assay.
[0324] [000324] In the assay used for Candida, two pools of magnetic particles are used for the detection of each species of Candida. In the first pool, a species-specific oligonucleotide probe that is conjugated to the magnetic particles. In the second pool, an additional species-specific oligonucleotide probe is coupled to the magnetic particles. In hybridization, the two particles will hydride to two distinct species-specific sequences within the sense strand of the target nucleic acid, separated by approximately 10 to 100 nucleotides. (Alternatively, the two capture oligonucleotides can be conjugated to an isolated particle pool, resulting in individual particles that have specificity for the first and second regions as well). Magnetic particles decorated with oligonucleotides are designed to aggregate in the presence of nucleic acid molecules from a particular species of Candida. Thus, unlike the inhibition assays used for cretinin and tacrolimus, the Candida assay represents an increase in agglomeration in the presence of the target Candida nucleic acid molecules. The hybridization-mediated agglomerative assay architecture is described in Figure 7C.
[0325] [000325] Magnetic carboxylated particles are used in Candida assays. Magnetic particles were conjugated to oligonucleotide capture probes to create oligonucleotide-particle conjugates. For each target amplicon, two populations of oligonucleotide-particle conjugates were prepared. Particle oligonucleotide conjugates were prepared using the standard EDC chemical between aminated oligonucleotides and carboxylated particles, or, optionally, coupling biotin-TEG-modified oligonucleotides to streptavidin particles. Coupling reactions were typically carried out at a particle concentration of 1% solids.
[0326] [000326] Densities of functional, post-conjugation oligonucleotides were measured by hybridizing the Cy5 labeled complements to the particles, washing the particles three times to remove the unhydridized oligo; and eluting by heating at 95 ° C for 5 minutes. The amount of oligonucleotide labeled by Cy5 was quantified by fluorescence spectroscopy.
[0327] [000327] The coupling reactions were carried out overnight at 37 ° C with continuous mixing using an oscillator or roller. The resulting particle conjugates were washed twice with 1x reaction volume of Millipore water; twice with 1x reaction volume of 0.1 M Imidazole (pH 6.0) at 37 ° C for 5 minutes; three times with a 1x reaction volume of 0.1 M sodium bicarbonate at 37 ° C for 5 minutes; then twice with a 1x reaction volume of 0.1 M sodium bicarbonate at 65 ° C for 30 minutes. The resulting particle conjugates were stored at 1% solids in TE (pH 8), 0.1% Tween®20).
[0328] [000328] The panel of Candida species detected includes C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis. The sequences are amplified using universal primers recognizing highly conserved sequence within the genus Candida. The capture oligonucleotides were designed to recognize and hybridize to species-specific regions within the amplicon.
[0329] (i) Uma amostra de sangue total foi misturada com um volume em excesso (1,25×, 1,5×, ou 2×) de solução de lise hipotônica de cloreto de amônio. A adição de solução de lise rompe todos RBCs, porém não rompe WBC, levedura, ou células bacterianas. A matéria celular foi centrifugada em 9000 rpm durante 5 minutos e o lisado foi removido. As células intactas foram reconstituídas com 100 μl de TE (tris EDTA, pH = 8) a um volume final de cerca de 100 pl; e (ii) Para aproximadamente 100 μl de amostra, 120 mg de 0,5 mm de contas foram adicionados. A amostra foi agitada durante 3 minutos em cerca de 3K rpm, desse modo formando um lisado. [000329] An aliquot of a blood sample was first subjected to lysis as follows: (i) A whole blood sample was mixed with an excess volume (1.25 ×, 1.5 ×, or 2 ×) of ammonium chloride hypotonic lysis solution. The addition of lysis solution disrupts all RBCs, but does not disrupt WBC, yeast, or bacterial cells. The cell matter was centrifuged at 9000 rpm for 5 minutes and the lysate was removed. The intact cells were reconstituted with 100 μl TE (tris EDTA, pH = 8) at a final volume of about 100 pl; and (ii) For approximately 100 μl of sample, 120 mg of 0.5 mm beads were added. The sample was stirred for 3 minutes at about 3K rpm, thereby forming a lysate.
[0330] [000330] An aliquot of approximately 50 μl of lysate was then subjected to PCR amplification by adding the lysate to a main PCR mixture including the nucleotides; buffer ((NH4) 5mM SO4, 3.5mM MgCl2, 6% glycerol, 60mM Tricine, pH = 8.7 at 25 ° C; primers (4 × excess sense initiator (300mM sense; antisense to 0.75mM) to allow the production of asymmetric single filament in the final product) and thermostable polymerase (HemoKlenTaq (New England Biolabs)). After an initial incubation at 95 ° C for 3 minutes, the mixture is subjected to PCR cycles : annealing at 62 ° C; elongation at 68 ° C; 95 ° C - for 40 cycles Note: there is a difference at 6 ° C in the temperatures of annealing and elongation; annealing and elongation can be combined in one step to reduce the response time of the total amplification.
[0331] [000331] The PCR amplicon, now ready for detection, is combined with two populations of particles in a sandwich assay.
[0332] [000332] The PCR primers and capture probes that can be used in the Candida assay are provided below in Table 6.
[0333] [000333] Optionally, the test is performed in the presence of a control sequence, together with magnetic particles decorated with probes to confirm the presence of the control sequence. Example 11. Non-agglomerative methods.
[0334] [000334] This process has been demonstrated using aminosilane-treated nickel metal foam with 400 μm pores decorated with anticretinin antibodies and shown to specifically bind magnetic particles derived from cretinine. A 1 cm square piece of nickel metal foam (Recemat RCM-Ni-4753.016) was washed by incubating at room temperature for 1 hr in 2M HCL, rinsed completely in deionized water, and dried at 100 ° C for 2 hours . The nickel foam was then treated with 2% 3-aminopropyltriethoxysilane in acetone at room temperature overnight. The nickel metal foam was then washed extensively with deionized water and dried for 2 hours at 100 ° C. The aminosilane-treated nickel metal foam was treated with 2% gluteraldehyde in water for 2 hours at room temperature and washed extensively with deionized water. Then, the metal foam was exposed to 100μg / ml of anticretinin antibody (14H03) (see Example 6) in PBS overnight, washed extensively with PBS, and treated with Surmodics Stabilguard to stabilize and block non-specific binding. Two-mm square pieces of the derived metal foam were cut using a new razor blade taking care not to damage the foam structure. A piece of the derived metal foam was placed in a PCR tube in 20 μl of assay buffer (100 mM glycine (pH = 9.0), 150 mM NaCl, 1% BSA, 0.05 ProClin® %, and 0.05% Tween®). Twenty microliters of control particles (which should not bind to ABX1-11 metal foam) in 0.2mMFe were added to the tube to bring the final volume to 40ul and the final particle concentration to 0.1mM Fe (1 × 106 - 1 × 108 particles / tube). A separate PCR tube with the exact particle and buffer, without the metal foam was similarly prepared. The PCR tube containing the derived metal foam and control particles was subjected to magnetic assisted agglomeration in a gradient field for one minute, and then placed in contact with a manual demagnetizer, and subjected to magnetic assisted agglomeration for another minute. , removed in contact with a manual demagnetizer and subjected to magnetic assisted agglomeration for another minute and vortexed (three magnetic exposures of 1 minute). Thirty μl of sample was removed from both PCR tubes, heated to 37 ° C in a concession block heater for 5 minutes and the T2 read using the MR Reader (see Example 6). The T2 of the foamless sample read 39.2, and the PCR tube samples containing the foam read 45.1, demonstrating a low level of particle depletion due to NSB. The derived metal foam was demagnetized, vortexed and rinsed in assay buffer. It was placed in a new PCR tube with 20 μl of assay buffer and 20 μl of AACr2-3-4 particles derived with cretinin with a final particle concentration of 0.1mMFe. A duplicated PCR tube without the derived metal foam was similarly established as in the control experiment. The PCR tube with the metal foam was cyclized twice by magnetic assisted agglomeration just like the control experiment (3 exposures of one minute with demag after each exposure, and final vortex). Thirty μl samples from both tubes were removed and heated to 37 ° C for 5 minutes and then read in the MR reader. The PCR tube sample with the derived metal foam read 41.5, and the PCR tube sample with the derived metal foam with the anticretinin antibody read 324.2, thereby demonstrating specific binding / depletion of the derived magnetic particles. of cretinin appropriate to the reading of aqueous volume by the MR reader. Example 12. Detection of simple nucleotide polymorphisms.
[0335] [000335] There are numerous methods by which T2 measurements could detect simple nucleotide polymorphisms.
[0336] [000336] The simplest request would involve the discrimination of imbalances by a thermophilic DNA ligase (Tth ligase). This assay would require lysis of the sample material followed by DNA shear. Adapters can be attached over sheared DNA if universal amplification of genomic DNA was required. The SNP would be detected by creating capture probes attached to the superparamagnetic particle that flank the SNP such that the 5 'end of the 3' amino capture probe would be perfectly complementary to a particular SNP allele and subsequent treatment with Tth ligase would result in the binding of the two capture probes connected to the particle. The bond, therefore, would close the particles in an agglomerated state. Hybridization, repeated fusion cycles will result in signal amplification in cases where amplification of genomic DNA is not desired because of the risk of amplification deviation. The same 5 'amino capture probe can be used in all cases while the 3' amino probe can be generated to produce 4 distinct pools (one A, G, C, or T) at the extreme 5 'end. Detection would require splitting the sample into the 4 pools to determine which nucleotide (s) was (were) present at the polymorphic site within that particular individual. For example, a strong T2 change in the G detection tube would only indicate that the individual was homozygous for G at that particular sequence site, while a change in G and A would indicate that the individual is heterozygous for G and A at that SNP site private. The advantage of this method is that Tth polymerase has been shown to have superior discrimination capacity even to distinctive G-T imbalances (a particular permissive imbalance and likewise the most common) 1: 200 times the correct complement. While ligase detection reactions as well as oligonucleotide ligase assays have been employed in the past to define nucleotide sequences at known polymorphic sites, all the amplification required before or after ligation; in this particular example the signal could be amplified by a linkage-induced increase in the size of the resulting agglomerated particle complex and thereby increases in the measured relaxation times (T2).
[0337] [000337] A modification to this procedure could include the hybridization of a capture probe attached to the particle that flanks the hybridization of a biotinylated probe. When a perfectly complementary duplex is formed by hybridizing the probe bound to the particle, the ligase would covalently link the biotin probe to the magnetic particle. Again repeated circles of heat denaturation followed by annealing and bonding should produce a high proportion of long biotinylated oligos on the magnetic particle surface. A wash to remove any free probe would be conducted followed by the addition of a second superparamagnetic particle labeled by streptavidin. Agglomeration would only result if the biotinylated probes were attached on the surface of the first particle.
[0338] [000338] A hybridization discrimination method can also be employed. In this example, amine oligonucleotide complements adjacent to known SNPs would be generated. These amino-oligonucleotides would be used to derivatize the surface of a 96-well plate with 1 SNP detection reaction conducted per well. The genomic DNA would then be sheared, attached to adapters, and asymmetrically expanded. This amplified genomic DNA would then be applied as well as a biotinylated SNP detection probe. The amplified genomic DNA would hybridize to the well-connected capture probe and the SNP detection probe would then bind to the strung genomic DNA. The wash would be conducted to remove the free SNP detection probe. A magnetic streptavidin (SA) particle would then be added to each well. Washing again would be required to remove particles without SA. T2 detection can be conducted directly into the wells by biotinylated superparamagnetic particles added to produce the agglomerated particles tied to the surface, or the SA magnetic particles can be eluted from each well in the array and incubated in detection reactions with biotinylated magnetic particles.
[0339] [000339] Finally, a primer extension reaction can be coupled with the detection of T2 to discriminate which nucleotide is present in a polymorphic site. In this assay, a pool of dideoxynucleotides would be employed with one nucleotide per pool that has a biotin (ie, ddA, ddT, ddbiotin-C and / or ddG). A superparamagnetic particle that supports a capture probe whose last base in hybridization is adjacent to an SNP would be used.
[0340] [000340] The sheared genomic DNA would be divided and incubated in four separate primer extension reactions. An exo DNA polymerase would then catalyze the addition of a dideoxy complementary to the nucleotide present in the SNP. Again this reaction can be cyclized if a thermophilic polymerase is employed to ensure that most of the capture probes on the particle will be extended. A magnetic separation followed by a washing of the particles would be conducted followed by incubation with superparamagnetic streptavidin particles. The clustering would continue proportional to the extent of the biotinylated capture probe on the surface of the first particle. If the two didesoxipools generated a gain in T2 (that is, to facilitate particle agglomeration), the patient would be a heterozygote. If only one pool produced and increased by T2, the patient would be a homozygote.
[0341] [000341] A final method for detecting SNPs employs allele-specific PCR primers, wherein the 3 'end of the primer comprises SNP. Since strict amplification conditions are employed, if the white sequence is not perfectly complementary to the primer, PCR amplification will be compromised with little or no product generated. In general, multiple sensory primers would be designed (one perfectly complementary to each allele) along with a single antisense primer. The amplicon would be detected using two or more superparamagnetic particles attached to the capture probe to induce agglomeration reactions based on hybridization. An advantage of this method is that it influences some work already carried out on T2 in PCR within raw samples, and would only require primers designed to cover known SNPs. A disadvantage with this method is that it cannot determine SNP locations again.
[0342] [000342] An additional method that can be used is simply depending on the discrimination capabilities of particle-particle crosslinking due to hybridization to a short nucleic acid target. Base pair imbalances for oligonucleotides have been shown to dramatically change the state of particle agglomeration, and the T2 signal measured, due to reduced hybridization efficiencies from the presence of a single base imbalance. Example 13. Candida Diagnosis panel.
[0343] [000343] The test was performed during the 45-day course. Reference strains of C. albicans and C. krusei as well as clinical isolates of C. albicans were grown and maintained for the duration of the study. Materials:
[0344] [000344] Nanoparticles of C. albicans and C. krusei: Two particle populations were generated for each species, the particles carrying covalently conjugated oligos complementary to species-specific sequences within the ITS2 region (see Example 10). The particles were stored at 4-8 ° C in TE (pH 8), 0.1% Tween and were diluted in 0.097 mM Fe in DNA hybridization buffer immediately before use.
[0345] [000345] Candida strain: Panels were made using the reference strain of C. albicans MYA 2876 (GenBank FN652297.1), reference strain of C. krusei 24210 (GenBank AY939808.1), and clinical isolates of C. albicans . The five isolated C. albicans used were grown in YPD at room temperature. Isolated colonies were selected, washed 3 times with PBS, and then quantified by hemocytometer to prepare whole blood boosters. The samples were stored as glycerol raw materials frozen at -80 ° C.
[0346] [000346] Human whole blood: Whole blood was collected from healthy donors and treated with K2EDTA and reinforced with serially diluted Candida cells washed in concentrations ranging from 1E5 to 5 cells / mL. Cell boosters prepared in fresh blood were stored at -20 ° C.
[0347] [000347] Erythrocyte Lysis Buffer: A hypotonic lysis buffer containing 10 mM potassium bicarbonate, 155 mM ammonium chloride, and 0.1 mM EDTA was sterilized in a filter and stored at room temperature before use. Alternatively, an erythrocyte lysis agent can be used, such as a non-ionic detergent (for example, a mixture of Triton-X 100 and igepal, or Brij-58).
[0348] [000348] PCR master mix: A master mix containing buffer, nucleotides, primers, and enzyme was prepared (20 μL of 5 * reaction buffer, 22 pL of water, 2 pL of 10 mM dNTP, 3 pL of primer sense at 10 μΜ, 3 pL of antisense initiator at 2.5 μΜ, 10 pL of HemoKlenTaq, and 40 pL of beaded lysate) and stored at room temperature.
[0349] [000349] Particle hybridization master mix: A master mix consisting of nanoparticle conjugates, salts, surfactant, and formamide was prepared (78 pL formamide, 78 pL 20 * SSC, 88.3 pL 1 * TE + 0.1% Tween, 7.5 pL CP 1 - 3 ', and 8.2 pL CP 3 - 5') immediately before use.
[0350] [000350] Glass beads (0.5 mm), used in mechanical lysis of Candida, were washed in acid and autoclaved and stored at room temperature before use. PCR protocol:
[0351] [000351] A general workflow scheme for detecting a pathogen (eg, Candida) in a whole blood sample is shown in Figure 20. The protocol was as follows: (i) enhanced human whole blood samples were allowed warm to room temperature (~ 30 minutes); (ii) 1 ml of erythrocyte lysis buffer was aliquoted in each tube; (iii) each tube was centrifuged at 9000 g for 5 minutes and the lysed blood was discarded; (iv) 100 µL of 0.2 micron filtered TE were aliquoted in each tube; (v) 120 mg of acid-washed glass beads were added to each tube; (vi) each tube was vortexed for 3 minutes at maximum speed (~ 3000 rpm); (vii) 50 µl of lysed sample was aliquoted in a tube containing PCR master mix; (viii) cycle PCR reactions as follows: (initial denaturation: 95 ° C, 3 minutes; 30-40 cycles at 95 ° C, 20 seconds; 30-40 cycles at 62 ° C, 30 seconds; 30-40 cycles at 68 ° C, 20 seconds; final extension: 68 ° C, 10 minutes; final imbibition: 4 ° C); (ix) each sample was centrifuged briefly after thermocycling to form clotted blood from the sediment; (x) 5 μL of main particle mixture were aliquoted in the tube for each 10 μL of amplified sample; (xi) the resulting mixture was mixed well and the sample denatured at 95 ° C for 3 minutes; (xii) the sample was hydridized at 60 ° C for 1 hour with gentle agitation; (xiii) the sample was then diluted to 150 µL with particle dilution buffer, and equilibrated at 37 ° C in a heating block for 1 minute; and (xiv) the T2 of the sample was measured using a T2 MR reader. Test Results
[0352] [000352] Repeated detection of Candida albicans in human whole blood: To determine the repeat of the T2 measurement in human whole blood infected with C. albicans, a study was conducted on day eight in which the amplified and reinforced donor sample was hydridized to the superparamagnetic particles (n = 3) each day and the resulting T2 values were recorded.
[0353] [000353] The precision cycle is shown in Figure 19A and in general it is strict with the CVs of all measurements less than 12%. The repetition observed over the eight-day course is shown in Figure 19B with CVs less than 10% for the Candida concentration range and 6% for the negative control. A two-tailed Student's t-test from two populations was applied to determine whether the difference in means between false Candida-infected blood at 10 cells / mL and healthy donor blood was significant. The resulting P value was less than 0.0001, confirming that the differences were statistically significant.
[0354] [000354] Influence of the sample matrix on detection and reproducibility in Candida albicans and Candida krusei: Healthy blood from 6 donors was reinforced with a range of cells from C. albicans or C. krusei (1E5 cells / mL to 0 cell / mL) . From the reinforced blood of Candida albicans, sixteen independent experiments were conducted. Each experiment consisted of PCR amplification of blood reinforced with 1E5 to 0 cell / mL with each amplification reaction subjected to three replicate T2 detection experiments; thus for C. albicans, a total of 48 T2 values were recorded at each concentration tested (see Figure 21A). At the lowest test concentration (10 cells / mL), we did not detect Candida albicans 37% of the time (6 out of 16 experiments); however, 100 cells / mL of Candida albicans was detected 100% of the time. This suggests that LOD for C. albicans is above 10 cells / mL, but below 100 cells / mL. More concentrations will be tested between 10 CFU at 100 cells / mL to better define the LOD; however, it is not expected that any main matrix effects will be observed on the performance of the assay. This is confirmed by the CVs of the T2 measurements, which are as follows: 12.6% in 1E5 cells / mL in 6 blood donors, 13.7% in 1E4 cells / mL, 15% in 1E3 cells / mL, 18% in 1E2 cells / ml, and 6% in 0 cell / ml. This suggests that the assay can strongly detect at concentrations of C. albicans greater than or equal to 100 cells / mL without major inhibition of the performance introduced by donor blood samples.
[0355] [000355] The same experiment was conducted using a reference strain of C. krusei. In this case, 7 independent experiments were conducted as the remaining boosted blood was reserved for blood culture analysis. We did not detect 10 cells / mL in any of the experimental cycles, but we detected 100 cells / mL for all experimental cycles. This suggests the LOD between 10 and 100 cells / ml. Again, a titration of cell concentrations between 100 and 10 cells / ml will need to be conducted to better define the LOD. The CV of the measurements by the concentration range was: 10.5% in 1E5, 9% in 1E4, 12% in 1E3, 20% in 1E2, 6.4% in 10, and 5.2% in 0 cell / mL. The results are shown in Figure 21B.
[0356] [000356] Preliminary determination of the limit of detection: Five clinical isolates of Candida albicans were boosted in 6 different blood samples from donor at concentrations of 1E4, 1E3, 5E2, 1E2, 50, 10, 5, and 0 cells / mL. Each isolate was boosted in a minimum of two different donor blood samples. Amplification reactions were detected by measuring T2 with the results plotted in Figure 22. It is important to note that no data has been removed for the source within this study. C. albicans was not detected 50% of the time in 5 cells / ml or 10 cells / ml; however, 50 cells / mL of C. albicans was detected 95% of the time. These data were generated using different clinical isolates; each isolate contains a different number of rDNA repeats and the number of these repeats can vary up to 4 times from strain to strain (ie ~ 50 units for 200 units). Since the input target copy numbers will vary slightly from strain to strain and certainly from species to species, there will be subtle differences in the absolute T2 values observed at very low cell numbers (ie, 10 cells / mL). Based on our very preliminary study, the data suggest a timeframe of 10 cells / mL; however, this determination cannot be made in the absence of final reagent formulations as well as the instrument / cartridge. This suggests that defining the C5-C95 range is difficult because at 10 cells / mL each reaction contains only 4 cells. Titration in cell numbers lower than this becomes challenging with this volume of blood input. The use of the Poisson distribution to calculate the number of reactions that would contain 0 cells to 10 cells / mL indicates that only 2% of the reactions would not contain cells; however, in 5 cells / mL, 13% of the reactions will not contain any Candida cells, and in 2 cells / mL, ~ 37% of the reactions would not contain Candida cells. To increase the sensitivity of the assay to 95% in 10 cells / mL, we could increase the amount of lysate added to the PCR reactions from 40 μL to 50 μL and we could increase the amount of patient's blood from 400 μL to 800 μL / reaction.
[0357] [000357] Preliminary determination of sensitivity / specificity: Initially, the quantification of incoming Candida colony forming units was accomplished using a hemocytometer; however, in this case, the operator counted daughter cells sprouting as separate cells. As our data are reported in colony-forming units / mL and not cells / mL, shoots should not be quantified. Because of this error, fewer cells / mL of Candida are present at various booster concentrations and our sensitivity at 10 cells / mL was only 90%, while specificity was 100%. In 25 cells / mL or more, 100% sensitivity and 100% specificity are observed. In all cases, blood culture vials inoculated with Candida cells were positive for blood culture on day 8. It should be noted that the basic parameter for blood culture is incubation for 5 days; however, it is necessary to extend this incubation time like many of the inocula required by> 5 days of incubation. As an example, Table 7 shows the inoculation time for positive culture recorded for four different clinical isolates of C. albicans inoculated in blood culture.
[0358] [000358] The results of T2 measurements carried out in 800 μL aliquots of these samples of reinforced whole blood are shown in Table 8. In all cases, it can be detected in 25 cells / mL, or more, however, it cannot be - detecting clinical isolate C3 in 12 cells / mL. It is important to note that CFUs were quantified by hemocytometer and not the Coulter counter for this particular method to compare the experiment. In total, 51 vials of blood culture were inoculated with clinical isolates of Candida albicans quantified by hemocytometer and 35 vials of negative blood culture were included in the experiment. The results for inoculum greater than 25 cells / mL are shown in the contingency table in Table 8.
[0359] [000359] Estimated specificity = 100x [TN / (FP + TN] = 100% (95% confidence interval = 90 to 100%)
[0360] [000360] Standardization of CFU quantification has improved our assay sensitivity and reproducibility. Preliminary results from 27 blood culture flasks are shown in Table 10. These preliminary results indicate that we have 100% sensitivity and specificity in 10 cells / mL or more. We have additionally started method comparisons using C. krusei. Preliminary results (from 36 vials) are shown in Table 11. The results indicate that we have a sensitivity / specificity of 88% / 100% at 10 cells / mL or more and 100% sensitivity / 100% specificity at 33 cells / mL or more for Candida krusei. Another important change that was instituted before the new blood culture according to comparisons was the use of a particle with multiple probes. In this case, the T2 cluster reactions for detection of C. albicans were conducted using multifunctional particles of albicans / parapsilosis / tropicalis while C. krusei was detected using the multifunctional particles of glabrata / krusei.
[0361] [000361] Preliminary assessment of clinical accuracy: Clinical accuracy is defined as the ability to discriminate between two or more clinical states, for example, Candidemia versus no Candidemia. Characteristic Plans of the Receiver Operator describe the test performance graphically illustrating the relationship between sensitivity (true positive fraction) and specificity (true negative fraction). Clinical accuracy (sensitivity / specificity pairs) is displayed for the entire spectrum of decision levels. Using data generated from 10-cell / mL and 50-cell / mL clinical isolate boosted whole blood samples, two ROC plans were generated and are shown in Figures 23A and 23B. The area under the usa curve is often used to quantify diagnostic accuracy; in this case our ability to discriminate between a Candidemic patient with an infection of 10 cells / mL or 50 cells / mL versus a patient without Candidemia. At 10 cells / mL, the area under the curve is 0.72 which means that if the T2 assay was conducted on a randomly chosen person with Candidemia at an infection level of 10 cells / mL, there is a 72% chance of your T2 value is higher than a person without Candidemia. The clinical accuracy of the test is much higher at 50 cells / mL with the area under the curve at 0.98. Again indicating that in a person with Candidemia at this level of infection, the T2 test would give a higher value than a sample from a patient without Candidemia 98% of the time. This is excellent clinical accuracy for infection levels of 50 cells / mL. ROC plans were not prepared for samples of 100 cells / mL or higher since the area would be translating to 100% of clinical diagnostic accuracy. The final clinical accuracy is determined from actual patient samples on the clinical platform.
[0362] [000362] Test return time: The primary test steps with calculated times are: (i) hypotonic lysis / centrifugation / counting (8 min); (ii) PCR (120 min); (iii) amplicon hybridization for particles (30 min.); (iv) subjected to magnetic assisted agglomeration in a homogeneous field (10 min.); and (v) download and read (10 seconds). The process time for the assay is estimated at ~ 178 minutes (~ 3 h), excluding the reagent and equipment preparation. This is the workflow used for qualification; however, the following modified workflow with shorter hybridization and PCR steps has been shown to produce the same detection sensitivity (see Figure 24) (albeit with a reduction in the amount of amplicon generated for some species of Candida (ie, glabrata) and consequently a smaller T2 delta between sick and normal): (i) hypotonic lysis / centrifugation / counting (8 min.); (ii) PCR (70 min.); (iii) amplicon hybridization for particles (30 min.); (iv) subjected to magnetic assisted agglomeration in a homogeneous field (10 min.); and (v) download and read (10 seconds). This modified flow generates a TAT of 133 minutes or 2 hours and 13 minutes (and this is without migration to a faster thermal cycler). Conclusions
[0363] [000363] This test demonstrates a current T2-based molecular diagnostic assay for Candidemia with the following metrics: (i) detection of Candida albicans within whole blood in a range that covers 5-1E5 cells / mL (5-log); (ii) detection of Candida krusei within whole blood in a range that covers 10 cells / mL in 1E5 cells / mL; (iii) sensitivity / specificity of 100% / 100% at> 25 cells / mL; (iv) diagnostic accuracy greater than 98% for concentrations> 50 cells / mL; (v) assay compatibility with whole blood (no main matrix effect observed using twelve different donor blood samples); (vi) repetition of T2 measurements (less than 12% within the same day and less than 13% for eight days); and (vii) total test return time reduced by 2 hours 3 minutes.
[0364] [000364] Higher inlet volumes of human blood have been tested and efficient hypotonic lysis has been found to be obtainable with these larger blood volumes; it also increased the reproducibility of detection by 10 cells / ml.
[0365] [000365] Contamination was observed within 2 samples of the 50 titrations. To reduce contamination issues, the PCR steps can be separated from the detection steps. In addition, chemical / biochemical methods can be used to make amplicons non-amplifiable. For example, uracils can be incorporated into the PCR product, and a pre-PCR incubation can be conducted with uracil N glycosylase.
[0366] [000366] The advantages of the systems and methods of the invention include the ability to analyze whole blood samples without separating non-target proteins and nucleic acids from the sample. Because no loss in target nucleic acids is suffered by DNA purification (eg, Qiagen fluorescent column after lysis and before amplification results in> 10 × loss of sensitivity; and use of whole blood interferes with optical detection methods in concentrations above 1%), variability from sample to sample and deviations (which can be introduced by DNA purification) are minimized and sensitivity is maximized.
[0367] [000367] Over 10% of patients with septic shock have Candida; this is the third most prevalent pathogen after S. aureus & E. coli, and there is a mortality rate of approximately 50% for patients with septic shock infected with Candida. Candida is the fourth leading cause of infections. Acquired in hospital. Rapid identification of these patients is critical for selecting proper treatment regimens. Example 14. Viral assay.
[0368] [000368] CMV genomic DNA was boosted and samples of healthy donor blood without CMV, 40 μL of this boosted blood were aliquoted in a 100 μL total volume PCR reaction. Amplification was conducted using a whole blood-compatible thermophilic DNA polymerase (T2 Biosystems, Lexington, MA) and exemplary universal primers that were designed as follows: CMV-specific 24-mer C6-sequence end, the exact sequences were as follows : 5'-CAT GAT CTG CTG GAG TCT GAC GTT A-3 '(SEQ ID NO. 11, universal tail probe # 1) 5'-GCA GAT CTC CTC AAT GCG GCG-3 (SEQ ID NO. 12, probe universal tail # 2) 5'-CGT GCC ACC GCA GAT AGT AAG-3 (SEQ ID NO. 13, CMV sense initiator US8) 5'-GAA TAC AGA CAC TTA GAG CTC GGG-3 (SEQ ID NO. 14 , CMV US8 antisense initiator)
[0369] [000369] The primers were designed such that the probe capture (i.e., the nucleic acid that decorates the magnetic particle) would anneal to the 10mer region (10mers are different at the 5 'or 3' end). The concentration of final initiator in the reaction tube was 300 nM and PCR master mix which included 5mM (NH4) 2SO4, 3.5mM MgCl2a, 6% glycerol, 60mM Tricine (pH 8.7)). Five separate sample reaction tubes were assembled. Cycle PCR reactions followed an initial denaturation of 95 ° C for 3 minutes, and each cycle consisted of 95 ° C, 20 seconds; 55 ° C, 30 seconds; and 68 ° C, 20 seconds. At 30, 33, 36, 39, and 42 cycles, the reaction tubes were removed and kept at 4 ° C. As soon as all samples were ready, 5 μL of main particle mixture (6 × SSC, 30% formamide, 0.1% Tween) was aliquoted in the tube for each 10 μL of amplified sample; the resulting mixture was mixed well and the sample denatured at 95 ° C for 3 minutes; the sample was hydridized at 45 ° C for 1 hour with gentle agitation; the sample was then diluted in 150 μL with particle dilution buffer (PBS, 0.1% Tween, 0.1% BSA), subjected to magnetic assisted agglomeration in a homogeneous field for 10 minutes, and equilibrated at 37 ° C in a heating block for 1 minute; and the T2 relaxation time for each of the five separate samples was measured using a T2 MR reader (see Figure 25).
[0370] [000370] The primers were designed to allow magnetic particles decorated with capture probes to anneal to the 10mer region (10mers are different at the 5 'or 3' end), providing particles with a universal architecture for aggregation with specific amplification primers.
[0371] [000371] The results provided in Figure 25 shows that the methods and systems of the invention can be used to perform real-time PCR and provide quantitative information about the amount of the target nucleic acid present in a whole blood sample. Example 15. Real-time PCR
[0372] [000372] Previous results showed that when the particles are present in the PCR reaction, amplicon production was inhibited. We hypothesize that the movement of the particles next to the reaction tube during thermocycling will allow the production of amplicon. A simple PCR block / magnetic separator insert (Figure 26) was designed to maintain the nanoparticles on the side walls during the PCR reaction, thereby minimizing particle interference and exposure to the PCR reaction components. When removing the magnetic field, the particles can be completely resuspended in the reaction mixture.
[0373] [000373] In an experiment, we tested the rate at which the particles can be isolated next to the tube and returned to the solution. In this experiment, 100 μL of the particle mixture of C. albicans (3 'and 5') in TE 1x (reference of non-clustered T2 of ~ 150 msec.) Went through a grouping / non-grouping process at 95 ° C three times. This was followed by the following protocol: (i) vortex, incubate at 37 ° C for 1 min, measure T2; (ii) heat at 95 ° C for 5 min when inserting magnetic PCR; (iii) incubate at 37 ° C for 1 min, measure T2; (iv) vortex 15 seconds, incubate at 37 ° C for 1 min, measure T2; and (v) proceed to step (ii). The results of this experiment are shown in Table 12 below.
[0374] [000374] As shown in Table 12, the fully reversible nanoparticle cluster was demonstrated at 95 ° C when using the tested magnetic separator. The particles are stable at 95 ° C for at least 3 grouping / non-grouping cycles.
[0375] [000375] Next, we tested the PCR efficiency in the presence of nanoparticles in the reaction solution. PCR was performed under two conditions: (1) nanoparticles are completely dispersed in the solution; and (2) nanoparticles are concentrated on the side walls of the PCR test tube using the magnetic insert.
[0376] [000376] Three PCR reactions (with nanoparticles concentrated on the test tube wall; completely dispersed in the solution; and no nanoparticles) were established using C. albicans genomic DNA as a starting material. Successful target DNA amplification has been validated using gel electrophoresis. Seramag particles decorated with a capture probe were used.
[0377] [000377] Asymmetric PCR reactions (4: 1) were initiated using pre-made PCR mix and 100 copies of genomic C. albicans DNA as a starting material. Mixture of C. albicans capture particles (3 'and 5') in 1x TE was added to reactions (1) and (3) (reference ~ 150ms). The control reaction (2) has no nanoparticles added (Figure 27).
[0378] [000378] No difference was observed in PCR product formation when nanoparticles were present in the solution (dispersed in the solution or concentrated on the side walls of the test tube by magnetic field) during PCR. Therefore, nanoparticles modified with capture probes do not interfere with PCR. Comparable amounts of product were generated in the reactions with and without the nanoparticles present in the solution as evidenced by gel electrophoresis. Likewise, the magnetic concentration of nanoparticles on the side walls of the test tube during the PCR process has no effect on the PCR. Example 16. Candida assay and clinical data.
[0379] [000379] A fast, accurate and reproducible molecular diagnostic test has been developed for the detection of five species of Candida directly within all human blood with a detection limit (LOD) of 10 cells / mL and a time to result of less than 2 hours. The clinical performance of the trial was determined using 32 covered clinical specimens and in this study we observed 100% positive and 100% negative according to blood culture, while accurately identifying the causative Candida species within 100% of the candidate patient samples . The test is also applied to blood specimens taken from Candida positive patients and we observed a decrease in the detection of Candida according to the course of antifungal treatment. This diagnostic method is fast, amenable to automation, and offers clinicians the opportunity to detect multiple human pathogens within complex biological specimens. Magnetic Resonance Relaxometer
[0380] [000380] A compact magnetic resonance (MR) system was designed and built for accurate T2 relaxation measures to perform the planned test under described conditions. This system was maintained at 37 ° C by temperature control and contains a samarium cobalt permanent magnet of approximately 0.5 T, which corresponds to an operating proton frequency of 22-24 MHz. All standard MR components: radio frequency probe, low-noise preamplifier and electronic transmitters, spectrometer panel, as well as temperature control hardware are packaged in the system. The system uses the standard AC power input and connects to an external computer via Ethernet. A friendly graphical user interface allows users to set the experimental parameters.
[0381] [000381] The system is designed to accept samples in standard 0.2 ml PCR tubes. The electronics as well as the spiral have been optimized to improve the accuracy of the measurement of the applicable sample volumes, while allowing us to achieve single scan cycle-to-cycle CVs in T2 below 0.1%. Instrument-to-instrument variability is below 2% with minimum tolerance requirements on system components and without calibration. Nanoparticle Sensor Characterization and Conjugation
[0382] [000382] 800 nm of superparamagnetic particles of carboxylated iron oxide, consisting of numerous iron oxide nanocrystals embedded in a polymer matrix including a total particle diameter of 800 nm (see Demas et al., New J. Phys. 13: 1 (2011)), were conjugated to amino-DNA oligonucleotides using standard carbodiimide chemistry. DNA-derived nanoparticles were stored at 4 ° C in 1 × Tris-EDTA (pH 8), 0.1% Tween-20. The iron concentration of the nanoparticle conjugates was measured by dissolving the particle with 6M HCl followed by the addition of hydroxylamine hydrochloride and 1.10 O-phenanthroline and subsequent spectrophotometric detection as described in Owen et al., J Immunol Methods, 73: 41 (1984). The oligonucleotide-derived particles are then subjected to a functional performance test by conducting the agglomeration reactions induced by hybridization using the identical diluted synthetic oligonucleotide targets following the fungal ITS2 sequences of the five different Candida species within a buffer of 4 × SSPE sodium phosphate hybridization (600 mM NaCl, 40 mM sodium phosphate, 4 mM EDTA). The reversibility of the agglomeration reaction was confirmed by submitting the agglomerated reactions to a heat denaturation step at 95 ° C, conducting a T2 measurement, and repeated hybridization at 60 ° C followed by a second T2 measurement. Design of nanoparticle capture probe and PCR initiator
[0383] [000383] Universal Pan Candida PCE primers were designed to complement the 5.8S and 26S rRNA sequences that amplify the intervening transcribed spacer 2 (ITS2) region of the Candida genome. A pair of oligonucleotide capture probes was designed to complement the sequence nested at the 5 'and 3' ends of the asymmetrically amplified PCR product, respectively. The capture probe that hybridizes to the 5 'end of the amplicon was 3' aminated while the capture probe that hybridizes to the 3 'end of the amplicon was 5' aminated. A poly-T linker (n = 9 to 24) is added between the amino group and the first nucleotide base of the capture probe sequence. HPLC purified PCR primers and capture probes were obtained from IDT Technologies (Coralville, IA). Inhibition Control Project
[0384] [000384] A PCR inhibition control has been designed to co-amplify with Candida species and monitor factors within whole blood specimens that inhibit PCR amplification. A synthetic model was designed to contain 30 identical nucleotide flanking sequences in sequence to the 5.8S and 26S regions of the Candida rRNA operon. The internal sequence within this model consists of a randomized mixed C. albicans amplicon. Capture probes were designed to complement the excess amplified filament within the asymmetric Candida PCR reactions. Synthetic oligonucleotide ultramers were obtained from identical IDT (Coralville, IA) following the inhibition control. The oligonucleotides were annealed at a concentration of 5 μΜ in 2 × SSC and cloned in HindII / EcoRV digested pBR322 (NEB, Ipswich, MA) using standard methods. The transformation was carried out by electroporation of 1 μL of the binding reaction in electrocompetent E. coli K12 cells and the transformers were placed on Luria Bertani agar plates (LB) containing 100 μg / mL ampicillin. Two ampicillin-resistant colonies were selected and cultured in 2 ml of LB ampicillin media. Plasmid mini-preps were conducted followed by restriction enzyme mapping to confirm the clones contained in the correct insert. Sanger's dideoxy sequencing was then conducted (SeqWright, Houston, TX) to confirm successful control cloning and DNA max-preps were conducted on clones carrying correct insertion. Inhibition control titrations in the presence of increasing concentrations of all 5 species of Candida were conducted to determine the lowest concentration of inhibition control that could be reproducibly detected. Confirmation of the function of the inhibition control was demonstrated by conducting the PCR reactions in the presence of titrations of known PCR interferers (SDS, heparin, ethanol) and demonstrating that the amplification of the control was inhibited. Candida cultivation and enhanced in vitro sample preparation
[0385] [000385] MYA-2876, ATCC 2001, ATCC 24210, ATCC 66029, and ATCC 22019 were laboratory reference strains of C. albicans, C. glabrata, C. krusei, C. tropicalis, and C. parapsilosis (ATCC, Manassas , VA) used to prepare in vitro enhanced whole blood specimens. Yeasts were cultured on yeast peptone dextrose agar (YPD) plates and incubated at 25 ° C. Isolated colonies were selected and suspended in phosphate buffered saline (PBS). The species were verified by ITS2 sequencing at Accugenix (Newark, Delaware). The cells were then subjected to a low speed centrifugation (3000 g for 2 minutes) and washed three times with fresh PBS. An aliquot of the cells washed with PBS was then diluted in ISOTON II diluent (Beckman Coulter, Brea, CA) inside a 20 mL Accuvette and the cells were quantified in a Multisizer 4 Coulter Counter (Beckman Coulter, Brea, CA) following the manufacturer's instruction. The cells were then serially diluted in concentrations ranging from 500 to 5 cells / 100 μL of PBS buffer. Fresh healthy human donor blood drawn by sterile collection in K2EDTA Vacutainer tubes (BD Diagnostics, Franklin Lakes, NJ) was obtained from ProMedX. Typically, five milliliters of human blood were boosted with 100 μL of quantified Candida cells. Samples reinforced with whole blood are then immediately used in the assay. Whole Blood PCR
[0386] [000386] Erythrocyte lysis was conducted within 1 ml of the whole blood sample using previously described methods (see Bramley et al., Biochimica et Biophysica Acta (BBA) - Biomembranes, 241: 752 (1971) and Wessels JM, Biochim Biophys Acta. , 2: 178 (1973)), a low spin speed is then conducted and the supernatant was removed and discarded. One hundred µL of Tris EDTA (TE) buffer pH 8.0 containing 1500 copies of the inhibition control was then added to the collected sediment and the suspension was subjected to mechanical lysis (see Garver et al., Appl. Microbiol., 1959. 7 : 318 (1959); Hamilton et al., Appl. Microbiol., 10: 577 (1962); and Ranhand, JM, Appl. Microbiol., 28:66 (1974)). Fifty μL of lysate was then added to 50 μL of a PCR master mix containing deoxynucleotides, PCR primers and a thermophilic DNA polymerase compatible with whole blood (T2 Biosystems, Lexington, MA). Thermocycling was conducted using the following cycle parameters: heat denaturation at 95 ° C for 5 minutes, 40 cycles consisting of a 30 second heat denaturation step at 95 ° C, a 20 second annealing step at 62 ° C, and a 30 second stretching step at 68 ° C, and a final extension at 68 ° C for 10 minutes. Agglomeration tests induced by hybridization
[0387] [000387] Fifteen microliters of the resulting amplification reaction were aliquoted in 0.2 ml thin-walled PCR tubes and incubated inside a sodium phosphate hybridization buffer (4 × SSPE) with pairs of oligonucleotide-derived nanoparticles in one final iron concentration of 0.2 mM iron per reaction. Hybridization reactions were incubated for 3 minutes at 95 ° C followed by 30 minutes incubation at 60 ° C inside a shaking incubator set at a shaking speed of 1000 rpm (Vortemp, LabNet International). The hybridized samples are then placed in a heating block at 37 ° C to balance the temperature with that of the MR reader for 3 minutes. Each sample is then subjected to a 5 second vortexing step (3000 rpm) and inserted in the MR reader for T2 measurement. Sample collection protocol for a patient with Candida.
[0388] [000388] Descartes of blood specimen that had been taken in K2EDTA vacutainers (BD) on the same day as specimens taken for blood culture (T = 0) were obtained from the clinical hematology laboratory at Massachusetts General Hospital (MGH) or Houston University Hospital. Specimens were collected and cataloged from patients who have positive blood culture results. Samples were stored inside the original vacutainer at -80 ° C and the covered specimen collection was transported overnight on dry ice in T2 Biosystems. Clinical sample collection protocols have been reviewed by the appropriate Human Research Committees. Statistical analysis
[0389] [000389] For each species, the detection limit was determined using the model per unit of probability. For each species, the 90% level of detection and 95% fiduciary intervals were calculated. Each crude T2 signal was transformed as T2_mseg at the base of the assay. SAS V. 9.1.3 (Cary, NC) was used in the statistical calculations for the analysis for detection limit, according to specimens reinforced with culture, sensitivity and specificity in clinical specimens, and consecutive tests to measure the release of Candida. T2 MR detection agreement of Candida with blood culture
[0390] [000390] The current gold standard for diagnosing Candida is blood culture. Specimens of whole blood from healthy donor reinforced in vitro were prepared using the laboratory reference strains for C. albicans and C. krusei and clinical isolates of C. albicans in concentrations of 0.33 and 100 cells / mL. Pediatric BACTEC blood culture vials (BACTEC Peds Plus / F vials, Beckton Dickenson) were inoculated with an aliquot of in vitro reinforced specimens evaluated by T2MR. Blood culture vials inoculated with Candida cells were positive for blood culture by day 8 in all cases. In total, 133 blood culture vials were inoculated with 90 Candida boosted blood samples (33 cell / mL inoculum) or 43 negative blood samples. Ninety-eight percent positive agreement and 100% negative agreement were observed between blood culture and T2MR. Clinical Specimen Data
[0391] [000391] K2 EDTA whole blood patient specimens were obtained to test the clinical performance of the Candida T2MR assay. Patients presented with symptoms of septicemia and blood were removed for culture. Blood samples were stored at 4 ° C in the hematology laboratory and selected for T2MR if the result was positive blood culture for Candida, positive blood culture for bacteremia, or negative blood culture to better represent the spectrum of the samples that would be conducted on the platform. Fourteen of the samples were from candidate patients, eight were from bacteremic patients, and ten were from blood culture negative patients. Figure 29 shows the T2 values measured for all 32 patient samples. A simple PCR reaction was conducted using 1 ml of each specimen. 750 copies of the internal inhibition control were added to each PCR reaction. Among the negative Candida samples, the average internal control (CI) signal was 279 ms with a CV on the 18 negative Candida specimens of 25%. In no case was the IC signal below the decision threshold (128 ms, 5 standard deviations added to mean T2 measured in negative Candida detection reactions) suggesting that all negatives were real negatives and no inhibitory substances were present with the samples. whole blood. Detection reactions were multiplexed based on IDSA standards, such that three results were reported as follows: C. albicans or C tropicalis positive; C.krusei or C. glabrata positive; and C. parapsilosis positive. The mean T2 measured in Candida negative specimens is 114 ms, the CV for these averages was 2.4%, and the decision threshold (calculated by adding five times the standard deviation measured in negative Candida detection reactions plus the mean T2 measured in negative Candida specimens) was 128 ms. In Candida positive specimens, the IC signal was suppressed due to competition for amplification reagents. In cases of elevated C. albicans, some cross-reactivity was observed for detection with C. parapsilosis particles (eg, patient sample # 3), however, this signal is not significantly above the reduction (20 ms) and does not leads to a difference in antifungal therapy when both C. albicans and C. parapsilosis are susceptible to fluconazole.
[0392] [000392] T2MR successfully identified fourteen samples of C. albicans, C. parapsilosis, or C. krusei that were confirmed positive by blood culture followed by biochemical card Vitek 2. In addition, the detection was specific for Candida spp. As bacteremic patient samples with Escherichia coli, Enterococcus sp., Staphylococcus aureus, Klebsiella pneumoniae, coagulase negative Staphylococcus, or alpha hemolytic Streptococcus remained negative.
[0393] [000393] Serially extracted samples were tested from two patients who exhibited symptoms suggestive of candidemia, such as persistent fever after receiving antibiotics to demonstrate the usefulness of the assay in monitoring Candida clearance. The withdrawals of santue for T2MR occurred on the same day as the withdrawals of blood for blood culture. Surveillance cultures were then performed in a nine-day course for Patient A and in a five-day course for Patient B. Figure 3 shows the results obtained with the T2MR method for both patients. Patient A had the blood removed for culture (t = 0), was diagnosed with candidemia and intravenously administered micafungin (C. glabrata) the following day through blood culture (t = 1). Whole blood specimens were tested with T2MR at t = 0 days, t = 3 days, t = 7 days, t = 8 days, t = 9 days. The T2MR values obtained were 320 ms at t = 0, 467 ms at t = 3, 284 ms at t = 7, 245 ms at t = 8, and 117 ms (below the reduction) for t = 9. Subsequent blood culture withdrawals on day 3 and day 8 took 24 and 48 hours for positive culture, respectively. A series of specimens serially removed was obtained from Patient B. C. albicans was correctly detected with T2MR on day 0 (T2 = 426 ms). Blood culture was positive on day 2 with subsequent identification of C. albicans. One day after the patient was administered micafungin, an abrupt decrease in C. albicans T2MR was evident (T2 = 169 ms) and three or more days after the antifungal treatment was started and no detectable C. albicans was observed. All tests were completed in a total processing time of two hours, using a quick-lock PCR thermocycler and a three-step thermocycling procedure that was not optimized for speed. Conclusions
[0394] [000394] A full-blood Candida T2MR assay capable of detecting five clinically important Candida species that influences the advantages of non-optical detection to eliminate the purification of the analyte was developed and validated, thus enabling faster inversion times to be enabled and more reproducible results. Asymmetric PCR was used to specifically amplify the ITS2 region of the Candida genome directly in whole blood to obtain clinically relevant detection sensitivities. A T2 detection method has been developed in which two groups of oligonucleotide-derived nanoparticles hybridize to each end of the single-stranded amplicon. Amplicons thus serve as interparticle moorings and induce nanoparticle agglomeration that produces a measurable and reproducible change in the gyro-gyro (T2) relaxometry of protons in water molecules. An internal inhibition control was also built and implemented to monitor for PCR inhibitors that may be present in patient samples.
[0395] [000395] The trial was evaluated using reference strains and clinical isolates quantified by Coulter Counter and which reached the maximum in whole blood from healthy donors. The repetition capacity of the assay was measured using reinforced blood of C. albicans (same sample, same operator, same instrument) during the course of 10 days and CVs smaller than 12.8% (n = 30) were observed in the range of whole dynamic response (0 to 1E5 cells / mL). Analytical sensitivity and detection limit of <10 cells / mL were measured for C. albicans, C. tropicalis, C. krusei, and C. parapsilosis and> 10 cells / mL with 92.5% detected at 10 cells / mL for C. glabrata. Although not proven, a possible cause of the highest LoD observed in C. glabrata may be that the copy number of the rDNA operon is reduced in C. glabrata when compared to another Candida spp questioned since C. glabrata is known to exist in nature as a haploid, while the other Candida species are diploid. According to the gold standard for Candida, the diagnosis was increased with 98% positive and 100% negative according to the observed for 133 samples of C. albicans and C. krusei that reached their maximum in vitro. It should be noted that the time for the result was 2 hours for the T2 Candida test while the time for positive blood culture was typically 2 days for C. albicans and ~ 1 day (18 to 24 hours ) for Candida krusei.
[0396] [000396] The 32 clinical specimens are similar to blood culture results. The measured T2 was above a reduction established in five standard deviations from the T2 values measured in the negative Candida specimens added to its mean. In this case, the threshold was 128 ms (n = 54). In no case did we observe inhibition of the PCR reaction, when internal control was detected in all 32 reactions with a reduced IC signal observed in Candida positive patients and a 25% CV (mean T2 of 279 ms) in Candida negative specimens (n = 18). The assay is highly specific for the detection of Candida when no cross-reactivity was observed with any of the bacteremic specimens (n = 8). The positive specimens of Candida were precisely identified, Candida spp. causative was precisely identified, and all within a 2 hour response time.
[0397] [000397] The potential for this assay to provide rapid detection of Candida clearance after administration of antifungal therapy has also been demonstrated. Two groups of patient samples were taken and submitted to T2MR (Figure 3). Moderate to elevated T2 signs for C. glabrata were observed in patient A on day 0 and day 3 with antifungal agents administered on day 1. A decrease in sign of C. glabrata was observed on subsequent days without any detectable after eight days of treatment antifungal. A strong signal of C. albicans was measured for patient B on day 0, and an abrupt decline (delta T2 of 306 ms) in signal of T2 was observed one day after administration of antifungal without any detectable after two days of antifungal treatment. Although preliminary, these data suggest that the test can be used to monitor the effectiveness of Candida treatment and clearance in a real-time mode.
[0398] [000398] At the conclusion, a sensitive and specific test was developed for the diagnosis of candidemia caused by the five most commonly found Candida species. Early clinical results have been encouraging and show that rapid diagnosis and species identification are achievable and may not only facilitate early treatment with the appropriate antifungal, but also provide a way to monitor Candida clearance. We anticipate that this nanoparticle-based T2MR method can be widely applied to diagnoses of infectious disease in a variety of specimen types and pathogens. Example 17. Tacrolimus assay using Fab.
[0399] [000399] The tacrolimus assay is a homogeneous competitive immunoassay performed using an EDTA sample of whole blood extracted to release tacrolimus from red blood cells and bind proteins. An essential component of the assay is a high-affinity tacrolimus antibody, a safe extraction method, and enhancement of selected buffer systems to promote specific aggregation and minimize non-specific aggregation. This version of the assay uses a recombinant monovalent Fab with high affinity for tacrolimus.
[0400] [000400] The tacrolimus assay was evaluated using whole blood calibrators, commercial whole blood controls, peak samples and patient samples.
[0401] [000401] Assay reagents included: (a) 244 nm of particle conjugated to sequential BSA, and monovalent Fab antibody and blocked with mPEG-thiol + NEM (particle is diluted to 0.2 mM Fe in assay buffer); (b) C22-modified tacromimus conjugated to BSA in the ratio of 10: 1 tacrolimus to BSA consumption (diluted to 600 ng / ml in assay buffer); (c) 100 mM Glycine buffer buffer pH 9.0, 1% BSA, 0.05% Tween 80, 150 mM NaCl, and 0.05% Procline; and (d) 70% MeOH extraction reagent, 60 mM ZnSO4 in dH20.
[0402] [000402] Whole blood calibrators were prepared using 1 mg / ml of Sigma FK506 Stock in 100% MeOH. EDTA whole blood peaked at varying levels with the tacrolimus solution. The reinforced blood was incubated at 37 ° C with gentle mixing and then stored overnight at 4 ° C before aliquoting and freezing. The target levels were 0, 1, 2, 5, 10, 20, 50, 100, and 250 ng / ml tacrolimus. The calibrators were supplied to an external laboratory for designation of value by the Architect Tacrolimus assay. The samples were tested by mass spectroscopy. The results show a correlation of 0.9998 for designation of theoretical versus actual value.
[0403] [000403] Quality controls consisted of 3 levels of UTAK Immunosuppressive Matrix Controls. Patient samples were obtained from patients transplanted on tacrolimus therapy.
[0404] (i) Permitir todas as amostras, calibradores, QC e reagentes para equilibrarem em temperatura ambiente, misturar por inversão suave. (ii) Pipetar 200 μL de amostra, calibrador, ou material de QC em um tubo de micrófogos de 1,5 mL. Adicionar 200 uL de reagente de extração e vortexar durante 30 segs. Permitir a amostra incubar durante 2 minutos em temperatura ambiente, e centrifugar durante 5 minutos em 10.000 rpm. Transferir o sobrenadante limpo para um tubo limpo e preparar uma diluição de 2,5× usando tampão de ensaio. (iii) pipetar 10 μL do extrato diluído e 10 μL de partícula diluída no tubo de reação, vortexar a mistura e incubar durante 15 minutos a 37°C. Pipetar 20 μL de conjugado de BSA-tac no tubo de reação, vortexar a mistura e incubar durante 15 minutos a 37°C. Amostra submetida à aglomeração assistida magnética em um campo de gradiente durante 6 ciclos (12 min.). Vortexar a mistura, incubar durante 5 minutos a 37°C e ler na leitora T2 a 37°C. [000404] The test protocol was as follows: (i) Allow all samples, calibrators, QC and reagents to equilibrate at room temperature, mix by gentle inversion. (ii) Pipette 200 μL of sample, calibrator, or QC material into a 1.5 mL microphotograph tube. Add 200 μL of extraction reagent and vortex for 30 sec. Allow the sample to incubate for 2 minutes at room temperature, and centrifuge for 5 minutes at 10,000 rpm. Transfer the clean supernatant to a clean tube and prepare a 2.5 × dilution using assay buffer. (iii) pipette 10 μL of the diluted extract and 10 μL of diluted particle in the reaction tube, vortex the mixture and incubate for 15 minutes at 37 ° C. Pipette 20 μL of BSA-tac conjugate into the reaction tube, vortex the mixture and incubate for 15 minutes at 37 ° C. Sample subjected to magnetic assisted agglomeration in a gradient field for 6 cycles (12 min.). Vortex the mixture, incubate for 5 minutes at 37 ° C and read in reader T2 at 37 ° C.
[0405] [000405] The calibrators were tested in triplicate for each test cycle (total of 6 cycles). The individual cycle data was fitted with a 5PL model using GraphPad Prism 5 for Windows, version 5.02, GraphPad Software, San Diego California USA. Calibrator 0 was entered as 0.01 ng / ml and used in the curve model. The resulting calibration curves (Run Calibration) were used to recalculate the tacrolimus concentration for all calibrators, whole blood boosters, QC and patient samples contained in the cycle.
[0406] [000406] In addition, a Main Calibration curve was obtained by adjusting the data by the study of 3 whole days (n = 18) for each calibrator. All samples were recalculated using the Main Curve and the resulting tacrolimus levels compared to those obtained using Cycle Calibration.
[0407] [000407] A reproducibility panel consisting of 13 members (9 calibrators, 3 controls and 1 sample of reinforced whole blood) was tested in triplicate for 3 days with 2 cycles per day for a total of 18 replications. Calibrators were stored at -80 ° C while controls and whole blood booster were stored at 4 to 8 ° C for the duration of the study.
[0408] [000408] Sample concentrations were predicted using the cycle calibration curve, as well as the main curve in GraphPrism. The precision within the cycle, within the day, day to day and total was calculated by ANOVA using MiniTab15.
[0409] [000409] Data predicted using the Cycle Calibration method showed total inaccuracy <25% CV over a tacrolimus concentration range of ~ 3 - 210 ng / ml.
[0410] [000410] The analytical sensitivity was calculated by the 2SD method. The standard deviation of 18 replicates of calibrator 0 was determined. The tacrolimus level at the maximum T2 (top asymptote of the curve adjustment) -2SD was then calculated and the concentration predicted using the Main Calibration Curve. The analytical sensitivity is 0.8 ng / ml.
[0411] [000411] During tacrolimus antibody evaluation and development, antibody specificity was evaluated against five tacrolimus metabolites. ELISA inhibition was performed with each of the 5 metabolites and compared to free tacrolimus for five affinity matured clones and seven cross affinity matured clones. Data for two of the crossed clones and a prior art murine monoclonal RUO antibody are shown below. The only cross-reactivity observed was mild reactivity to the 15-O-desmethyl metabolite.
[0412] [000412] A summary of tacrolimus assay performance is tabulated below.
[0413] [000413] Preparation of isolated probe particles: 800 nm of superparamagnetic particles of carboxylated iron oxide, consisting of numerous iron oxide nanocrystals embedded in a polymer matrix including a total particle diameter of 800 nm (see Demas et al., New J. Phys. 13: 1 (2011)) were washed using a magnetic rack before use. The magnetic particles were resuspended in 66 μL of nuclease-free water, 20 μL of 250 mM MES buffer pH 6, and 4 μL of amine probe (obtained from IDT), at a concentration of 1 mM per mg of particle to be prepared . A 3 'amine probe particle and a 5' amine probe particle were prepared (for example, the probe for C. parapsilosis). The probe was added to the particle and the suspension was vortexed using a vortexer equipped with a foam support to support the tube. The vortexer was fixed at a speed that keeps the particles well suspended without any spraying. N-ethyl-N '- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) was then dissolved in water and immediately added to the vortex probe particle mixture. The tube was then closed and incubated with rotation in an incubator at 37 ° C for 2 hours. The tube was then placed on a magnetic shelf and the reaction fluid was removed. The particles were washed with a series of washes (125 μL / mg of particle) as follows: water, water, 0.1M imidazole, pH 6.0 with a 5 minute incubation with rotation at 37 ° C, water, 0 , 1 M sodium bicarbonate, pH 8.0 with a 5 minute incubation with water rotation at 37 ° C. The particles were then subjected to a heat stress of 1 hour at 60-65 ° C in 0.1M sodium bicarbonate pH 8.0 with rotation. After heat stress, the bicarbonate was removed by placing the tube on a magnetic shelf. The particles were then resuspended in the storage buffer (Tris-EDTA, 0.1% tween 20) and vortexed. The storage buffer was removed and the final 100 μl of the storage buffer was added to the particle preparation. The particles were stored at 2-8 ° C, qualified using an iron test to determine the iron concentration of the particles, and tested against target nucleic acid (for example, ITS2 oligo titration of C. paraplsilosis). In the Candida assay, the particles are diluted in 8 × SSPE supplemented with 0.09% sodium azide as a preservative.
[0414] [000414] Preparation of dual probe particles: For the preparation of a dual probe particle, the procedure is the same as above, unless the equal volumes of a second probe (eg C. albicans amine 3 ') and the first probe (eg, C. tropicalis 3 'amine) are mixed prior to addition to the magnetic particles. Similarly, equal volumes of the 5 'amino probes were mixed prior to addition to the magnetic particles. Example 19. Improvements to the Candida assay.
[0415] [000415] The detection limit for the Example 16 Candida assay was improved by washing the pellet. 2.0 mL of whole blood was combined with 100 μL of TRAX erythrocyte lysis buffer (ie, a mixture of nonyl phenoxy-polyethoxyethanol (NP-40) and 4-octylphenol polyethoxylate (Triton-X100)) and incubated for about 5 minutes. The sample was centrifuged for 5 minutes at 6000g and the resulting supernatant was removed and discarded. To wash the pellet, the pellet was mixed with 200 μL of Tris EDTA (TE) buffer pH 8.0 and vortexed. The sample was centrifuged again for 5 minutes at 6000g and the resulting supernatant was removed and discarded. Following the washing step, the pellet was mixed with 100 μL of TE buffer and subjected to bead beating (for example, as with 0.5 mm glass beads, 0.1 mm silica beads, 0, 7 mm silica beads, or a mixture of beads of different sizes) with vigorous stirring. The sample was spun again. Fifty μL of the resulting lysate was then added to 50 μL of a master mix of asymmetric PCR containing deoxynucleotides, PCR primers and a thermophilic DNA polymerase compatible with whole blood (T2 Biosystems, Lexington, MA). The agglomeration assays induced by thermocycling and hybridization were conducted as described in Example 16 to produce T2 values characteristic of the presence of Candida in the blood sample. The test can produce (i) a coefficient of variation in the T2 value less than 20% in positive Candida samples; (ii) at least 95% of correct detection less than or equal to 5 cells / mL in samples boosted in 50 blood samples from an individual healthy patient; (iii) at least 95% of correct detection less than or equal to 5 cells / mL in samples boosted in 50 blood samples from an individual sick patient; and / or (iv) greater than or equal to 80% of correct detection in clinically positive patient samples (ie, Candida positive by another technique, such as by cell culture) starting with 2mL of blood.
[0416] [000416] This application claims priority for US Series Order number 12 / 910,594, filed on October 22, 2010, and benefit from claims in US Provisional Patent Application No. 61 / 414,141, filed on November 16, 2010, Application for US Provisional Patent No. 61 / 418,465, filed on December 1, 2010, and US Provisional Patent Application No. 61 / 497,374, filed on June 15, 2011, each of which is incorporated herein by reference. Other Modalities
[0417] [000417] All publications, patents, and patent applications mentioned in this specification are hereby incorporated by reference to the same extent as if each independent publication or patent application was specifically and individually indicated to be incorporated by reference.
[0418] [000418] While the invention has been described with respect to the specific modalities thereof, it will be understood that it is capable of other modifications and this application is intended to cover any variation, uses, or adaptations of the invention following, in general, the principles of the invention and including such divergences from the present description which are included in the known or usual practice within the technique to which the invention belongs and can be applied to the essential aspects mentioned above, and follows the scope of the claims.
[0419] [000419] Other modalities are within the claims.

权利要求:
Claims (21)
[0001]
Method for detecting the presence of a nucleic acid analyte in a liquid sample, characterized by the fact that it comprises: (a) contacting a solution with magnetic particles to produce a liquid sample comprising from 1x106 to 1x1013 magnetic particles per milliliter of the liquid sample, where the magnetic particles have an average diameter of 700 nm to 950 nm, a relaxation T2 per particle of 1x109 at 1x1012 mM-1s-1, and have binding portions on their surface, the binding portions operative to alter an aggregation of the magnetic particles in the presence of the nucleic acid analyte; (b) placing the liquid sample in a device, the device comprising (i) a support defining a cavity retaining the liquid sample comprising the magnetic particles and the nucleic acid analyte, and having an RF spiral arranged around the cavity, the spiral RF configured to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence, and optionally (ii) a removable cartridge sized to facilitate insertion into and removal of the device, wherein the removable cartridge is a modular cartridge comprising a reagent module for retaining one or more test reagents and a detection module comprising a detection chamber for retaining a liquid sample comprising the magnetic particles and the nucleic acid analyte ; (c) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (d) after step (c), measure the signal; and (e) based on the result of step (d), detect the presence or concentration of the nucleic acid analyte, preferablyi) in which the magnetic particles are substantially monodispersed and / or exhibit non-specific reversibility in the absence of the analyte and multivalent binding agent, or ii) in which step (d) comprises measuring the relaxation response T2 of the liquid sample, and in which the increase in agglomeration in the liquid sample produces an increase in the observed relaxation rate T2 of the sample, or iii) in which said target nucleic acid is extracted from a leukocyte or pathogen, or in which the magnetic particles have an average particle diameter between 700 and 900 nm, more preferably between 700 and 850 nm.
[0002]
Method for detecting the presence of a pathogen in a whole blood sample, characterized by the fact that it comprises: (a) providing an individual's whole blood sample; (b) mixing the whole blood sample with an erythrocyte lysis agent to produce broken red blood cells; (c) after step (b), centrifuge the sample to form a supernatant and sediment, discard some or all of the supernatants, and resuspend the sediment to form an extract, optionally washing the sediment before resuspending the sediment and optionally repeat step (c); (d) lyse the extract cells to form a lysate; (e) placing the lysate from step (d) in a container and amplifying a target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid, where the target nucleic acid is characteristic of the pathogen to be detected; (f) after step (e), mix the amplified lysate solution, with 1x106 to 1x1013 magnetic particles per milliliter of the amplified lysate solution to form a liquid sample, where the magnetic particles have an average diameter of 700 nm at 950 nm, a relaxation T2 per particle from 1x108 to 1x1012 mM-1s-1, and binding portions on its surface, the binding portions operative to alter the aggregation of magnetic particles in the presence of the target nucleic acid or a multivalent binding agent ; (g) placing the liquid sample in a device, the device comprising a support defining a cavity to hold the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF spiral arranged around the cavity, the configured RF spiral to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (h) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (i) after step (h), measure the signal from the liquid sample, wherein the liquid sample optionally comprises whole blood proteins and non-target oligonucleotides; and (j) based on the result of step (i), detect the pathogen, in which optionally the pathogen is bacteria or fungi, and in which the method is able to detect a concentration of pathogen of 10 cells / mL, in the blood sample total, preferably in which steps (a) to (i) are completed in 3 hours and / or step (i) is carried out without any previous purification of the amplified lysate solution.
[0003]
Method for detecting the presence of a virus in a whole blood sample, characterized by the fact that it comprises: (a) providing a plasma sample from an individual; (b) mixing 0.05 to 4.0 ml of the plasma sample with a lysis agent to produce a mixture comprising the disrupted virus; (c) placing the mixture (b) in a container and amplifying a target nucleic acid in the filtrate to form an amplified filtered solution comprising the target nucleic acid, where the target nucleic acid is characteristic of the virus to be detected; (d) after step (c), mix the amplified filtered solution with 1x106 to 1x1013 magnetic particles per milliliter of the amplified filtered solution to form the liquid sample, where the magnetic particles have an average diameter of 700 nm to 950 nm, a relaxation T2 per particle from 1x108 to 1x1012 mM-1s-1, and binding portions on its surface, the binding portions operative to alter the aggregation of magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (e) placing the liquid sample in a device, the device comprising a holder defining a cavity for holding a detection tube comprising the magnetic particles and the target nucleic acid, and having an RF spiral arranged around the cavity, the configured RF spiral to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing the liquid sample to a polarizing magnetic field and an RF pulse sequence; (g) after step (f), measure the signal from the liquid sample; and (h) based on the result of step (g), detect the virus, in which the method is capable of detecting less than 100 copies of viruses in the whole blood sample, preferably in which steps (a) to (g) are completed in 3 hours.
[0004]
Method for detecting the presence of a pathogen in a whole blood sample, characterized by the fact that it comprises: (a) providing an individual's whole blood sample; (b) mixing the whole blood sample with an erythrocyte lysis agent to produce the ruptured red blood cells; (c) after step (b), centrifuge the sample to form a supernatant and pellet, discard some or all of the supernatant, and resuspend the pellet to form an extract, optionally wash the pellet before resuspending the pellet and optionally repeat step (c); (d) lyse the extract cells to form a lysate; (e) placing the lysate from step (d) in a detection tube and amplifying a target nucleic acid in the lysate to form an amplified lysate solution including the target nucleic acid, where the target nucleic acid is characteristic of the pathogen to be detected; (f) after step (e), add the detection tube of 1 × 106 to 1 × 1013 magnetic particles per milliliter of the amplified lysate solution, where the magnetic particles have an average diameter of 700 nm to 950 nm and portions binding on its surface, the binding portions operative to alter the aggregation of magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (g) placing the detection tube in a device, the device comprising a holder defining a cavity for holding the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF spiral arranged around the cavity, the RF spiral configured to detect a produced signal by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (h) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (i) after step (h), measure the signal from the detection tube; and (j) based on the result of step (i), detect the pathogen, preferably in which steps (a) to (i) are completed in 3 hours and / or step (i) is carried out without any previous purification of the amplified lysate solution.
[0005]
Method for detecting the presence of a target nucleic acid in a whole blood sample, characterized by the fact that it comprises: (a) providing one or more cells from an individual's whole blood sample; (b) lyse said cells to form a lysate; (c) amplifying a target nucleic acid in the lysate to form an amplified lysate solution comprising the target nucleic acid; (d) after step (c), add the amplified lysate solution and from 1x106 to 1x1013 magnetic particles per milliliter of the amplified lysate solution to a detection tube, where the magnetic particles have an average diameter of 700 nm at 950 nm and binding portions on its surface, the binding portions operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (e) placing the detection tube in a device, the device comprising a holder defining a cavity for holding a detection tube comprising the magnetic particles and the target nucleic acid, and having an RF spiral arranged around the cavity, the RF spiral configured to detect a produced signal by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (h) after step (f), measure the signal from the detection tube; and (i) based on the result of step (h), detecting the target nucleic acid, preferably wherein said target nucleic acid is purified before step (d).
[0006]
Method for detecting the presence of a target nucleic acid in a whole blood sample, characterized by the fact that it comprises: (a) providing an extract produced by lysis of red blood cells in a whole blood sample from an individual, centrifuge the sample to form a supernatant and sediment, discard some or all of the supernatants, and resuspend the sediment to form an extract, optionally wash the pellet before resuspending the pellet and optionally repeating the centrifugation, disposal, and resuspension steps; (b) lyse the cells in the extract to form a lysate, optionally comprising combining the extract with beads to form a mixture and stirring the mixture to form a lysate; (c) placing the lysate from step (b) in a detection tube and amplifying the nucleic acids therein to form an amplified lysate solution comprising the target nucleic acid; (d) after step (c), add 1 × 106 to 1 × 1013 magnetic particles per milliliter of the amplified lysate solution to the detection tube, where the magnetic particles have an average diameter of 100 nm to 950 nm and portions binding on its surface, the binding portions operative to alter aggregation of the magnetic particles in the presence of the target nucleic acid or a multivalent binding agent; (e) placing the detection tube in a device, the device comprising a support defining a cavity for securing the detection tube comprising the magnetic particles and the target nucleic acid, and having an RF spiral arranged around the cavity, the RF spiral configured to detect a produced signal by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (f) exposing the samples to a polarizing magnetic field and an RF pulse sequence; (g) after step (f), measure the signal from the detection tube; and (h) based on the result of step (g), detecting the target nucleic acid, where step (g) is performed without any previous purification of the amplified lysate solution.
[0007]
Method according to any one of claims 2 to 6, characterized in that said magnetic particles comprise one or more populations having a first probe and a second probe conjugated to their surface, the first probe operative to connect to a first segment of the target nucleic acid and the second probe operative to bind to a second segment of the target nucleic acid, where the magnetic particles form aggregates in the presence of the target nucleic acid.
[0008]
Method according to any of claims 2 to 6, characterized in that said magnetic particles comprise a first population having a first bonding portion on its surface and a second population having a second bonding portion on its surface, and the said multivalent attachment portion comprising a first probe and a second probe, the first probe operative to attach said first attachment portion and the second operative probe to attach to a second attachment portion, the attachment portions and attachment portion multivalent operative to alter an aggregation of magnetic particles in the presence of the target nucleic acid.
[0009]
Method to detect the presence of a species of Candida in a liquid sample, characterized by the fact that it comprises: (a) lyse Candida cells in the liquid sample; (b) amplifying a nucleic acid to be detected in the presence of a sense primer and an antisense initiate, each of which is universal for multiple Candida species to form a solution comprising a Candida amplicon; (c) contacting the solution with the magnetic particles to produce a liquid sample comprising from 1x106 to 1x1013 magnetic particles per milliliter of the liquid sample, where the magnetic particles have an average diameter of 700 nm to 950 nm, a relaxation T2 per particle of 1x109 at 1x1012 mM-1s-1, and the bonding portions on its surface, the bonding portions operative to alter the aggregation of the magnetic particles in the presence of Candida amplicon or a multivalent bonding agent; (d) placing the liquid sample in a device, the device comprising a holder defining a cavity for holding the liquid sample comprising the magnetic particles and Candida's amplicon, and having an RF spiral arranged around the cavity, the RF spiral configured for detecting a signal produced by exposing the liquid sample to a polarizing magnetic field created using one or more magnets and an RF pulse sequence; (e) exposing the sample to a polarizing magnetic field and an RF pulse sequence; (f) after step (e), measure the signal; and (g) based on the result of step (f), determine if the Candida species was present in the sample, preferably i) where the sense primer comprises the 5'-GGC ATG CCT GTT TGA GCG TC-3 'oligonucleotide sequence (SEQ ID NO: 1) and / or the antisense primer comprises the 5'-GCT TAT TGA TAT oligonucleotide sequence GCT TAA GTT CAG CGG GT-3 '(SEQ ID NO: 2), or ii) where: (a) the Candida species is Candida albicans, and where the first probe comprises the 5'-ACC CAG CGG TTT GAG GGA GAA AC-3 'oligonucleotide sequence (SEQ ID NO: 3), and the second probe comprises the oligonucleotide sequence 5'-AAA GTT TGA AGA TAT ACG TGG TGG ACG TTA-3 '(SEQ ID NO: 4); (b) where the Candida species is Candida krusei, and where the first probe and the second probe comprise an oligonucleotide sequence selected from 5'-CGC ACG CGC AAG ATG GAA ACG-3 '(SEQ ID NO: 5), 5'-AAG TTC AGC GGG TAT TCC TAC CT-3 '(SEQ ID NO: 6), and 5'-AGC TTT TTG TTG TCT CGC AAC ACT CGC-3' (SEQ ID NO: 15); (c) where the Candida species is Candida glabrata, and where the first probe comprises the 5'-CTA CCA AAC ACA ATG TGT TTG AGA AG-3 'oligonucleotide sequence (SEQ ID NO: 7), and the second soda comprises the 5'-CCT GAT TTG AGG TCA AAC TTA AAG ACG TCT G-3 'oligonucleotide sequence (SEQ ID NO: 8), or (d) where the Candida species is Candida parapsilosis or Candida tropicalis, and where the first probe and the second probe comprise an oligonucleotide sequence selected from: 5'-AGT CCT ACC TGA TTT GAG GTCNitIndAA-3 '(SEQ ID NO : 9), 5'-CCG NitIndGG GTT TGA GGG AGA AAT-3 '(SEQ ID NO: 10), 5'-AAA GTT ATG AAATAA ATT GTG GTG GCC ACT AGC-3' (SEQ ID NO: 16), 5 '-ACC CGG GGGTTT GAG GGA GAA A-3' (SEQ ID NO: 17), 5'-AGT CCT ACC TGA TTT GAG GTC GAA-3 '(SEQ ID NO: 18), and 5'-CCG AGG GTT TGA GGG AGA AAT-3 '(SEQ ID NO: 19).
[0010]
Method according to claim 9, characterized by the fact that i) steps (a) to (h) are completed in 3 hours, or ii) the magnetic particles comprise two populations, a first population carrying the first probe on its surface, and the second population carrying the second probe on its surface, or iii) said magnetic particles comprise one or more populations having a first probe and a second probe conjugated to its surface, the first probe operating to connect to a first segment of Candida's amplicon and the second probe operating to connect to a second segment of the Candida amplicon, in which the magnetic particles form aggregates in the presence of the Candida amplicon, or iv) said magnetic particles comprise a first population having a first attachment portion on its surface and a second population having a second attachment portion on its surface, and said multivalent attachment portion comprising a first probe and a second probe, a first operative probe to bind said first binding portion and the second operative probe to bind a second binding portion, the binding portions and multivalent binding portion operative to alter an aggregation of the magnetic particles in the presence of Candida amplicon .
[0011]
System for the detection of one or more analytes, characterized by the fact that it comprises: (a) a first unit comprising (i) a permanent magnet defining a magnetic field; (ii) a support defining the cavity to hold a liquid sample, the cavity comprising magnetic particles having an average particle diameter between 700 and 900 nm, and having an RF spiral arranged around the cavity, the RF spiral configured to detect a signal produced by exposing the liquid sample to a polarizing magnetic field created using the permanent magnet and an RF pulse sequence; and (iii) one or more electrical elements in communication with the RF spiral, the electrical elements configured to amplify, rectify, transmit, and / or digitize the signal; and (b) a second unit comprising a removable cartridge sized to facilitate insertion into and removal from the system, where the removable cartridge is a modular cartridge comprising (i) a reagent module to retain one or more test reagents; and (ii) a detection module comprising a detection chamber for holding the liquid sample comprising the magnetic particles and the one or more analytes, and, optionally, (iii) a sterilizable input module, wherein the reagent module and the detection module, and optionally the sterilizable input module, can be mounted on the modular cartridge before use, and where the detection chamber is removable from the modular cartridge, preferably i) where the system also comprises a computer system with a processor to implement an assay protocol and store assay data, and where the removable cartridge also comprises (i) an interpretable label indicating the analyte to be detected, (ii) an interpretable label indicating the assay protocol to be implemented, (iii) an interpretable label indicating a patient identification number, (iv) an interpretable label indicating the position of assay reagents contained in the cartridge, or (v) an interpretable label comprising instructions for the programmable processor, and / or ii) in which the one or more analytes are nucleic acids, more preferably, wherein the magnetic particles have an average particle diameter between 700 and 900 nm, even more preferably between 700 and 850 nm.
[0012]
Removable cartridge sized to facilitate insertion and removal of a system of the invention, characterized by the fact that it comprises one or more chambers to hold a plurality of reagent modules to retain one or more test reagents, where the reagent modules include (i) a chamber holding from 1x106 to 1x1013 magnetic particles having an average diameter of 700 nm to 950 nm, a T2 relaxation per particle from 1x109 to 1x1012 mM-1s-1, and oligonucleotide binding portions on their surfaces, the connecting portions of oligonucleotides operative to alter the specific aggregation of magnetic particles in the presence of one or more analytes; and (ii) a chamber for retaining a buffer, in which said buffer optionally includes 0.1% to 3% (weight / weight) albumin, 0.01% to 0.5% non-ionic surfactant, a lysis agent, or a combination thereof.
[0013]
Removable cartridge according to claim 12, characterized by the fact that i) the magnetic particles and buffer are together in a single chamber inside the cartridge, and / or ii) the removable cartridge also comprises: (a) a chamber comprising beads to lyse cells; (b) a chamber comprising a polymerase; and / or (c) a chamber comprising an initiator.
[0014]
Method for amplifying a target pathogen nucleic acid in a whole blood sample, characterized by the fact that it comprises: (a) contacting a whole blood sample suspected of containing one or more pathogen cells with an erythrocyte lysis agent, thereby lysing the erythrocytes; (b) centrifuging the product from step (a) to form a supernatant and a sediment; (c) discarding part or all of the supernatant from step (b) and resuspending the pellet to form an extract; (d) combining the extract from step (c) with beads to form a mixture and agitating the mixture to form a lysate, the lysate containing both the nucleic acid of the individual's cells and the nucleic acid of the pathogen; and (e) supplying the lysate from step (d) in a detection tube and amplifying the pathogen nucleic acids to form an amplified lysate solution, wherein the amplification includes the polymerase chain reaction (PCR); wherein ten pathogen cells per milliliter of the whole blood sample are sufficient to allow amplification of the target pathogen nucleic acid, wherein the amplified lysate solution from step (e) comprises whole blood proteins and non-target oligonucleotides.
[0015]
Method for amplifying a target pathogen nucleic acid in a whole blood sample, characterized by the fact that it comprises: (a) contacting a whole blood sample suspected of containing one or more pathogen cells with an erythrocyte lysis agent, thereby lysing the erythrocytes; (b) centrifuging the product from step (a) to form a supernatant and a sediment; (c) discard part or all of the supernatant from step (b) and wash the pellet once; (d) centrifuging the product from step (c) to form a supernatant and a sediment; (e) discard part or all of the supernatant from the step (d) and mixing the sediment from step (d) with a buffer; (f) combining the product of step (e) with spheres to form a mixture and agitating the mixture to form a lysate, containing both the nucleic acid of the individual's cells and the pathogenic nucleic acid; and (g) supplying the lysate from step (f) in a detection tube and amplifying the pathogen nucleic acids by PCR, so as to form an amplified lysate solution; wherein ten pathogen cells per milliliter of the whole blood sample are sufficient to allow amplification of the target pathogen nucleic acid.
[0016]
Method according to claim 14 or 15, characterized by the fact that: (i) the lysis of step (a) is carried out by detergent lysis or hypotonic lysis, and / or (ii) PCR is an asymmetric PCR.
[0017]
Method according to claim 15, characterized by the fact that: (i) the amplified lysate solution from step (g) comprises whole blood proteins and non-target oligonucleotides; (ii) the sediment from step (c) is washed by mixing with TE buffer; (iii) the buffer of step (e) is TE buffer, optionally wherein the TE buffer has a volume of about 100 μL; and / or (iv) the buffer of step (e) comprises an inhibition control.
[0018]
Method according to any one of claims 14 to 17, characterized in that it further comprises the detection of the amplified target nucleic acid.
[0019]
Method according to any one of claims 14 to 18, characterized by the fact that the pathogen is a fungal pathogen, optionally in which the fungal pathogen is a species of Candida, optionally even in which the species of Candida is selected from the group constituted by Candida albicans, Candida krusei, Candida glabrata, Candida parapsilosis and Candida tropicalis, still optionally in which the amplification comprises the amplification of a Candida nucleic acid to be detected in the presence of a sense primer and an antisense primer, each of which is universal to several Candida species, to form a solution comprising a Candida amplicon.
[0020]
Method according to any of claims 14 to 18, characterized in that the pathogen is a bacterial pathogen, optionally in which the bacterial pathogen is selected from the group consisting of Acinetobacter sp., Bacteroides fragilis, Burkholderia cepacia, Campylobacter jejuni / coli, Clostridium pefringens, coagulase-negative Staphylococcus sp., Enterobacter aerogenes, Enterobacter cloacae, Enterobacteriaceae, Enterococcus faecalis, Enterococcus faecium, Escherichia coli, Haemophilus influenzae, Kingella kinge, Klebsbsiellaeylyneocyleae, Klein MRSA), Morganella morganii, Neisseria meningitidis, non-meningitidis Neisseria sp., Prevotella buccae, Prevotella intermedia, Prevotella melaninogenica, Propionibacterium acnes, Proteus mirabilis, Proteus vulgaris, Pseudomonas aeruginosa, Salmonella enterica, Staphylophyloscensis, Staphylophyloces as maltophilia, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus bovis, Streptococcus dysgalactiae, Streptococcus mitis, Streptococcus mutans, Streptococcus pneumoniae, Streptococcus pyogenes and Streptococcus pyogenes and bloodstream.
[0021]
Method according to claim 20, characterized by the fact that: (i) the bacterial pathogen is Borrelia burgdorferi; or (ii) the bacterium is selected from the group consisting of Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Pseudomonas aeruginosa and Escherichia coli, optionally in which the method comprises individually detecting three or more among Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Klebsiella pneumoniae and Escherichia coli.

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法律状态:
2019-05-28| B06T| Formal requirements before examination [chapter 6.20 patent gazette]|

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2019-08-27| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

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2020-04-07| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2020-08-25| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 19/10/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

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申请号 | 申请日 | 专利标题

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US12/910,594|US8563298B2|2010-10-22|2010-10-22|NMR systems and methods for the rapid detection of analytes|

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US12/910,594|2010-10-22|

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US41414110P| true| 2010-11-16|2010-11-16|

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US41846510P| true| 2010-12-01|2010-12-01|

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