METHOD OF TRAINING A SENSOR FOR ONE OR MORE ANALYTICS

专利摘要:
method for optimizing sensitivity and / or selectivity of a sensor for one or more analytes, method for forming a sensor for one or more analytes, kit for detecting one or more analytes, method of identifying an aptamer, composition of matter and aptamers . sensors for target entities having functionalized at least one aptamer specific to the target entity, and method of making and using them are described for use in monitoring glycated protein and / or biomarkers.
公开号:BR112014018831B1
申请号:R112014018831-9
申请日:2013-01-31
公开日:2021-04-27
发明作者:Brent D. Cameron；Dong-Shik Kim
申请人:The University Of Toledo；
IPC主号:

专利说明:

[0001] Inventors: Brent D. Cameron, Dong-Shik Kim
[0002] CROSS REFERENCE TO RELATED REQUESTS
[0003] This order claims the benefit of United States Provisional Order Serial No. 61593,054 filed on January 31, 2012, the entire disclosure of which is expressly incorporated herein by reference.
[0004] NOTE CONCERNING RESEARCH SPONSORED BY THE FEDERAL GOVERNMENT
[0005] The invention was not made with any government support and the government has no right to the invention.
[0006] SEQUENCE LISTING
[0007] The present application contains a Sequence Listing that was submitted on the EFS-web and is hereby incorporated by reference in its entirety. The ASCII copy, created on January 31, 2013, is called 420_53354_SEQ_LIST_D2012-15.txt, and is 3,888 bytes in size.
[0008] TECHNICAL FIELD AND INDUSTRIAL APPLICABILITY
[0009] The present invention relates to aptamer-functionalized surface plasmon resonance sensors (SPR), methods for making and methods for using them.
[0010] FUNDAMENTALS
[0011] The following description provides a summary of information relevant to the present disclosure and is not a concession that any information provided or publications referred to herein are state of the art for the claimed invention.
[0012] The direct detection of blood proteins can benefit numerous scientific and clinical applications, such as in the monitoring of the specific protein glycation ratio in diabetes, biomarkers for drug research and environmental monitoring, cancer diagnosis and treatment, and the like. Current clinical and laboratory measurement techniques for blood proteins are boronate affinity immunoassay, high performance liquid chromatography (HPLC), mass spectrometry and capillary-based systems, which are time-consuming and expensive.
[0013] More efficient and quick response measurement methods could benefit and greatly improve related application areas, especially to develop the next generation of hand-held portable diagnostic devices capable of real-time analysis. Several optical-based diagnostic techniques, such as near-infrared spectroscopy, polarimetry, optical coherence tomography, surface plasmon resonance (SPR), Raman spectroscopy and fluorescence, have recently been investigated to monitor blood components. Many of these optical methods, however, are limited in their usefulness due to the effects of confounding substances that may be present in the sample under investigation.
[0014] A method for the in vitro selection of nucleic acid molecules that are capable of binding with high specificity to target molecules is generally known as SELEX (Systematic Evolution of Ligands by Exponential Amplification - Systematic Evolution of Ligands by Exponential Amplification) and is described in U.S. Pat. No. US5,475,096 entitled "Nucleic Acid Ligands" and "Pat. No. US5,270,163, entitled "Nucleic Acid Ligands" ("Nucleic Acid Ligands") each of which is specifically incorporated herein by reference.
[0015] Although the currently used SELEX processes are useful, there is always a need for improved processes that allow the selection of more selective aptamers to be generated from in vitro selection techniques.
[0016] SUMMARY
[0017] In a broad aspect, a sensor is provided here to detect the presence of a target entity, comprising an aptamer probe having an amine-terminated end or the like attached to a substrate, where, when the sensor is excited by a energy source: i) in the absence of specific interaction between the target entity and the aptamer probe, a baseline signal is emitted; or ii) in the presence of specific interaction between the target entity and the aptamer probe, a detection signal is emitted, in which the baseline signal is different from the detection signal, whereby the selective presence of the target entity is detected.
[0018] In certain embodiments, the aptamer probe includes a nucleotide sequence that interacts specifically with the target entity.
[0019] In certain embodiments, the target entity is one or more of: a large biomolecule, a small biomolecule, an organic molecule, a small molecule, a nucleic acid, a metal ion, a protein, an enzyme, a peptide, a drug, a dye, a cancer cell, a virus, a hormone, or a microorganism. In certain embodiments, the protein is a blood protein.
[0020] In certain embodiments, the aptamer probe comprises an aptamer and an attached amine moiety.
[0021] In certain embodiments, the aptamer probe includes a SAM binder between the substrate and the amine moiety.
[0022] In certain embodiments, the amine-terminated aptamer probe is attached to the substrate by 3-mercaptopropionic acid (MPA).
[0023] In certain modalities, the sensor has an adjustable detectable range capable of detecting from pM to nM, based on the characteristics of the binder.
[0024] Broadly speaking, a method is provided here to determine whether a target entity is present in a sample comprising: i) contacting the sample with a sensor as described here; ii) excite the sensor with a power source; and iii) determine the strength of the signal emitted, thereby determining whether the target entity is present in the sample.
[0025] In certain embodiments, the energy source is measured using surface plasmon resonance (SPR).
[0026] In certain modalities, the method has a response time of less than 1 minute. In certain embodiments, the method has a response time of less than 1 minute at about room temperature.
[0027] In another broad aspect, a kit is provided here for the detection of a target entity, comprising: a sensor as described here; and at least one container containing the sensor, where a sample can be added to the container.
[0028] In another broad aspect, a method for making a sensor is provided here, comprising: i) immobilizing a self-organized monolayer binder (SAM) to a substrate; and ii) immobilize an amine-terminated aptamer to the SAM linker.
[0029] In another broad aspect, a method is provided here for making a sensor, comprising: i) functionalizing a substrate with a self-organizing monolayer binder (SAM); ii) exposing the functionalized substrate from step i) to a composition having an amine moiety sufficient for the amine moiety to be immobilized on the SAM binder; iii) exposing the amine-functionalized substrate of step ii) to at least one aptamer sufficient for the aptamer to be immobilized in the amine portion; iv) optionally, removing aptamer not specifically immobilized; and v) exposing the functionalized amine-terminated aptamer substrate from step iii) or iv) to a sufficient blocking agent to block unoccupied SAM sites activated by the amine moiety.
[0030] In certain embodiments, the composition having the amine moiety is coupled to one or more of: N-hydroxysuccinimide (NHS) and N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC).
[0031] In another aspect, a method is provided here for detecting blood proteins using a sensor as described here.
[0032] In another aspect, a method for the detection and ultra-sensitive and selective measurement of glycated proteins for application in therapeutic guidance of diabetes is provided here. In a particular embodiment, the method includes the use of surface plasmon resonance spectroscopy.
[0033] In another embodiment, this method of functionalization is applicable to other detection modalities, including Raman spectroscopy and fluorescence, and can be used to further improve the performance of existing monitoring technologies.
[0034] In another aspect, a method is provided here to optimize the in vitro selection of aptamers to target specific glycated forms of blood proteins. In one embodiment, a method of surface functionalization is used to optimize sensitivity and selectivity based on the target characteristics.
[0035] In another aspect, a method is provided here to further reduce the effects of confounding substances that may be present in the sample under investigation.
[0036] In yet another aspect, a robust, economical and portable detection platform is provided here that is capable of achieving performance similar to existing large-scale clinical instrumentation. In addition, the integrated platform is useful in a diagnostic device capable of assessing compliance with insulin-dependent diabetes therapy. The integrated platform allows for a low-cost hand-held device that can be used in a doctor's office or in a home environment. The integrated platform also provides an immediate analysis of the collected data, thus allowing the caregiver and / or patient to assess the patient's glucose regulation compliance in the long term.
[0037] In another broad aspect, a method is provided here to identify aptamers targeting a defined site (eg, glycated protein site), comprising introducing a non-target candidate (eg, non-glycated candidate) in at least one round of a systematic evolution of ligands by exponential enrichment protocol (SELEX), and introducing the non-target candidate in at least a second round of SELEX protocol to remove aptamer candidates with affinity for both glycated and non-glycated protein forms.
[0038] In another broad aspect, a method of surface functionalization is provided here to optimize sensitivity and / or selectivity based on target and / or aptamer characteristics, comprising: using a self-organized monolayer (SAM) formation process binary using bonds having a desired spacing and / or bond length, where at least one of the bond spacing and / or length is chosen to optimize sensitivity and selectivity of surface plasmon resonance (SPR) based on target characteristics and / or aptamer.
[0039] In another aspect, a method is provided here to optimize sensitivity and / or selectivity of a sensor for one or more analytes, comprising linking one or more types of aptamers to a substrate with a self-organizing monolayer (SAM) bond , the SAM bond having a desired spacing and / or bond length to form a functionalized surface on the substrate. The desired spacing and / or binding length can be chosen to optimize at least one of the sensitivity and selectivity of surface plasmon resonance (SPR), Raman spectroscopy, or fluorescence spectroscopy based on analyte and / or aptamer characteristics.
[0040] In certain embodiments, at least one of the packing density and / or length of the SAM binding affects a surface plasmon resonance (SPR) signal.
[0041] In certain modalities, the connection is through a binary SAM and reducing desorption process.
[0042] In certain embodiments, the desorption process comprises exposing the functionalized surface of the substrate to a material resistant to protein adsorption to prevent non-specific adsorption of protein on the functionalized substrate.
[0043] In certain embodiments, the material resistant to protein adsorption comprises (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3).
[0044] In certain embodiments, the SAM bond comprises using a method of immobilizing SAM by thiol, wherein a thiol compound has a carboxy moiety capable of forming a stable bond with the aptamer.
[0045] In certain embodiments, the thiol compound comprises dithiobis-N-succinimidyl propionate (DTSP).
[0046] In certain embodiments, SAM binding is formed using dithiobis-N-succinimidyl propionate (DTSP) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3), where PEG3 prevents non-specific adsorption of proteins, and in which a carboxylic moiety in DTSP forms the stable bond with the aptamer.
[0047] In certain embodiments, a binary SAM thiol solution is used to bind the SAM.
[0048] In certain embodiments, the binary SAM thiol solution is prepared by mixing 1 mM ethanol solutions of 3-mercaptopropionic acid (MPA) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) , while maintaining a total concentration of binary SAMs of around 1 mM.
[0049] In certain modalities, MPA and PEG3 are present at a ratio of: about 20:80, about 50:50 or about 80:20.
[0050] In certain embodiments, the method additionally comprises eliminating MPA by reducing desorption, leaving PEG3 intact; and allowing dithiobis-N-succinimidyl propionate (DTSP) to make a covalent bond with an amino group on the aptamer, where the aptamer is attached to DTSP only, and at the same time, PEG3 forms no bond.
[0051] In certain embodiments, the aptamer comprises an amine modified aptamer capable of being immobilized in the MPA.
[0052] In certain embodiments, the surface has an optimal dynamics in the range of about 5 nM to about 1000 nM.
[0053] In certain embodiments, the sensor includes a spacer layer of mixed length.
[0054] In certain embodiments, the mixed length layer comprises 11-mercaptoundecanoic acid (MUA) combined with 3-mercaptopropionic acid (MPA).
[0055] In certain embodiments, a water-soluble amino acid containing thiol capable of binding directly to the surface to form the self-organized monolayer (SAM) is used. In certain embodiments, the amino acid comprises cysteine.
[0056] In another aspect, a method is provided here to form a sensor for one or more analytes, comprising: adsorbing binary components comprised of 3-mercaptopropionic acid (MPA) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) on a substrate; desorbing MPA reductively from the substrate of step a); immersing the substrate of step b) in a DTSP solution to form a layer of DTSP on the substrate; immobilize at least one type of aptamer on the substrate of step c); and removing unbound aptamer from PEG3 on the substrate of step d), thus leaving aptamer attached to the substrate's DTSP layer.
[0057] In another aspect, a method is provided here to form a sensor for one or more analytes, comprising: adsorbing binary components comprised of 3-mercaptopropionic acid (MPA) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) on a gold surface substrate in an ethanol solution; desorbing MPA reductively from the substrate in a 0.5 M KOH solution, where MPA adsorbed to a self-organized binary monolayer separated in phases (SAM) of MPA and PEG3 is selectively reduced by applying a potential of - 1.2 V for 30 min to the solution; immerse the substrate having the PEG3 layer on top of it in a 1 mM DTSP solution to form a layer of DTSP on top of it; immobilize at least one type of aptamer on the substrate; and removing aptamer from PEG3 in the substrate, thereby leaving aptamer attached to the substrate's DTSP layer.
[0058] In certain embodiments, the substrate has a gold surface.
[0059] In certain embodiments, the analyte comprises a glycated form of a protein in blood or serum.
[0060] In certain embodiments, the method comprises determining a fraction of a specific glycated protein from a level of total whey protein.
[0061] In certain embodiments, the analyte comprises one or more non-glycated and / or glycated forms of human hemoglobin, albumin, including human serum albumin (HSA), immunoglobulin G (IgG), immunoglobulin M (IgM), fibrinogen, and / or fragments thereof.
[0062] In certain embodiments, the analytes comprise at least one first analyte having a different half-life than at least one second analyte, and the method additionally comprises quantifying the first and second analytes to provide a retrospective judgment related to levels of the first and according to analytes over one or more periods of time.
[0063] In certain embodiments, the first analyte comprises hemoglobin and the second analyte comprises IgM.
[0064] In certain embodiments, the analytes comprise at least a first analyte, at least a second analyte and at least a third analyte, each of the first, second and third analytes having different half-lives, the method further comprising: quantifying the first, second and third analytes to provide a retrospective judgment related to levels of the first, second and third analytes over one or more periods of time.
[0065] In certain embodiments, the first analyte comprises hemoglobin, the second analyte comprises IgM and the third analyte comprises albumin.
[0066] In certain embodiments, the method is useful for monitoring past average glucose levels, the method comprising: contacting a sensor formed by a method described here with a blood sample; determine an amount of the glycated form of the protein in the blood; and correlating an amount of the protein present in the blood sample in glycated form to a glucose control level for a given time interval.
[0067] In certain embodiments, the amount of the glycated form of the protein is determined using surface plasmon resonance (SPR).
[0068] In another aspect, a sensor is provided here to detect the presence of one or more analytes, in which the sensor is formed by any of the methods described here.
[0069] In certain embodiments, the aptamer includes a sequence of nucleotides capable of interacting with a specific analyte.
[0070] In certain modalities, the sensor is able to interact with one or more analytes selected from: a large biomolecule, a small biomolecule, an organic molecule, a small molecule, a nucleic acid, a metallic ion, a protein, an enzyme , a peptide, a drug, a dye, a cancer cell, a virus, a hormone, or a microorganism.
[0071] In certain modalities, the analyte is one or more of: a biological sample, an environmental sample, a chemical sample, a pharmaceutical sample, a food sample, an agricultural sample, and a veterinary sample.
[0072] In certain embodiments, the protein is a blood protein.
[0073] In certain modalities, the sensor has an adjustable detectable range capable of detecting from pM to nM, based on the characteristics of the binder.
[0074] In certain modalities, the sensor has a response time of less than 1 minute.
[0075] In certain modalities, the sensor has a response time of less than 1 minute at about room temperature.
[0076] In another aspect, a kit is provided here for the detection of one or more analytes, comprising: a sensor as described here; and at least one container containing the sensor, where a sample can be added to the container.
[0077] In another aspect, a method is provided here to reduce an effect of at least one confoundable substance that may be present in a sample, comprising: incorporating one or more hydrophilic groups at sufficient non-binding sites on the substrate to substantially reduce / prevent non-specific adsorption of the confoundable substance, attaching an aptamer to the substrate with a self-organizing monolayer (SAM) bond, the SAM bond having a desired spacing and / or bond length to form a functionalized surface on the substrate, and detecting response from connection to aptamer by SPR sensor the separation distance beyond the normal SPR detection limit.
[0078] Also described here is a method that uses surfaces of surface plasmon resonance spectroscopy (SPR) and functionalized sensor based on aptamer developed specifically to detect and / or quantify one or more target molecules, or fragments thereof, in a test environment. The method allows the detection and / or measurement of such molecules with a wide range of half-lives, including, but not limited to, target molecules with shorter half-lives than that of hemoglobin. In addition, the method can be conducted without the use of labels or markers, such as fluorescent dyes, or photoentangling. The method also has low sample consumption, and provides a quick response time (usually seconds), making it useful for application in the assessment of glycemic compliance.
[0079] In another aspect, a sensor is provided here, comprising: one or more types of aptamers attached to a substrate with a self-organizing monolayer (SAM) bond, the SAM bond having a bond spacing and / or length desired to form a functionalized surface on the substrate, the desired spacing and / or bond length being chosen to optimize at least one of sensitivity and selectivity of surface plasmon resonance (SPR), Raman spectroscopy, or fluorescence spectroscopy based on characteristics analyte and / or aptamer.
[0080] In certain embodiments, at least one of the packing density and / or length of the SAM binding affects a surface plasmon resonance (SPR) signal.
[0081] In certain modalities, the connection is made through a binary SAM and reducing desorption process.
[0082] In certain embodiments, the functionalized surface of the substrate was exposed to a material resistant to protein adsorption sufficient to inhibit non-specific protein adsorption on the functionalized surface.
[0083] In certain embodiments, the protein-resistant material comprises 1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3).
[0084] In certain embodiments, the SAM bond comprises a thiol compound having a carboxy moiety capable of forming a stable bond with the aptamer.
[0085] In certain embodiments, the thiol compound comprises dithiobis-N-succinimidyl propionate (DTSP).
[0086] In certain embodiments, SAM binding is formed using dithiobis-N-succinimidyl propionate (DTSP) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3), where PEG3 prevents non-specific adsorption of proteins, and in which a carboxylic moiety in DTSP forms a stable bond with the aptamer.
[0087] In certain embodiments, a binary SAM thiol solution is used to bind the SAM.
[0088] In certain embodiments, the binary SAM thiol solution is prepared by mixing 1 mM ethanol solutions of 3-mercaptopropionic acid (MPA) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) , while maintaining a total concentration of binary SAMs of around 1 mM.
[0089] In certain modalities, MPA and PEG3 are present in a ratio of: about 20:80, about 50:50 or about 80:20.
[0090] In certain modalities, MPA was eliminated by reducing desorption, leaving PEG3 intact; and dithiobis-N-succinimidyl propionate (DTSP) attached to an amino group in the aptamer, and PEG3 forms no bond.
[0091] In certain embodiments, the aptamer comprises an amine-modified aptamer capable of being immobilized in 3-mercaptopropionic acid (MPA).
[0092] In certain embodiments, the surface has an optimal dynamics in the range of about 5 nM to about 1000 nM.
[0093] In certain modalities, the sensor includes a spacer layer of mixed length.
[0094] In certain embodiments, the mixed length layer comprises 11-mercaptoundecanoic acid (MUA) combined with 3-mercaptopropionic acid (MPA).
[0095] In certain embodiments, the SAM bond comprises a water-soluble amino acid containing thiol capable of binding directly to the substrate surface.
[0096] In certain embodiments, the amino acid comprises cysteine.
[0097] In another aspect, a sensor is provided here where at least the surface of the substrate is gold.
[0098] In certain modalities, the sensor is configured to detect an analyte comprised of a glycated form of a protein in blood.
[0099] In certain embodiments, the sensor is configured to determine a fraction of a specific glycated protein from a level of total whey protein.
[00100] In certain embodiments, the analyte comprises one or more of: human hemoglobin, albumin, including human serum albumin (HSA), immunoglobulin G (IgG), immunoglobulin M (IgM), fibrinogen, and / or fragments thereof, the analyte being either glycated or non-glycated.
[00101] In certain embodiments, the analytes comprise at least one first analyte having a different half-life than at least a second analyte.
[00102] In certain embodiments, the first analyte is comprised of hemoglobin and the second analyte is comprised of immunoglobulin M (IgM); and, in which the first analyte or the second analyte is present in a glycated or non-glycated form.
[00103] In certain embodiments, the analytes comprise at least a first analyte, at least a second analyte and at least a third analyte, each of the first, second and third analytes having different half-lives.
[00104] In certain embodiments, the first analyte is comprised of hemoglobin, the second analyte is comprised of IgM, and the third analyte is comprised of albumin; wherein one or more of the first analyte, second analyte or third analyte is present in a glycated or non-glycated form.
[00105] In another aspect, a use of any of the sensors described here is provided here to monitor past average levels of analyte in the blood, through: contact of a sensor formed by a method described here with a blood sample; determining an amount of the glycated form of the analyte in the blood; and correlating an amount of the analyte present in the blood sample analyte in a glycated form at a control level for a time interval.
[00106] In certain embodiments, the amount of the glycated form of the protein is determined using surface plasmon resonance (SPR).
[00107] In certain embodiments, the aptamer includes a sequence of nucleotides capable of interacting with a specific analyte.
[00108] In certain modalities, the sensor is capable of interacting with one or more analytes selected from: a large biomolecule, a small biomolecule, an organic molecule, a small molecule, a nucleic acid, a metal ion, a protein, an enzyme , a peptide, a drug, a dye, a cancer cell, a virus, a hormone, or a microorganism.
[00109] In certain modalities, the analyte is one or more of: a biological sample, an environmental sample, a chemical sample, a pharmaceutical sample, a food sample, an agricultural sample, and a veterinary sample.
[00110] In certain modalities, the analyte is a blood protein.
[00111] In certain modalities, the sensor has an adjustable detectable range capable of detecting pM to nM, based on the characteristics of the binder.
[00112] In certain modalities, the sensor has a response time of less than 1 minute.
[00113] In certain embodiments, the sensor has a response time of less than 1 minute at about room temperature.
[00114] In certain embodiments, the sensor includes an aptamer where the aptamer comprises a DNA sequence having at least 70% identity with the entire sequence of any one of SEQ ID NOS: 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.
[00115] In another aspect, a kit is provided here for the detection of one or more analytes, comprising: any one or more of the sensors described here; and at least one container including the sensor, where a sample can be added to the container.
[00116] In certain embodiments, the kit additionally comprises one or more solid supports, one or more separation agents to separate the sensor from an element, and one or more reagents to separate an aptamer from the sensor.
[00117] In another aspect, a method is provided here for identifying a single target site binding aptamer from a pool of nucleic acids having a single target site binding aptamers and non-target protein binding aptaments therein, comprising:
[00118] adding a single site target protein complex to the nucleic acid pool, in which both the non-target protein binding aptamers present in the pool and the non-target protein binding aptamers present in the pool bind to the target protein complex of single site, and form a single target site binding aptamer + non-target protein binding aptamer + single site target protein complex;
[00119] separating the aptamer from binding to a single target site + aptamer from binding to non-target protein + single site target protein complex from the pool;
Eluting aptamers binding to a single target site and aptamers binding to non-target protein from the single site target protein complex;
[00121] add a non-target protein complex to the previous step elute, in which the non-target protein binding aptamers present in the step c) elute bind to the non-target protein complex, and form a protein binding aptamer. non-target + non-target protein complex;
[00122] separating the non-target protein + non-target protein complex from the elute from the previous step, leaving the binding aptamer to a single target site on the elute; and,
[00123] separating the aptamers of binding to a single elution target site; optionally further amplifying the binding aptamers to a single target site.
[00124] In certain embodiments, aptamers binding to a single target site are used to select one of: hemoglobin, immunoglobulin G (IgG), immunoglobulin M (IgM) and albumin.
[00125] In certain embodiments, aptamers binding to a single target site are selected from: SEQ ID NOs: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14 and 15.
[00126] In certain embodiments, the single site target protein is immobilized on a solid support.
[00127] In certain embodiments, the non-target protein complex is immobilized on a solid support.
[00128] In certain embodiments, the solid support comprises a magnetic sphere, a chromatographic matrix, a microtiter plate or a matrix.
[00129] In another aspect, there is provided here an aptamer that binds to glycated hemoglobin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4 and 5.
[00130] In certain embodiments, glycated hemoglobin is human hemoglobin.
[00131] In certain embodiments, the aptamer has a dissociation constant for human hemoglobin of 100 nM or less.
[00132] In another aspect, an aptamer described here is provided that binds to glycated hemoglobin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 4 and 5, and one or more of: a 5 'linker and a 3' linker.
[00133] In certain modalities, the ligand is a self-organized monolayer (SAM).
In another aspect, an aptamer with at least 70% identity to the entire sequence of any one of SEQ ID NOs: 4 and 5 and which binds to human glycated hemoglobin is provided here.
In another aspect, a composition of matter comprising a self-organized monolayer (SAM) conjugated to a nucleic acid aptamer molecule comprising a sequence of polynucleotides capable of specifically binding to a region of glycated hemoglobin is provided here. that the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 4 and 5.
[00136] In another aspect, an aptamer is attached here that binds to non-glycated hemoglobin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7.
[00137] In certain embodiments, non-glycated hemoglobin is human hemoglobin.
[00138] In certain embodiments, the aptamer has a dissociation constant for human hemoglobin of 100 nM or less.
[00139] In another aspect, an aptamer is attached here that binds to non-glycated hemoglobin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 6 and 7, and one or more of: a linker 5 'and a 3' linker.
[00140] In certain modalities, the ligand is a self-organized monolayer (SAM).
In another aspect, an aptamer with at least 70% identity to the entire sequence of any of SEQ ID NOs: 6 and 7 and which binds to human non-glycated hemoglobin is provided here.
[00142] In another aspect, there is provided here a composition of matter comprising a self-organized monolayer (SAM) conjugated to a nucleic acid aptamer molecule comprising a sequence of polynucleotides capable of specifically binding to a region of non-glycated hemoglobin, wherein the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 6 and 7.
[00143] In another aspect, an aptamer is attached here that binds to glycated serum albumin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 8 and 9.
[00144] In certain embodiments, glycated serum albumin is human glycated serum albumin.
[00145] In certain embodiments, the aptamer has a dissociation constant for human glycated serum albumin of 100 nM or less.
[00146] In another aspect, an aptamer is attached here that binds to glycated serum albumin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 3, 8 and 9, and one or more of: a 5 'linker and a 3' linker.
[00147] In certain modalities, the ligand is a self-organized monolayer (SAM).
In another aspect, an aptamer with at least 70% identity to the entire sequence of any one of SEQ ID NOS: 3, 8 and 9, and which binds to human glycated serum albumin, is provided here.
In another aspect, a composition of matter comprising a self-organized monolayer (SAM) conjugated to a nucleic acid aptamer molecule comprising a sequence of polynucleotides capable of specifically binding to a region of glycated serum albumin is provided here. , in which the polynucleotide sequence is selected from the group consisting of SEQ ID NOs: 3, 8 and 9.
[00150] In another aspect, there is provided here an aptamer that binds to non-glycated serum albumin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14 and 15.
[00151] In certain embodiments, non-glycated serum albumin is human glycated serum albumin.
[00152] In certain embodiments, the aptamer has a dissociation constant for human non-glycated serum albumin of 100 nM or less.
[00153] In another aspect, an aptamer is attached here that binds to non-glycated serum albumin, wherein the aptamer comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 10, 11, 12, 13, 14 and 15, and one or more of: a 5 'linker and a 3' linker.
[00154] In certain modalities, the ligand is a self-organized monolayer (SAM).
[00155] In another aspect, an aptamer with at least 70% identity is provided here with the entire sequence of any one of SEQ ID NOS: 10, 11, 12, 13, 14 and 15, and which binds albumin of human non-glycated serum.
In another aspect, a composition of matter comprising a self-organizing monolayer (SAM) conjugated to a nucleic acid aptamer molecule comprising a sequence of polynucleotides capable of specifically binding to a region of non-serum albumin is provided here. glycated, in which the polynucleotide sequence is selected from the group consisting of SEQ ID Nos. 10, 11, 12, 13, 14 and 15.
[00157] In certain embodiments, the aptamer comprises at least one chemical modification.
[00158] In certain embodiments, the modification is selected from the group consisting of: a chemical substitution in a sugar position, a chemical substitution in an internucleotide bond, and a chemical substitution in a base position.
[00159] In another aspect, a test reagent is provided here comprising an effective amount of an aptamer described herein, or a salt thereof, and a support for it.
[00160] In another aspect, a kit is provided here comprising at least one aptamer as described here.
[00161] In certain embodiments, the aptamer is PEGylated.
[00162] In certain embodiments, the PEGylated aptamer molecule includes 1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3).
[00163] In certain embodiments, the SAM bond is formed using dithiobis-N-succinimidyl propionate (DTSP) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3).
[00164] In certain embodiments, the aptamer comprises at least one chemical modification.
[00165] In certain embodiments, the modification is selected from the group consisting of: a chemical substitution in a sugar position, a chemical substitution in an internucleotide bond, and a chemical substitution in a base position.
[00166] A test reagent comprising an effective amount of one or more aptamers described herein or a salt thereof, and a support for them.
[00167] A kit comprising one or more aptamers described here.
[00168] In another aspect, purified and isolated non-naturally occurring DNA sequences selected from the group consisting of SEQ ID NOs are provided here: 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13 , 14 and 15.
[00169] In another aspect, a method is provided here to reduce an effect of at least one confoundable substance that may be present in a sample, comprising: a) incorporating one or more hydrophilic groups in sufficient non-binding locations on the substrate to reduce / substantially prevent non-specific adsorption of the confoundable substance, b) attaching an aptamer to the substrate with a self-organizing monolayer (SAM) bond, the SAM bond having a desired spacing and / or bond length to form a functionalized surface on the substrate, and c) detecting aptamer bond response by SPR sensor at separation distance beyond the normal SPR detection limit.
[00170] Other systems, methods, resources, and advantages of the present invention will or will become apparent to someone skilled in the art upon examination of the following figures and detailed description. It is intended that all such systems, methods, resources, and additional advantages are included within this description, are within the scope of the present invention, and are protected by the appended claims.
[00171] BRIEF DESCRIPTION OF THE FIGURES
[00172] The patent or application deposit may contain one or more figures executed in color and / or one or more photographs. Copies of this patent or publication of a patent application with color figures and / or photograph (s) will be provided by the patent office upon request and payment of the required fine.
[00173] Fig.1: Schematic diagram of a sensor surface functionalization method.
[00174] Fig. 2: Impedance spectrum Nyquist plots obtained in 100 mM PB (f 7.2) solution containing 5 mM Fe (CN) 6 3- / Fe (CN) 6 4- (Column A) Bare Au; (Column B) Au / MPA / EDC-NHS / EA / PPA; (Column C) Au / MPA / EDC-NHS / EA / PPA / APT1. The plot on the right shows the (Ret) of each layer. The impedance spectrum was collected in the frequency range from 0.1 Hz to 100 kHz with a potential amplitude of 5 mV rms at 10 points per decade.
[00175] Fig. 3: Graph showing proportion of aptamer / thrombin binding in mol by the magnetic granule coupling method.
[00176] Fig. 4: Graphics showing bare Au SPR response and aptamer-modified sensors. All data points were weighted from 3 readings of experimental data. Samples were thrombin only (upper plot) and thrombin with 400 nM BSA (lower plot). The embedded plots are the same data plotted on a logarithmic scale to allow better visualization at lower concentrations.
[00177] Fig. 5: Graph showing Suprs Response. different sensing surfaces for 400 nM BSA (BSA Group), 500 nM thrombin (Thrombin Group), and 500 nM thrombin with 400 nM BSA (Thrombin + BSA Group). The error bars represent the standard deviation of the values determined from three freshly prepared samples.
[00178] Fig. 6: Graph showing SPRs response of different sensing surfaces for 50 nM, 250 nM, 500 nM thrombin with and without 400 nM BSA, upper axis (APT1), lower axis (APT2); the zero position of the lower axis was intentionally shifted to better distinguish between overlapping data points.
[00179] Fig. 7: Schematic illustration of an excited surface plasma.
[00180] Fig. 8a: Schematic illustration of SPR with Kretschmann Configuration.
[00181] Fig. 8b: Schematic illustration of displacement at an angle of resonance due to the change in refractive index.
[00182] Fig. 9: Schematic illustration of HbA1c binding with aptamer immobilized on a SAM surface affixed to a SPR sensing surface (top); and, schematic illustration of displacement at resonance angle due to the change in refractive index (lower).
[00183] Fig. 10: Graphs showing SPR Response for HSA at different levels of glycation (% proportions in percentage; glycated / total protein). Note: the total protein concentration of each sample is constant at a total protein level at 1μg / mL. (Green) Functionalized aptamer surface (Red) Bare Au surface.
[00184] Figs. 11a-11e: Schematic illustration of reductive desorption for binary DTSP-PEG3 formation SAM: (Fig. 11a) coadsorption of MPA and PEG3 in Au; (Fig. 11b) reductive MPA desorption; (Fig. 11c) adsorption of DTSP; (Fig. 11d) aptamer immobilization; and, (Fig. 11e) removing PEG3 aptamer.
[00185] Fig. 12: Schematic illustration of a Collection opposite MB for a SELEX Process
[00186] DETAILED DESCRIPTION
[00187] Throughout this disclosure, various publications, patents and published patent specifications are referenced by an identification citation. The disclosures of these publications, patents and published patent specifications are hereby incorporated by reference in the present disclosure to more fully describe the state of the art to which this invention belongs.
[00188] Definitions
[00189] All publications, published patent documents, and patent applications cited in this specification are indicative of the level of the person (s) in the technique to which the invention belongs. All publications, published patent documents, and patent applications cited in this document are hereby incorporated by reference to the same extent as if each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference.
[00190] As used in this specification, including the claims, the singular forms "one," "one," and "o / a" include plural references, unless the content clearly dictates otherwise, and are used interchangeably with "at least one" and "one or more." That is, a reference to "an aptamer" includes mixtures of aptamers, reference to "nucleic acids" includes mixtures of nucleic acids, and the like.
[00191] As used in this document, the term "about" represents an insignificant change or variation in numerical values such that the basic function of the item to which the numerical value relationship is unchanged.
[00192] As used herein, the terms "comprises," "comprising," "includes," "including," "contains," "containing," and any variations thereof, are intended to cover a non-exclusive inclusion, as that a process, method, process by product, or composition in question that comprises, includes, or contains an element or list of elements not only includes those elements, but may include other elements not expressly listed or inherent in that process, method, process by product, or composition in question.
[00193] The term "aptamers" as used here indicates molecules of oligonucleic acid or peptide that are capable of binding a specific target entity. In general, aptamers are artificial oligonucleotides that can serve as antibody mimics because of their high affinity and selectivity for various target compounds ranging from small cells, such as drugs and dyes, to complex biological molecules such as enzymes, peptides, and proteins. Customized aptamers can be identified from random oligonucleotide libraries for specific target compounds by an in vitro iterative process called Systematic Evolution of Ligands by Exponential Amplification (SELEX). For examples of SELEX Processes see Pat. No. U.S. 5,270,163; 5,475,096; and 5,567,588, which are incorporated herein by reference in their entirety.
[00194] Aptamers can form a 3D structure serving as specific receptors for their antibody-like target compounds. Aptamers also have a number of advantages over antibodies such as tolerance to wide ranges of f and salt concentrations, heat stability, ease of synthesis, and cost efficiency. The specificity and affinity of aptamers is comparable, if not higher, to antibodies. Aptamers are also able to be reversibly denatured for the release of target compounds, which makes receptor aptamers especially useful for biosensing applications.
[00195] For example, aptamers may be composed of single-stranded oligonucleotides (ss) and / or be chemically synthesized peptides that have been designed through repeated rounds of in vitro selection, or equivalent techniques identifiable by a person skilled in the art, to connect to multiple targets.
[00196] An "aptamer" or "nucleic acid ligand" is a set of copies of a type or species of nucleic nucleic acid molecule that has a particular nucleotide sequence. An aptamer can include any suitable number of nucleotides. "Aptamers" refer to more than such a set of molecules. Different aptamers can have either the same number or a different number of nucleotides. Aptamers can be DNA or RNA and can be single-stranded, double-stranded, or contain double-stranded regions.
[00197] It should be understood that interactions of affinity between and aptamer and an analyte or target are a matter of degree. That is, the "specific binding affinity" of an aptamer to its target means that the aptamer binds to its target generally with a much higher degree of affinity than that aptamer can bind to other, non-target components in a mixture or sample.
[00198] As used in this document the term "amplification" or "amplify" means any process or combination of process steps that increases the quantity or number of copies of a molecule or class of molecules.
[00199] As used in this document, "pool" is a mixture of nucleic acids of differentiated sequence from which to select a desired ligand. The source of a pool can be from naturally occurring nucleic acids or fragments thereof, chemically synthesized nucleic acids, enzymatically synthesized nucleic acids or nucleic acids made by a combination of the prior art. Modified nucleotides, such as nucleotides with a detectable tag, reactive groups or other modifications, can be incorporated into the pool. In certain embodiments, a SELEX process and / or the improved SELEX method described in this document can be used to produce a pool. A pool can also comprise nucleic acids with one or more common structural moieties, such that the nucleic acids can be separated by structure, rather than by chemistry, size, or other method of separation. As used in this document, a pool is also sometimes referred to as a "library" or a "candidate or mixture of nucleic acid." For example, an "RNA Pool" refers to a candidate mixture made up of RNA.
[00200] As used herein, "nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides of any length, and such nucleotides may include deoxyribonucleotides, ribonucleotides, and / or analogous or chemically modified deoxyribonucleotides or ribonucleotides. The terms "polynucleotide," "oligonucleotide," and "nucleic acid" include single or double branched molecules, as well as triple helical molecules.
[00201] The term "sensor" as used in this document indicates a device that measures a physical quantity and converts it into a signal that can be read by an observer or an instrument. As is understood, a sensor is calibrated against known standards. Therefore, a sensor can be used to capture a target entity when exploring aptamer affinity for the target entity, and can be detected using techniques identifiable by a person skilled in the art by reading the present disclosure.
[00202] The term "detect" or "detection" as used in this document indicates the determination of the existence, presence or fact of a target or signal in a limited portion of space, including, but not limited to a sample, a reaction mixture , a molecular complex and a substrate including a platform and a matrix. Detection is "quantitative" when it refers to, relates to, or involves the measurement of quantity or quantity of the target or signal (also referred to as quantification), which includes but is not limited to any analysis designed to determine the quantities or proportions of the target or sign. Detection is "qualitative" when referring to, relating to, or involves identifying a quality or type of target or signal in terms of abundance relative to another target or signal, which is unquantified. An "optical detection" indicates detection carried out using visually detectable signals: spectrum or images from a target of interest or a test affixed to the target.
[00203] The term "labeling agent," "tag," or "detectable portion", or "detectable element" or "detectable component" refers to one or more reagents that can be used to detect a target / complex molecule. aptamer. A detectable portion or mark is capable of being detected directly or indirectly.
[00204] The terms "target," "target entity" and "analyte" can be used interchangeably in this document, and generally refer to a substance, compound or component whose presence or absence in a sample must be detected. Analytes include but are not limited to biomolecules and in particular biomarkers. The term "biomolecule" as used in this document indicates a compound of a substance or component associated with a biological environment, including, but not limited to, sugars, amino acids, peptides, proteins, oligonucleotides, polynucleotides, polypeptides, inorganic molecules, haptens, epitopes, biological cells , parts of biological cells, vitamins, hormones and the like. the term "biomarker" indicates a biomolecule that is associated with a specific state in a biological environment, including, but not limited to, a cell cycle phase, health and disease state. The presence, absence, reduction, overregulation of the biomarker is associated with and is indicative of a particular state. The terms "polypeptides," "peptides," and "proteins" are intended to encompass polymers of amino acids of any length, either linear or branched, which may or may not be modified naturally or by intervention, such as by glycation, lipidation, acetylation , phosphorylation, disulfide bond formation, conjugation, or other manipulation or modification.
[00205] The term "solid support" means any substrate having a surface to which molecules can be attached, directly or indirectly, through either covalent or non-covalent bonds. The substrate materials can be naturally occurring, synthetic, or a modification of a naturally occurring material. Solid support materials can include magnetic granules, or any other materials that are capable of having one or more functional groups, such as any of an amino, carboxyl, thiol, or hydroxyl functional group, for example , incorporated in his / her surface. The solid support can take any of a variety of configurations ranging from simple to complex and can have any of a number of shapes, including granules, discs, particles, plates, rods, strips, tubes , walls, and the like. The surface can be relatively flat (for example, a blade), spherical (for example, a granule), cylindrical (for example, a Co luna), or ridged.
[00206] The term "separation" means any process whereby one or more components of a mixture are separated from other components of the mixture. For example, bound aptamers of the target molecule can be separated from other nucleic acids that are not bound to target molecules and from non-target molecules. That is, a separation process or step allows for the separation of all nucleic acids in a candidate mixture into at least two pools based on their relative affinity and / or dissociation rate for the target molecule. The separation process can be achieved by several methods. For example, magnetic granules in which target molecules are conjugated can also be used to separate aptamers in a mixture. As another example, surface plasma resonance (SPR) technology can be used to separate nucleic acids in a mixture by immobilizing a target on a sensor chip and flowing the mixture over the chip, in which those nucleic acids having affinity to the target can be linked to the target, and the remaining nucleic acids can be washed away.
[00207] The term "sample" in this document refers to a mixture, gas or substance that may or may not include a target or analyte. Examples include but are not limited to biological samples, such as blood, sputum, respiration, urine, semen, saliva, amniotic fluid, meningeal fluid, glandular fluid, nipple aspirate, lymphatic fluid, bronchial aspirate, joint aspirate, synovial fluid, extract cell, cerebrospinal fluid, homogenized solid material from stool or tissue samples, bacterial culture, viral culture or experimentally separated fractions.
[00208] The term "non-target" refers to molecules in a sample that form a specific complex with an aptamer. It will be appreciated that a molecule that is a non-target for a first aptamer can be a target for a second aptamer. Likewise, a molecule that is a target for a first aptamer can be a non-target for a second aptamer.
[00209] Overview
[00210] The methods and devices described provide a system that has both the desired high sensitivity and specificity to be able to detect glycated proteins in a desired test environment and at sensitive concentrations.
[00211] In a particular aspect, the method includes determining the fraction of a specific glycated protein from the level of total serum proteins. Non-limiting examples of such proteins include: human hemoglobin, albumin (such as human serum albumin (HSA)) and IgM proteins.
[00212] Two common glycated proteins found in the body are hemoglobin A1c (HbA1c) and immunoglobulin M (IgM), (which is a basic antibody present in B cells). Both HbA1c and IgM have different half-lives in the body; for example, ~ 6-8 weeks for HbA1c and ~ 1 week for IgM. Therefore, quantification of these glycated proteins in the serum provides a retrospective judgment on glycemic control over both a shorter and longer term. The present method overcomes one of the primary deficiencies of other tests where only one type of whey protein could be detected; and therefore, any conformity assessment with regard to glucose control was limited to only a fixed period of time. It should also be noted that the present method overcomes other deficiencies that limit the test results, such as interferences of hemoglobinopathies, hemolysis and / or anemia.
[00213] It should be understood that, in other certain modalities of the devices / methods described in this document, one or more molecules or fragments thereof, such as other glycated proteins, can be accurately tested. Since the present method facilitates the detection and measurement of glycated blood proteins other than site-specific hemoglobin or HbA1c, the method is also useful for other technologies for the assessment of glycemic control.
[00214] In certain modalities, a target historical record of glycated proteins from a period of about a few days to about six weeks is possible depending on the specific glycated protein evaluated because different glycated proteins have different half-lives in the blood. In contrast, previous tests are limited to evaluating only a fixed period of time.
[00215] This method and the platform using this method are highly miniaturized and are useful on a portable device to provide analysis and detection in real time.
[00216] The method has the necessary sensitivity to be useful in medical analyte tests.
[00217] The method additionally allows the evaluation of different types of proteins, such as glycated hemoglobin and other forms of blood protein glycates.
[00218] In a method described here, surface plasma resonance is used with a highly functionalized aptamer sensing surface in order to provide an accurate, fast and relatively inexpensive method for assessing glycemic compliance by measuring the levels of certain proteins blood glucose levels.
[00219] Determination of Aptamers
[00220] The method described in this document is useful for detecting different types of aptamers. In one embodiment, in order to isolate and identify the specific oligonucleotides (aptamers) for hemoglobin, albumin and glycated IgM / non-glycated proteins, a systematic evolution of ligands, using the exponential enrichment protocol (SELEX) can be used.
[00221] While the standard SELEX protocol allows the screening of ligands that are specific to a particular protein of interest, an improved SELEX method is described here that identifies the secondary aptamers that are capable of detecting and capturing the two versions of protein (forms ie, glycated and non-glycated), as further explained in this document.
[00222] In an embodiment described in this document, the identification of the secondary aptamer is used to determine the percentage glycation that can be correlated with glucose levels for a given period of time.
[00223] Detection Platform: Protein Sensing Spectroscopy and Plasma Resonance (SPR)
[00224] For the detection of proteins, self-assembled monolayers (SAMs) are used to attach specific aptamers to gold SPR sensing surfaces. SPR spectroscopy itself is related to a phenomenon that occurs at the interface between conductors and dielectrics. At this interface, surface plasma may exist which are charge density oscillations in the electron structure. These surface plasmas are most commonly excited by light in the visible to the near infrared spectrum. This excitation can occur as surface plasmas propagating freely on a continuous propagating metal surface or as a localized effect using metal based nanoparticle structures. In an embodiment described in this document, a freely propagating surface plasma approach is used.
[00225] Briefly, valence electrons are disassociated from the atomic nucleus and in essence behave like an electron gas in the presence of an external electric field; it can be shown that the surface plasma is a wave coupled with a corresponding wave vector equal to: 2 ~~~~. 2 2π εmetafc sample
[00226]
(1) Y metal s' sample
[00227] where À is the wavelength of light, and εmetal and the sample are the relative permittivity constants of the medium, respectively. Therefore, the transfer of energy to the surface plasma will occur (that is, it will be excited) if the incident light has an electric field vector with a transverse polarization component with an energy close to ksp.
[00228] As shown in Fig. 7, the incident light vector has a component, kx, which can be represented by the equation:
[00229]
(two)
[00230] where n is the index of refraction of the incident medium and θi is the incident angle of the incoming light coming into contact with the metal surface. Surface plasma resonance is highly sensitive to local variations in the sample's refractive index due to the dependence of εmetal and ε Samples on the wavelength À of the incident light. Changes in the refractive index can be measured using a reflectance-based approach. The light reflected at the interface of two dielectric media, as shown in Fig. 7, generates an evanescent field with maximum intensity on the surface that will resonate with the free electrons (ie, surface plasma). This results in light energy, being transferred to the surface plasma with a corresponding reduction in the degree of reflected light. The angle at which this decrease occurs is commonly called the resonance angle.
[00231] A Kretschmann instrumentation configuration used to measure the resonance angle is illustrated in Fig. 8a. In this configuration, the light passes through a prism that is reflected in the metal-glass interface. An expanded version of the interaction at the metal-light interface is shown in Fig. 7. Any change in the refractive index at the metal / sample interface will result in a corresponding change or deviation in the resonance angle, as shown in Fig. 8b.
[00232] The present method overcomes the disadvantages of using SPR alone, which is often negatively affected by this lack of specificity problem. In addition, in the use of SPR alone, if the sensing analyte does not elicit at least a moderate change in the refractive index, the SPR is also negatively affected by issues of lack of sensitivity.
[00233] The present method overcomes these adverse issues by using the selective aptamers described in this document and by using self-assembled monolayers (SAMs) with SPR. The present method provides advantages such as high sensitivity and selectivity, cost effectiveness, chemical and thermal stability, easy synthesis and storage.
[00234] The aptamer-based sensing method described here is especially useful as a sensing element in biosensor applications. The nucleic acid nature of aptamers also makes immobilization and regeneration easier. In a SPR application modality, receptors (i.e., aptamers) are immobilized on solid substrates of various types to capture target analytes or molecules (see Fig. 9).
[00235] In addition, the method and apparatus described at this time overcome past problems with non-specific adsorption of proteins that had been associated with SAMs where such non-specific adsorption was detrimental to the sensor activity. In particular, the specific adsorption of matrices from complex samples, such as blood, urine or other clinical samples, was a major factor that limited sensitivity.
[00236] Other limiting factors were the biophysical and chemical properties of the adsorbed surface itself. In such SAMs, these properties needed to be suppressed in order to guarantee the specific affinity interaction with the analyte of interest. In addition, proteins adsorbed on a SAM surface partially lose their bioactivity due to conformational changes in the secondary structure and / or non-ideal orientation and distribution on the surface. Furthermore, the protocols for surface preparation and the conditions of mass transport significantly influence the protein adsorption response. Therefore, a quantitative comparison of data obtained in different laboratories was difficult and often inaccurate.
[00237] EXAMPLES
[00238] The present invention is further defined in the following examples, in which all parts and percentages are by weight and degrees are Celsius, unless otherwise stated. It should be understood that these examples, while indicating preferred embodiments of the invention, are given by way of illustration only. From the above discussion and these examples, one skilled in the art can determine the essential features of the present invention, and without departing from the spirit and scope of the same, can make several changes and modifications of the invention to adapt it for various uses and conditions. All publications, including patents and non-patent literature, referred to in this specification are expressly incorporated by reference. The following examples are designed to illustrate certain preferred embodiments of the invention and are not to be construed to limit the scope of the invention, as defined in the claims, unless otherwise specified.
[00239] Example 1
[00240] Materials
[00241] The identified aptamers were synthesized by integrated DNA technologies (Coralville, IA), including a 15bp aptamer (APT1): 5 '-NH2- (CH2) 6-GGTTGGTGTGGTTGG-3' [SEQ ID NO: 1], and a 34bp aptamer (APT2): 5 '-NH2- (CH2) 6- CTATCAGTCCGTGGTAGGGCAGGTTGGGGTGACT-3'. [SEQ ID NO: 2].
[00242] Magnetic tosyl activated granules (MBs) were purchased from Invitrogen (Carlsbad, CA). All other chemicals were purchased from Sigma-Aldrich (Carlsbad, CA) in the highest purity available. Aptamer solutions were prepared with 1 M of pH 8 phosphate buffer. The solution of 3-mercaptopropionic acid (MPA) was prepared in ethanol. Protein sample solutions were prepared using a 0.1M PBS buffer solution of pH 7.2 with 5 mM KCl and 1 mM MgCl2. The phosphoric acid (PPA) used was 100 mM. All other solutions were prepared in deionized water (DI).
[00243] Instrumentation
[00244] SPR measurements were performed using a commercial-grade SensiQ Discovery system (ICx Technologies, Arlington, VA) at 25 ° C. This sensor is based on a Kretschmann configuration, in which the light from a light-emitting diode (LED) ) integrated with a prism is first polarized and then internally reflected from a gold surface. The angle of light reflection and the relative intensity were measured with a photodiode matrix. When the sample solution was applied to the sensor surface, the minimum SPR profile (also known as the SPR angle) shifted as a function of the refractive index of the loaded sample, giving a real-time refractive index reading (although, by itself) the sensor is not specific / selective for any given target). The SPR response profile was recorded by the SensiQ software and then processed inside MATLAB ®.
[00245] Electrochemical impedance spectroscopy (EIS) measurements were performed using a Gamry Reference 600 potentiostat (Warminster, PA) in 5 mM Fe (CN) 6 3- / Fe (CN) 6 4- solution with KCl as a support electrolyte. All experiments were carried out at room temperature with the solutions purged with nitrogen gas for 15 minutes and the nitrogen blanket was kept during the experiments. The experiments were performed at 25 ° C. Impedance spectra were collected in the frequency range from 0.1 Hz to 100 kHz, with a potential amplitude of 5 mVrms at 10 points per decade. EIS results were analyzed when fitting the experimental impedance data for equivalent electrical circuit models. Parameters of the equivalent electrical circuits were obtained by fitting the impedance function to the Bode and Nyquist diagrams measured with a nonlinear complex minimum square program (CNLS) built on the Gamry EIS 300 electrochemical impedance spectroscope.
[00246] Aptamer binding capacity was determined as follows: 10 nmol of amine modified aptamer was coupled to 10 mg of washed magnetic granules (MBs) in a shaker incubator at 37 ° C for 18 hours. Unoccupied binding sites were blocked by bovine serum albumin (BSA). The MBs coupled aptamers were washed thoroughly, and then 10 nmol of thrombin was mixed with the MBs coupled aptamer for 2 hours in a shaker at room temperature. The control group was prepared by exactly the same method, except for the absence of aptamers. Total and unlimited proteins were measured with a SPR sensor functionalized by carboxyl provided by SensiQ.
[00247] To demonstrate the use of an aptamer-based SPR sensor to detect blood proteins, thrombin and antithrombin aptamer were chosen. Gold slides were prepared by physical vapor deposition (PVD) forming a 1 nm layer of titanium and a 50 nm layer of gold on previously cleaned microscope layer slides. These were then washed with large amounts of DI water and ethanol. They were dried in nitrogen gas before use.
[00248] To make the gold slides functional, they were immersed in the 10 mM MPA solution for 30 min and then washed with ethanol and DI water. After the slides were dried, they were then immersed in a solution of N-Hydroxysuccinimide (NHS) and N- (3-dimethylaminopropyl) -N-ethylcarbodiimide hydrochloride (EDC) (0.2M NHS, 0.05M EDC ) for 30 min. the slides were then washed with DI water and then immersed in the 5 μM aptamer solution. Finally, the slides were washed with PBS buffer to unclog non-specifically adsorbed proteins. Then, the slides were ready for measurement. In certain modalities, this two-stage surface functionalization process is applicable not only in SPR, but also Raman and fluorescence spectroscopy. The surface functionalization process is illustrated schematically in Fig. 1.
[00249] SensiQ base sensors uncoated (i.e., there is no gold) have been customized modified with gold developed based on SPR sensor surfaces. Specifically, gold substrates immobilized by freshly prepared aptamer were coupled to the peeled sensors with an index corresponding to optical oil. This was then followed by loading 100 μL of 1M ethanolamine (EA), with a flow rate of 20 μ l / min to block the unoccupied MPA sites activated by EDC / NHS, followed by an injection of 100 μL of 100 mM phosphoric acid (PPA) at 50 μL / min to remove non-specific binding. Running buffer was 0.1 M pH of 7.2 PBS. The sensor was first normalized with the buffer for 10 min and then the thrombin sample (25 μL) in concentrations of 5 nM, 25 nM, 50 nM, 250 nM 500 nM, 1000 nM, 2000 nM were loaded in 5 μ l / min. Samples with BSA were prepared with 400 nM BSA. All data was recorded at 290s, 300s and 310s after sample and average injection. Sensor regeneration was performed by injecting 100 μL PPA at 50 μL / min, followed by washing with the running buffer.
[00250] Results for example 1
[00251] EIS measurement
[00252] The successful immobilization of each functionalized layer was confirmed through EIS measurements. Fig. 2 shows the Nyquist diagram of impedance spectra at different electrodes. The bare gold electrode represented a very small circle at high frequencies, indicating a very low electron transfer resistance for the redox probe dissolved in the electrolytic solution (curve A). When the MPA was immobilized on the electrode and treated with EA and PPA, the electron transfer resistance (Ret) increased to 125 Q, (curve B). Then, when 5 μM of the APT1 aptamer was added and linked to the SAM, Ret increased to 600 Q (curve C). In this modality, the reactive sites on the gold electrode were blocked by EA (ethanolamine) to prevent the non-specific adsorption of aptamers on the gold surface, thus ensuring that the aptamers were only attached to the SAM. The Ret increase is caused by the electrostatic repulsion between the immobilized aptamer and the redox probe, causing a barrier to the interfacial electron transfer. These results show successful immobilization of the SAM layer to the gold surface and stable bonding of the aptamer to the SAM
[00253] Magnetic granules (MB) - based on maximum bonding capacity
[00254] After the MBs coupled aptamers were carefully washed, thrombin was added and the change in concentration was measured using a modified carboxyl SPR sensor. The refractive index is controlled only by changing the concentration of added thrombin. Other experimental variables such as temperature and protein degeneration had minor influences on the results of SPR and, therefore, were not considered to influence the results.
[00255] As shown in Fig. 3, the change in thrombin concentration was insignificant for the control group (less than 3%) which was not functionalized by the aptamer. This shows that the change in concentration in the two experimental groups was mainly due to the link between aptamer and thrombin. For the APT1 and APT2 groups, the mixture of MB functionalized aptamer and thrombin solution was allowed to react for 18 hours and the reaction was considered to be complete based on the MB manufacturer's specifications. Thus, the final concentration reflects the maximum mol / mol binding capacity of aptamer to thrombin.
[00256] The results showed that the proportion of APT1 binding (57.1%) has a better capacity than APT2 (55.2%). Both aptamers had more than the 50% mol / mol binding ratio of thrombin, indicating that they are good candidates for receptors for thrombin sensing applications. It should be understood that, in certain modalities, not all aptamers can bind to MBs and, therefore, the actual binding capacity of the binding aptamers towards target compound / s may be slightly greater.
The control group was comprised of MBs without functionalization of aptamers and all binding sites blocked by BSA. The groups containing aptamers were: APT1 - and APT2-MBs functionalized by the respective aptamers with unoccupied binding sites blocked by BSA. The error bars represent the standard deviation of the values determined from three samples.
[00258] SPR Results
[00259] Two different aptamers were immobilized on gold surfaces and the bonding performance of each was compared. For reference, samples of different thrombin concentrations (5 nM, 25 nM, 50 nM, 250 nM, 1000 nM, 2000 nM) were individually loaded onto a bare Au sensor, an APT1 sensor and an APT2 sensor, respectively. A secondary experiment was then performed using the same concentrations of thrombin; however, with a 400nM BSA confounding component added for each thrombin sample for comparison. As shown in the "Thrombin only" experiment in Fig. 4, the SPR displacements were very low for the bare Au sensor surface even for the relatively high concentrations of thrombin.
[00260] In contrast, for the sensors modified by aptamers the SPR displacements were significantly improved and the ideal detection range was 5 nM to 1000 nM (linear scale). The "Thrombin + 400 nM BSA" data (shown in Fig. 4) shows where a large 400nM BSA confusion concentration component was added for each thrombin sample concentration. Compared to the single thrombin group, the responses are almost identical, indicating that aptamer-modified sensors APT1 and APT2 are highly specific for single thrombin.
[00261] This is further illustrated in Fig. 5, which shows the change in SPR displacement to the concentration of thrombin 500 nM with and without, 400 nM BSA. Addition of BSA to the sample had a minimal effect on the SPR response for aptamer-modified sensors, indicating good sensor selectivity towards thrombin. This is in contrast to the bare Au sensor, which experienced a significant change between the thrombin samples with and without BSA. The modified APT1 sensor had a slightly larger change than the APT2 sensor for all concentrations of thrombin. The slope of the assembly line for APT1 is also slightly greater than APT2 in the linear response range (Fig. 6), once again demonstrating better sensitivity. These two aptamers bind to different thrombin sites, so the affinity for the target is different in both the interfacial and solution binding environments.
[00262] Antibody sensing
[00263] In the MBs Connection Tests, APT1 had a slightly higher connection capacity than APT2, which corresponds to the SPR results in terms of the sensitivity of the functionalized sensor. While not wishing to be bound by theory, it is believed that, in the present embodiment, this may be due to the lower aptamer having a greater probability of accessing target protein binding sites. Also, in certain embodiments, larger aptamers that have more complicated secondary structures may require extra spatial flexibility to form a bond with target compounds.
[00264] As example 1 in this document shows, the MPA layer has excellent gold coverage rate and is useful for antibody immunization for biosensitization purposes. These results also show that the amine-modified aptamer is readily immobilized in an MPA layer and the sensor performance was comparable to the antibody-based sensors.
[00265] Three sensing slides were prepared for each aptamer and also the control group. The sensor for sensor performance was consistent when using readily prepared samples, yielding relatively small errors for each measurement and an average of less than 2% standard deviation from the total signal (error bar shown in Fig. 5).
[00266] Adding BSA presented a slightly larger error and by reducing the flow rate and increasing the sample loading time, the error can be reduced although not considered significant enough to be considered. Most of the error is thought to be caused by the temperature variation; therefore, in some embodiments, placing the sensor at a controlled ambient temperature can help increase accuracy.
[00267] The sensor surface described here had an ideal dynamic range from 5 nM to 1000 nM, which is comparable to or greater than the largest dynamic ranges reported for aptamer-based sensing techniques. Since the range of thrombin concentration in human blood is reported to be within the low nanomolar low micromolar range, the method described at this point is suitable for quantitative detection of thrombin in vivo.
[00268] Sensor reversibility
[00269] To test the reversibility of the sensor, fixed sample concentrations were repeatedly loaded into the sensor 10 times. Sensor regeneration was done by PPA. The average SPR response with error bars for standard deviation using thrombin concentrations of 50 nM, 250 nM and 500 nM are shown in Fig. 6. All data were obtained from freshly prepared Sensing Slides. The SPR response in general decreased by about 0.5% for each load for the same sample concentration. All the Sensing Blades maintained more than 95% of the original SPR displacement response after loading 10th. Also, the second sample load generally had the biggest change in response compared to the following loads. With a longer PPA injection time, the sensor recovery rate can be increased, depending on the experimental requirements. The appearance of BSA decreased the sensitivity of the sensor (for example, in Fig. 6, the appearance of BSA reduced the slope slightly in the response curve), although this did not affect the reversibility of the sensor. Fig. 6 also demonstrates that the sensor maintained a linear response with and without the appearance of BSA in the sample range from 50 nM to 500 nM.
[00270] Example 2
[00271] Other Sensor Modalities
[00272] In another embodiment, the sensor may include a spacer layer of mixed length. In a non-limiting example, the mixed-length layer can be as 11-mercaptoundecanoic acid (MUA) combined with MPA, which can be used in certain embodiments to increase sensitivity and specificity.
[00273] In other embodiments, a mixed-length spacer can be included to help form and maintain the specific shape of the immobilized aptamers.
[00274] In another embodiment, a hydrophilic group such as ethylene oxide can be inserted at the -5c end of the aptamer in order to reduce nonspecific protein binding.
[00275] In certain modalities of the two-step immobilization method described here, spacing the aptamers can also be done by adjusting the density of SAM MPA, or by incubating ethanolamine and the aptamer in various molar ratios.
[00276] Detection of blood proteins
[00277] For the detection of different blood proteins, in order to find the aptamer that specifically and directly binds to the target protein of interest, a SELEX procedure can be used. Then, the aptamer developed can be amino-terminated and immobilized on the gold surface, using one of the methods described at this point in order to form a specific target sensor for almost any protein. As such, aptamers can be generated through SELEX for specific target compounds with advantages over antibodies.
[00278] The two-phase immobilization method described here is especially useful for immobilizing a SAM and amine-terminated aptamer on a gold SPR sensing surface. The SPR sensor currently described provides advantages such as low sample consumption, the lack of marking requirements, high sensitivity and fast response time. Additional advantages of the two-step immobilization method include demonstrable cost efficiency, good reversibility, uniform density and use as a robust and specific blood protein detection platform.
[00279] Example 3
[00280] Sensing of glycated albumin protein based and SPR aptamer
[00281] Glycosated human serum albumin (HSA) was both detected and quantified. The aptamer (thiolate, non-reduced), developed and used was 5'-SH- (CH2) 6- CCGAAACCAGACCACCCCACCAAGGCCACTCGGTCGAACCGCCAACA CTCACCCCA-3 ’[SEQ ID NO: 3].
[00282] Gold slides were prepared by physical vapor deposition (PVD) forming a 1 nm layer of titanium and a 50 nm layer of gold on previously cleaned microscope layer slides. The gold slides were then washed with large amounts of DI water and ethanol. The gold slides were dried in nitrogen gas before use.
[00283] The thiol aptamer was diluted by 1M pH 8 phosphate buffer and mixed with Cleland's REDUCTACRYLTM reagent in a cocktail shaker for 2 hours to reduce the double thiol binding in the aptamer sequence. Cysteine is an amino acid containing water-soluble thiol that can bind directly to the gold surface to form a self-assembled monolayer (SAM), which is then added to the aptamer solution to help space out of aptamers, fill in the spaces between aptamers and reduce non-specific surface absorption. The final concentration of the aptamer in this preliminary experiment was set to be 1 μM and the molar ratio aptamer: cysteine was 1:10. The gold slides were immersed in the aptamer / cysteine mixture solution at 37 ° C.
[00284] After the immobilization process, the gold slides were washed with 0.01 M of pH 7.4 PBS buffer. The functionalized surface was then coupled to the corresponding SPR sensor and HSA samples 1μg / mL of total proteins (that is, total = glycosated + non-glycosized) were prepared for the proportions (%) percent of certain glycosides (glycosized / total proteins): 2, 6, 10, 14 and 18%.
[00285] SPR responses were recorded for each respective sample. The results for the functionalized surfaces together with the bare Au surfaces are summarized in Fig. 10. The functionalized aptamer SPR surface responds directly to changes in the glycated protein content. It should be noted that the concentration of total proteins is constant at 1μg / mL between samples.
[00286] The non-functionalized surface (ie bare gold) exhibits an insignificant response, further illustrating the improved sensitivity on the functionalized surface. Although short in length (40-60nt), in certain modalities, aptamer sequences can differentiate targets based on size and charge, and affinity can be affected. While not wishing to be crushed by theory, the inventors here now believe that the 3D structure of aptamers can also play a role; non-limiting examples include the cytosine-cytosine overhang structure and the ACC (C), or (C) CCA themes.
[00287] Aptamers for glycosated and non-glycosized protein binding sites and for HbA1c, albumin and IgM
[00288] Aptamers were developed to attach self-assembled monolayers (SAMs). For certain modalities, the proteins hemoglobin, albumin and IgM are useful since the half-life of each one provides information covering historical records of short, intermediate and long term in glycemic control. A summary of the properties of some common blood proteins is provided in Table 1 below. Table 1: Related properties of blood proteins

[00289] G ication of the respective proteins can be performed by incubating (37 ° C) the respective proteins in PBS pH 7.4 containing 1m glucose and DTPA for two days. The glycosated proteins are then subjected to a dialysis process and can then be further enriched by affinity chromatography. In this step, the glycosated proteins can be separated from the respective non-glycosized forms using boric acid immobilized on polyacrylamide granules in the support column. Through this process, both bound and unbound fractions can be collected and further concentrated using filtration methods.
[00290] To achieve the isolation and identification of key oligonucleotides (aptamers) specific for hemoglobin, albumin and IgM in both glycosized and non-glycosized versions of proteins, an improved exponential systematic ligand evolution (SELEX) enrichment method can be used, as will be seen below and illustrated schematically in Fig. 12.
[00291] The improved SELEX method allows the screening of ligands that are particular to a protein of interest. The improved SELEX method can be conducted, generating a large library of randomized RNA sequences. This library normally contains 1014-1015 different RNA species that fold into different structures, depending on their particular sequence. This library is then incubated with the target protein of interest, and those RNAs contained in the library that bind to the proteins are then separated from those that are not. The retained RNAs are then amplified by RT-PCR and transcribed in vitro to generate a pool of RNAs that have been enriched for those that bind to the target of interest. This selection and amplification process can be repeated between 8 to 12 rounds until the RNA ligands with the highest affinity to the target protein are isolated. These aptamers are then cloned and sequenced.
[00292] Determination of Proportion of Glucose Protein to Total Protein
[00293] The percentage proportion of glycated protein for total protein measurement was related to the average blood glucose over a given time window.
[00294] Aptamers specific for the glycation sites of the target proteins can be generated. Also, aptamers that will bind to the glycated and non-glycated versions of the respective proteins have been generated. In one embodiment, glycosylated versions of hemoglobin, albumin and IgM proteins were used as the target in the SELEX protocol. The resulting reduced aptamer pool contains the non-glycation site and binding site specific aptamers. At this point and one (a) round (s) later, non-glycosized protein (ie normal protein) can then be introduced, in which aptamers present that recognize the glycation site do not bind and can be recovered by characterization . This method provides separate aptamers that are capable of binding both the glycoside / non-glycoside versions of the proteins, as well as those that are only specific for the glycoside versions only.
[00295] Optimization of functionalized surface Plasmon resonance based on self-assembled monolayer aptamer
[00296] The identified aptamer Then, it can be characterized initially for general performance, including binding properties, sensitivity, specificity and selectivity. Presented in Table 2 below are examples of target specifications based on performance levels. Table 2

[00297] In particular, a method for characterizing binding affinities is the use of a SPR method. Based on identified aptamer candidates, SPR is useful for generating the respective binding response curves. For example, certain devices (such as SensiQ, iCx Nomatics) are equipped with a dual microfluidic channel and have controllable flow rates. Tests can be performed using immobilization methods similar to those described in Fig. 1.
[00298] Modifications to facilitate immobilization
[00299] Also, in certain modalities, specific glycated and non-glycated aptamer candidates can be modified with an attachment 5'-NH2-C6 to facilitate immobilization on a gold SPR surface modified by -COOH. SPR measurements are used to characterize the respective affinity constants for aptamer candidates.
[00300] In addition to affinity tests, using aptamers immobilized by SPR chip, both specificity and selectivity can be evaluated. In such modalities, the respective aptamer chips were exposed for each target protein in the glycated and non-glycated forms. Cross-reactivity between the two forms for a given protein, as well as for different proteins (for example, albumin for an HbA1c aptamer chip) was thus determined. In certain preferred embodiments, the target cross-reactivity is desired to be less than about 6%. If it is determined that this criterion is not met, the SELEX protocol can be repeated with improved selection conditions (for example, by increasing the frequency of elimination rounds) in order to further improve cross-reactivity performance.
[00301] It is also understood that recognition of a good target can also be affected by the aptamer bonding process used for immobilization. In certain embodiments, the method may include the use of one or more alternative aptamer bonding methods. In certain embodiments, bonds can be made through 3'-amino, thiol, or other potential bonds.
[00302] It is also within the scope contemplated that such connections can be modified by, for example, controlling certain parameters, such as density and length. Thus, aptamer and bonding methods can be optimized to provide the maximum desired performance. In addition, the method described in this document for creating functionalized surfaces can be optimized to provide a desired level of surface uniformity, as well as to optimize the aptamer sensor response.
[00303] Self-assembling monolayer connections (SAM)
[00304] In addition to the connection methods described above, another method that can be used includes connection through a binary self-assembly monolayer (SAM) and reducing desorption process. Since packaging density of SAMs and lengths of SAMs affect the SPR signal, the density and length of binary SAMs can be controlled through a reducing desorption process.
[00305] In a given embodiment, synthesized dithiobis-N-succinimidyl propionate (DTSP) can be used with (1-mercapto-11-undecIl) tri (glycol ethyl) (PEG3) to sew a mixed SAM. PEG3, which is resistant to protein adsorption, can be used to prevent non-specific protein adsorption. In addition, the carboxylic group in DTSP will form a stable bond with the aptamer.
[00306] In a particular embodiment, a method of immobilizing SAM thiol using dithiobis-N-succinimidyl propionate (DTSP) was used in a phosphate buffer solution. DTSP is useful for SAMs due, at least in part, to its distinctive surface properties, such as hydrophilicity, wettability, chemical reactivity and an affinity for proteins such as hemoglobin and cytochrome c.
[00307] For the immobilization of SAM-binary, 3-mercaptopropionic acid (MPA) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) can be used. In certain embodiments, MPA is chosen because it has a lower redox potential than PEG3, which means that MPa can be easily eliminated by reducing desorption leaving PEG3 intact. DTSP is able to form a covalent bond with the amino groups of the aptamer while PEG3 does not, so that the aptamer will attach to the single DTSP.
[00308] Two-component thiol solutions can be prepared by mixing 1 mM of MPA and PEG3 ethanol solutions in various proportions, keeping the total concentration of the binary SAMs at 1 mM. The binary SAM of MPA and PEG3, whose proportions are 20:80, 50:50 and 80:20, can then be formed on a gold electrode by immersing the electrodes in the mixed thiol solution for 1 hour.
[00309] Referring now to the schematic illustration in Figs. 11a-11e, formation of a binary SAM and reducing desorption procedure is shown. First, the binary components of 3-mercaptopropionic acid (MPA) and PEG3 are adsorbed on the gold surface in an ethanol solution (Fig. 11a). The MPA reducing desorption of the gold electrode is performed with 0.5 M KOH solution. The MPA adsorbed on a separate phase MPA and PEG3 binary SAMs is selectively reduced by applying the -1.2 V potential for 30 min (Fig. 11b).
[00310] After reducing MPA desorption, the sample with the PEG3 layer is immersed in the 1 mM DTSP solution to form DTSP layers (Fig. 11c). Fig. 11d shows aptamer immobilization and Fig. 11e shows PEG3 removal aptamer.
[00311] The aptamer covalently couples to the DTSP SAM, exposing it to -COOH end groups. To form a covalent bond, aptamer (50 μ g / ml) in PBS is injected together with freshly prepared NHS and EDC. Aptamers having N-terminal amino acid groups and can be immobilized on the DTSP SAM through the formation of the CO-NH Amide bond. The proportion of DTSP and PEG3 will vary to control the packaging of the SAMs and as a result, the binding of the protein that gives the ideal SPR signal can then be achieved.
[00312] Measurement of surface coverage
[00313] Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) can be used to measure the surface coverage of immobilized SAMs and redox responses of the samples. The surface composition can be estimated from the peak areas of a cyclic voltamogram for the adsorbed thiols. Responses from the binary SAMs deposited on the modified electrode can be compared with those from the unmodified electrodes.
[00314] Cyclic voltamogram of the reducing desorption can be recorded in 0.5 mol dm -3 of phosphate buffer solution using an Ag-AgCl - saturated KCl electrode as the reference electrode and a platinum wire as the counter electrode. The CV curves of Aptamer SAMs + coated with gold electrode (Au + SAM + Aptamer) and reductive elimination SAMs and Aptamer on the gold electrode (Au + RD SAM + Aptamer), therefore, can be compared. CV curves can be recorded for a scan rate of 100 mV / s for reductive elimination. At each voltammogram, a low peak of SAM-reducing desorption is expected to appear at around 50mV.
[00315] Both the length and density of the SAM can be controlled to obtain the optimal SPR response. When the linker length is long, more aptamers can be immobilized, but the SPR dip can become broader, as the aptamers are further away from the surface. Likewise, when the linker density is high, more aptamers can be attached to the SAM, but then the SPR dip can become narrower and more difficult to detect. These aptamer-modified surfaces can be characterized by the methods used with the 5'-NH2-C6 / -COOH method.
[00316] Calibration and validation of functionalized SPR sensor surfaces developed
[00317] SPR sensing platforms for HbA1c, albumin and detection of glycated / non-glycated protein can initially be calibrated in tests using a saline buffer with known ratios of target proteins. Respective sample solutions can be prepared for fixed levels of total protein at levels of reasonable proportion compared to those observed in blood (see table 1).
[00318] For each sample, the proportion of glycosated protein to the total amount of protein can be varied over a desired range (for example, for HbA1c% levels between 6 to 15% correspond to mean glycemic index levels of 60 to 360 mg / dL, respectively). In such a modality, a range of 1 to 25% v / v would be appropriate. The SPR response in the respective samples can then be evaluated and a calibration model can be determined in relation to% glycation and the standard calibration error can be calculated. To additionally evaluate the accuracy of the developed SPR tests, independent samples (ie, those not used in the calibration) can be used to evaluate the test performance based on the respective calibration model (s). Both relative and absolute errors can be determined and compared to the ranges that would be required for the purpose of useful diagnosis.
[00319] Serum blood test
[00320] To evaluate performance in real blood serum, blood serum from a non-diabetic source can be used. Serum samples can be analyzed to determine respective fractions of total versus glycoside protein (for both protein targets) using a standard clinical test.
[00321] Using these values as references, individual samples can be doped with specific amounts of the respective glycosated protein (s). Test assessment similar to that used with saline tests can be repeated. It is understood that, due to high concentrations of certain target proteins in serum (for example, hemoglobin as shown in table 1), it may be desired to dilute the samples before performing the tests. In addition, other potentially confounding effects such as introductory variations in the sample composition, outside of the glycosated protein, can be tested as problems can arise due to the complex chemical composition of the serum.
[00322] Example 4
[00323] Improved SELEX method for identifying Aptamer targeted to glycosides and / or Non-glycosized protein sites
[00324] SELEX protocol has been improved in order to allow the identification of aptamers with an affinity for glycosated protein sites. This improved the SELEX protocol allowed for determining the percentage ratio of glycosated protein to total proteins.
[00325] Aptamers specific to the glycation sites of the target protein (s), in addition to those that will bind to both glycosized and non-glycosized versions of the respective proteins, have been generated. In order to generate such aptamers for a respective protein (for example, hemoglobin, albumin, IgM, etc.), in a first round of amplification, the SELEX protocol was applied to a glycosized version of the respective proteins. This first round of the SELEX protocol resulted in a reduced aptamer pool that contained both "non-glycation site specific" aptamers and "glycation site specific" aptamers.
[00326] A non-glycosized protein (ie normal protein) is introduced into the pool obtained in the first round of the SELEX amplification process. In at least a second round of amplification, the aptamers in the pool that bind to this non-glycosized protein are not eluted in this specific round of SELEX, and are therefore removed from the pool. This improved SELEX protocol improves the chance that aptamers specific to the glycosated sites will remain in the pool in progress. Such remaining aptamers can then be recovered for characterization in subsequent SELEX rounds as part of a standard SELEX process. It should be understood that, in other modalities, the uses of "glycosated" and "non-glycosized" proteins can be reversed; for example, where a "glycosated" protein is introduced into the pool obtained in the first round SELEX amplification process.
[00327] Determination of High Affinity Non-Glycoside and / or Glycoside Protein Aptamers
[00328] A protein molecule (eg, albumin) has several sites available for glycation. The glycation level generally refers to the percentage of a given protein concentration that has been glycated in relation to the total protein level, whereas the glycation rate refers to how many sites within a single protein molecule have a limit glucose or glucose derivatives. The 3D conformation and charge distribution are significantly different between a highly glycated and non-glycated protein molecule, but very similar between a slightly glycated protein molecule (i.e., single glycation point) and non-glycated protein molecule. Therefore, the development of a specific glycosized protein in a single site binding to aptamer that has a low affinity for the non-glycosized form is very challenging.
[00329] An example of the improved SELEX in vitro selection protocol is shown in Fig. 12, where a large pool of random DNA is initially mixed with a target of glycated protein immobilized in magnetic granules (MBs); that is, a primary or "MB target glycoside protein" complex.
[00330] Aptamers with high affinities with the glycated protein target will bind and form a "glycated aptamer target MB complex".
[00331] The "aptamer-target-protein-MB-glycosate" complex is separated from the initial DNA pool.
[00332] In a later step, the bound aptamers are eluted from the "target protein-MB-glycosated” complex (ie, the only or slightly glycosized form of the protein).
[00333] At this point, a control protein (ie, a non-glycosized form of the protein), which is coupled to a second set of MBs (a secondary or "target-protein-non-glycosized complex" ”") Is added to this first elution.
[00334] The "target protein-MB-non-glycosized" complex is used to remove "selective" aptamers in the first elution which also has an affinity for the non-glycosized protein form.
[00335] In a later step, "selective" aptamers are eluted from the "target protein-MB-non-glycosized" complex.
[00336] After removal of the complexes by "" MB-non-glycosated target protein "", the remaining "selective" aptamers are those aptamers that have a high affinity for single or target glycation sites.
[00337] At this point, a standard SELEX method can be used to amplify these remaining "selective" aptamers that have a very high affinity for the desired glycosated protein site.
[00338] Specifically, this improved SELEX method allows for the development of unique high-affinity glycation site aptamers that have a low affinity for the non-glycosized form of the protein. This improved SELEX method is also useful for generating aptamers that have an ability to distinguish analytes / molecules that have very similar chemical structures.
[00339] Examples of glycated and non-glycated Aptamers
[00340] Examples of useful aptamers are shown below, where XXX and YYY refer to any one or more of additional linking groups such as biotin, thiol, Amine, etc., which can be used to facilitate the development of a given monolayer self-assembly (SAM).
[00341] 5'-XXX-ATCCTTCATCCCATGGTTGCATATTGATTGCCGGTTCCTTAAAT- YYY-3 ’Aptamers [SEQ ID NO: 4]; and 5'-XXX- AGGGAAAGGTGTGGGTTAGGAGCTTGAAATCGAAAAGAGGGGC G-YYY-3 '[SEQ ID NO: 5].
[00342] Aptamers of non-Glycosated hemoglobin 5'-XXX- TTAGCGAGCTGCACACACAATGGACTCGTCATACCGTGCTGTTT- YYY-3 '[SEQ ID NO: 6]; and 5’-XXX- ATCTGCAGAATTCGCCCTTGCTGGTGCAGTACACACCCGGCGGG -YYY-3 ’[SEQ ID NO: 7].
[00343] Glycosate: Human Serum Albumin (HSA) Aptamers 5'-XXX-CTCACTCCATACTCACTTGCTGATTCGCCAACAACACACCCTTAA A CAGTC-YYY-3 '[SEQ ID NO: 8]; and 5’-XXX- CCGAAACCAGACCACCCCACCAAGGCCACTCGGTCGAACCGCC AACACT CAC -YYY-3 ’[SEQ ID NO: 9].
[00344] Non-glycosylated: Human Serum Albumin (HSA) Aptamers 5'-XXX-CTCTCCGGCCGCTGACCCAGTTTGGAGGGGGGAGGAGGCCGG GC-YYY-3 ’[SEQ ID NO: 10];
[00345] 5’-XXX- ACGGGCACTGGTTCCATCCGCATGAGATTGATGTGTCAACTTAT-YYY-3 ’[SEQ ID NO: 11]; 5’-XXX- CAATACCGATTGTTCTAAGGGAAAACGTGTAACTTTGGATCCTT- YYY-3 ’[SEQ ID NO: 12]; 5’-XXX- TAGCGACACACGTGGCCGCTGGTTGCCGGGCGCCACGGATCCT T-YYY-3 ’[SEQ ID NO: 13]; 5’-XXX- CCAGCTCGTAGTGGCGTCTTTTTTTCATTTGGTACTTATCGCAA - YYY-3 ’[SEQ ID NO: 14]; and 5’-XXX- AAATTTCATGTTCCCACACGTTCCATGCGCCCTCCTTCGAGTGC- YYY-3 ’[SEQ ID NO: 15].
[00346] Example 5:
[00347] Method of surface functionalization using SAMs for sensitivity and selectivity of optimization based on characteristics of the target
[00348] The sensitivity and selectivity of binary SAM training for mobilization of aptamer can be further improved. For example, to control bond spacing and distance between aptamer and SPR surface, two different types of self-assembling thiol molecules are deposited on the surface. A 1 mM ethanol solution of 11-mercaptoundecanoic acid (SH- (CH2) 5-COOH, MUA) and mercaptopropanol (SH- (CH2) 2-OH, MPL) are prepared separately. Each solution is mixed in the proportion of 1: 1 volume, maintaining the total concentration of the two components at 1 mM. A binary SAM of MUA and MPL is formed on a gold surface by immersing the gold surface in the mixed thiol solution for 1 hr. Then, the gold surface is subsequently washed with ethanol and DI water.
[00349] MPL density can be controlled by optimal signal transfer by applying an electrical potential to the gold surface in 0.5 M KOH solution (pH 13). The applied potential of -0.5 ~ 1.0 V for 30 min detaches a portion of MPL, resulting in a less dense layer of MPL that improves signal transfer. Then, the surface is immediately washed with DI water.
[00350] After the surface is dried, it is then treated with a solution of N-Hydroxysuccinimide (NHS) and - (3-dimethylaminopropyl) -N-ethylcarbodiimide-N (EDC) hydrochloride (0.2 M NHS, 0 , 05 M EDC) for 30 min to activate the MUA carboxyl group. The surface is then washed with DI water and then immersed in the 5 μM aptamer solution. Aptamers are covalently linked to activated MUA. Finally, the surface is rinsed with the PBS buffer.
[00351] This Surface functionalization method is applicable not only for SPR, but also to optimize the sensitivity and selectivity of other sensing modalities such as Raman and fluorescence spectroscopy. The method can be used to improve the performance of existing monitoring technologies.
[00352] Example 6
[00353] Methods to reduce effects of confusing substances present in samples
[00354] As part of the functionalization process, the MPL layer is hydrophilic in nature. This property can prevent non-specific adsorption of proteins to the surface. In another embodiment, the aptamer recognition element can be extended beyond the normal SPR sensing range (while still maintaining a desired sensitivity) through an extended bonding approach. In this embodiment, multiple bonds can be obtained through terminations, such as for thiols. Between the terminations, interfaces of gold nanoparticles can be made, exposing the surface with a solution of gold nanoparticles. This coupling of nanoparticles can allow the aptamer bonding response to be detected by the SPR sensor at separation distances beyond the normal SPR detection limit.
[00355] It should be noted that, as in non-aptamer locations, densely packed packaging connections outside the SPR range can be made which are null from coupling metal particles. Therefore, if non-specific adsorption of the protein or other confounding components are found in these locations, a corresponding SPR response will not occur, improving the selectivity performance for the sensor.
[00356] In another embodiment, a secondary physical vapor deposition (PVD) can be formed on subsequent MPA layers, followed by heat treatment to obtain a similar structure to extend the aptamers away from the SPR foundation surface, maintaining sensitivity across the layers. metal coupling connections.
[00357] Example 7
[00358] Detection of biomarker
[00359] The method and platform described here are also useful in the field of biomarker detection for disease assessment and diagnosis.
[00360] For example, for the proteins described in this document (eg, glycosated proteins), accurate detection can facilitate the treatment of diabetes and help to minimize the numerous associated health conditions, such as increased risk of cardiovascular disease, blindness , kidney failure and many others.
[00361] The method and platform in this document can be miniaturized in order to be easily integrated into a portable device, thus allowing the method and / or the platform to be used directly in medical offices, at home or in the field.
[00362] Measurements of glycated proteins (which are a measure of glycemic index compliance), instead of being only available during medical examinations through the analysis of externally premature, thus are readily available to the patient's donor or health care provider. a more easily assessable form. These more widely accessible measurements, in turn, would provide complementary information for self-monitoring blood glucose measurements to further help diabetics better manage their condition and mitigate potential long-term health complications.
[00363] Furthermore, if this information is available on a more frequent basis with expanded historical time windows, this can significantly impact the understanding of glucose regulation within and outside the diabetic community, which can lead to a better understanding of the control glycemic levels through the development, education and training of new and / or optimized therapeutic approaches to diabetes.
[00364] Example 8
[00365] Kits
[00366] The sensor described here can be provided in the form of kits of parts. Such kits include, but are not limited to, diagnostic kits, biomarker discovery kits, environmental testing kits, biohazard detection kits or biological weapon detection kits, and target detection kits in medical or analytical chemistry applications. As a non-limiting example title, the amino terminating aptamers can be included as a molecule alone or already attached to a substrate. Additional components may also be included and comprise microfluidic chips, reference standards and additional components identifiable by a person skilled in the art after reading this disclosure. In addition, the kit components can be provided, with appropriate instructions and other necessary reagents, in order to carry out the methods disclosed herein. In some embodiments, the kit may contain the compositions in separate containers. Instructions, for example, written or audio instructions, on paper or in electronic form, such as tapes or CD-ROMs, for carrying out the test, can also be included in the kit. The kit may also contain, depending on the particular method used, other reagents and packaged materials (such as wash buffers and the like).
[00367] While the invention has been described with reference to several and preferred modalities, it should be understood by those skilled in the art that various changes can be made and equivalents can be replaced by respective elements without departing from the essential scope of the invention. In addition, many modifications can be made to adapt a specific situation or material to the teachings of the invention without departing from its essential scope.
[00368] Therefore, it is intended that the invention is not limited to the specific modality disclosed herein contemplated by the realization of this invention, but that the invention will include all modalities falling within the scope of the claims.
[00369] Citation of any of the documents recited in this document is not intended as an admission that any of the above is relevant to the prior technique. All instructions regarding the date or representation of the contents of these documents are based on the information available to the applicant and do not constitute any admission as to the accuracy of the contents of those documents or dates.

权利要求:
Claims (12)
[0001]
1. Method of forming a sensor for one or more analytes, characterized by the fact that it binds one or more types of aptamers to a substrate with a self-organized monolayer bond (SAM), the SAM bond forming a functionalized surface in the substrate, where the SAM binding is a binary (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) and dithiobis-N-succinimidyl propionate (DTSP) binary SAM in the substrate, the sensor being formed by using the steps (a) adsorbing binary components of 3-mercaptopropionic acid (MPA) and (1-mercapto-11-undecyl) tri (ethylene glycol) (PEG3) on a substrate; (b) reductively desorbing MPA from the substrate of step (a); (c) forming a layer of dithiobis-N-succinimidyl propionate (DTSP) on the substrate of step (b); (d) immobilizing one or more types of aptamer on the substrate of step (c); and, (e) removing unbound aptamer from PEG3 on the substrate of step (d), thus leaving aptamer attached to the DTSP layer of the substrate.
[0002]
2. Method according to claim 1, characterized by the fact that one or more of the density and / or packaging length of the SAM bond affects a surface plasmon resonance (SPR) signal.
[0003]
3. Method according to claim 1, characterized by the fact that PEG3 prevents non-specific adsorption of proteins, and by the fact that a carboxyl moiety in DTSP forms a stable bond with the aptamer.
[0004]
4. Method according to claim 1, characterized by the fact that the binary components are absorbed on a gold surface substrate in an ethanol solution; the MPA is reductively desorbed from the substrate in a 0.5 M KOH solution, in which the adsorbed MPA is in a binary self-organized monolayer (SAM) separated by phase of MPA and PEG3 is selectively reduced by applying a potential of -1, 2 V for 30 min for the solution; and the substrate has the PEG3 layer in it and is immersed in a 1 mM DTSP solution to form the DTSP layer in it.
[0005]
5. Method according to claim 1 or 4, characterized by the fact that the analyte is a glycated form of a protein in blood.
[0006]
6. Method according to claim 1 or 4, characterized by the fact that it still determines a fraction of a specific glycated protein from a level of total serum protein.
[0007]
7. Method according to claim 1 or 4, characterized by the fact that the analyte is one or more of: human hemoglobin, albumin, including human serum albumin (HSA), immunoglobulin G (IgG), immunoglobulin M (IgM) , fibrinogen, and / or fragments thereof, the analyte being either glycated or non-glycated.
[0008]
8. Method according to claim 1 or 4, characterized by the fact that the analytes include a first analyte having a different half-life than a second analyte, and the method additionally includes quantifying the first and second analytes to provide a judgment retrospective with respect to the levels of the first and second analytes over one or more periods of time.
[0009]
9. Method according to claim 1 or 4, characterized by the fact that the analytes include a first analyte, a second analyte and a third analyte, each of the first, second and third analytes having different half-lives, the method additionally including: quantifying the first, second and third analytes to provide a retrospective judgment regarding the levels of the first, second and third analytes over one or more periods of time.
[0010]
10. Method according to claim 9, characterized by the fact that the first analyte is hemoglobin, the second analyte is IgM, and the third analyte is albumin; wherein one or more of the first analytes, the second analytes or the third analytes are present in a glycated or non-glycated form.
[0011]
11. Method according to claim 1 or 4, characterized by the fact that it is for use in monitoring past mean blood analyte levels, the method including: contacting a sensor formed by a method according to any of the previous claims with a blood sample; determining an amount of the glycated form of the analyte in the blood; and correlating an amount of the analyte present in the blood sample analyte in a glycated form from a control level to a time interval.
[0012]
12. Method according to claim 11, characterized in that the amount of the glycated form of the protein is determined using surface plasmon resonance (SPR).

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同族专利:

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公开号 | 公开日

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BR112014018831A2|2018-05-08|

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CN104379724A|2015-02-25|

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JP2017198698A|2017-11-02|

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US20150024957A1|2015-01-22|

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EP2809769A4|2016-01-13|

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KR20140143140A|2014-12-15|

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US10145844B2|2018-12-04|

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US20160299135A1|2016-10-13|

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CA2863551A1|2013-08-08|

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US9417234B2|2016-08-16|

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EP2809769B1|2018-09-05|

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HK1204648A1|2015-11-27|

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US9494582B2|2016-11-15|

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CN104379724B|2018-01-02|

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US20140335630A1|2014-11-13|

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KR102138106B1|2020-07-28|

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WO2013116527A1|2013-08-08|

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JP6502426B2|2019-04-17|

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EP2809769A1|2014-12-10|

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JP2015507199A|2015-03-05|

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JP6170508B2|2017-07-26|

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法律状态:
2018-05-22| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

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2018-12-18| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2021-02-09| B09A| Decision: intention to grant [chapter 9.1 patent gazette]|

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2021-04-27| B16A| Patent or certificate of addition of invention granted|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 31/01/2013, 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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US201261593054P| true| 2012-01-31|2012-01-31|

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US61/593,054|2012-01-31|

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PCT/US2013/024158|WO2013116527A1|2012-01-31|2013-01-31|Methods and devices for detection and measurement of analytes|

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