apparatus and methods for integrated sample preparation, reaction and detection

专利摘要:
apparatus and methods for integrated sample preparation, reaction and detection. these are cartridges for the isolation of a biological sample and biological tests downstream on the sample. in one embodiment, a sample of nucleic acid is isolated from a biological sample and the sample of nucleic acid is amplified, for example, through the polymerase chain reaction. the cartridges provided here can also be used to isolate samples of non-nucleic acid, for example, proteins, binding assays. instruments are also provided to carry out biological tests downstream and to detect the results of the tests.
公开号:BR112012021202B1
申请号:R112012021202
申请日:2011-02-23
公开日:2020-06-09
发明作者:A Johnson Bruce；richardson Bruce；Hilmer Bird Dylan；Ching Jesus；E Moravick Keith
申请人:Genturadx Usa Inc；Luminex Corp；
IPC主号:

专利说明:

Descriptive Report of the Invention Patent for APPARATUS FOR INTEGRATED SAMPLE PREPARATION, REACTION AND DETECTION.
CROSS REFERENCE TO RELATED ORDERS
[0001] The present request claims priority to the Request
U.S. Provisional serial number 61 / 307,281, entitled CASSETTE AND INSTRUMENT FOR INTEGRATED NUCLEIC ACID ISOLATION AND AMPLIFICATION, filed on February 23, 2010, hereby incorporated in its entirety for reference.
[0002] This request is a continuation-in part of the Request for
US patent with serial number 12 / 789,831, entitled CASSETTE FOR SAMPLE PREPARATION, filed on May 28, 2010, which consists of a continuation of US Application No. 11 / 582,651, entitled CASSETTE FOR SAMPLE PREPARATION, filed on October 17, 2006, which claims the benefit to US Provisional Application No. 60 / 728,569, entitled METHOD AND APPARATUS FOR ISOLATING NUCLEIC ACID, filed on October 19, 2005; U.S. Provisional Application No. 60 / 753,622, entitled CASSETTE FOR SAMPLE PREPARATION, filed December 22, 2005; and, U.S. Provisional Order No. 60 / 753,618, filed December 22, 2005, entitled CASSETTE FOR SAMPLE PREPARATION, each of which is incorporated herein in its entirety by reference. This application is also a part-continuation of the US Patent Application with serial number 12 / 821,446, entitled Instrument for Cassette for Sample Preparation, filed on June 23, 2010, which consists of a continuation of the US Patent Application with serial number 12 / 005,860, entitled Instrument for Cassette for Sample Preparation, filed on December 27, 2007, which claims priority to US Serial Provisional Order No. 60 / 882.150, entitled Instrument for Cassette for Sample Preparation, filed
Petition 870200011939, of 01/24/2020, p. 5/198
2/184 on December 27, 2006, each of which is incorporated herein in its entirety as a reference.
BACKGROUND
[0003] The modalities described here refer to an apparatus and methods for sample preparation, reaction and analysis. More particularly, the modalities described herein refer to a cartridge and instrument within which the isolation, amplification and analysis of the nucleic acid can be carried out in an integrated process.
[0004] Some known diagnostic procedures include isolation and analysis of nucleic acids, such as DNA or RNA. Generally, known methods for isolating nucleic acids within a sample include several steps, such as: (1) removing proteins from the sample by adding a protease (for example, Proteinase K);
(2) decompose the remaining bulky sample to expose the contained nucleic acids (also referred to as cell lysis); (3) precipitating the nucleic acid from the sample; and (4) washing and / or otherwise preparing the nucleic acid for further analysis.
[0005] In certain cases, amplification of the isolated nucleic acid (for example, replication of the nucleic acid to increase its volume) is desired for further analysis. The polymerase chain reaction (PCR) process is a known technique for amplifying portions of a nucleic acid molecule. During a PCR, an input sample containing the target DNA is mixed with reagents, which include the DNA polymerase (for example, Taq polymerase). The input sample can, for example, be the isolated nucleic acid sample produced by the procedure described above. The sample is then thermally cycled multiple times in an isolated chamber to complete the reaction. The temperatures and time periods of the thermal cycling are carefully controlled to ensure accurate results. After the DNA sequence is
Petition 870200011939, of 01/24/2020, p. 6/198
3/184 sufficiently amplified, it can be analyzed using various optical techniques.
[0006] Some systems known to perform nucleic acid isolation and amplification include different portions (for example, an isolation portion and an amplification portion) between which samples must be transferred during human intervention and / or processes that may compromise the integrity of the sample. Some systems known for performing nucleic acid isolation and amplification include complex control systems that require significant preparation and / or calibration by an experienced laboratory technician. Consequently, such known systems are not well suited for benchtop applications, high-volume diagnostic programs and / or use in a wide variety of laboratory settings.
[0007] In certain applications, multiple reaction stages may be desired, with one or more last stages requiring the addition of reagents between the reaction stages. For example, in a Reverse Transcription PCR, a reverse transcription reaction is generally completed before a PCR process is performed, with the PCR process requiring additional reagents. In some known systems, additional reagents required for a final reaction stage are generally transferred to the reaction chamber with human intervention and / or processes that may compromise the integrity of the sample. Consequently, such known processes can induce errors and contamination, and can also be expensive and / or difficult to implement for high volume applications.
[0008] Although some known systems include chambers that contain reagents, such chambers are generally integral to the cartridge and / or the reaction chamber. Consequently, when such systems
Petition 870200011939, of 01/24/2020, p. 7/198
4/184 and / or cartridges are used in connection with different reactions and / or assays, a completely different cartridge, cassette or other device is generally used to facilitate the use of the particular combination of reagents to conduct the desired reaction process. Therefore, such known systems and / or cartridges are generally not used interchangeably for different processes and / or reaction tests. [0009] Although some known systems include optical detection systems to detect one or more different analytes and / or targets within a test sample, such known systems generally include excitation light sources and / or emission light detectors in a portion of the device that is mobile in relation to the reaction chamber. For example, some known systems are configured to provide a beam of excitation light to the reaction chamber through a movable cover. Therefore, such known systems are susceptible to the detection variability that can result from the variation in the location of the excitation and / or detection light paths.
[00010] Therefore, there is a need for improved apparatus and methods for performing isolation and amplification of nucleic acids.
SUMMARY
[00011] Cartridges and instruments for sample isolation and downstream reactions are described. In some embodiments, an apparatus includes an isolation module, which can be used, for example, to isolate a nucleic acid sample, and a reaction module, which can be used, for example, to amplify the nucleic acid sample. The isolation module includes a first compartment and a second compartment. The first compartment defines a first chamber and a second chamber. At least the first chamber is configured to contain a sample, such as, for example, a sample containing a nucleic acid. THE
Petition 870200011939, of 01/24/2020, p. 8/198
5/184 second compartment includes a side wall and a piercable member that collectively defines a first volume configured to contain a first substance. The first substance can, for example, be a reagent, a wash buffer solution, a mineral oil and / or any other substance to be added to the sample. At least a portion of the second compartment is configured so that it is arranged within the first compartment in such a way that the first volume is in fluid communication with the first chamber when a portion of the piercable member is pierced. The reaction module defines a reaction chamber and a second volume configured to contain a second substance. The reaction module is configured so that it is coupled to the isolation module in such a way that the reaction chamber and the second volume are in fluid communication with the second chamber of the first compartment.
[00012] In some embodiments, a PCR is performed on the cartridge and / or instrument provided here. In an additional embodiment, the reaction is monitored in real time using a fluorescent probe, for example, a single-stranded DNA molecule comprising a minor groove ligand (MGB) and a fluorophore at the 5 'termination, and a non-fluorescent suppressor in its 3 'termination. In one mode, a PCR is performed on multiple targets, and the progress of reactions is monitored in real time. In some embodiments, the targets are genetic sequences of one or more of the following viruses: influenza A, influenza B, respiratory syncytial virus (RSV), herpes simplex virus 1 (HSV1) or herpes simplex virus 2 (HSV 2). In some modalities, before a PCR, a reverse transcription reaction is performed on the cartridge and / or instrument provided here. BRIEF DESCRIPTION OF THE DRAWINGS
[00013] Figures 1 and 2 are schematic illustrations of a
Petition 870200011939, of 01/24/2020, p. 9/198
6/184 cartridge according to one mode, in a first configuration and in a second configuration, respectively.
[00014] Figure 3 is a schematic illustration of a cartridge having a first module, a second module and a third module, according to an embodiment.
[00015] Figure 4 is a schematic illustration of a cartridge having a first module, a second module and a third module, according to an embodiment.
[00016] Figure 5 is a schematic illustration of a cartridge having a first module and a second module, according to an embodiment.
[00017] Figures 6 and 7 are schematic illustrations of a portion of a cartridge, according to one embodiment, in a first configuration and a second configuration, respectively.
[00018] Figure 8 is a side perspective view of a cartridge according to an embodiment.
[00019] Figure 9 is a top perspective view of the cartridge shown in Figure 8.
[00020] Figure 10 is a side cross-sectional view of the cartridge shown in Figure 8.
[00021] Figure 11 is an exploded side view of a portion of the cartridge shown in Figure 8.
[00022] Figures 12 and 13 are seen in perspective of a cartridge reagent module shown in Figure 8.
[00023] Figure 14 is a perspective view of a portion of the reagent module shown in Figures 12 and 13.
[00024] Figures 15 to 18 are seen in lateral cross-section of a cartridge isolation module shown in Figure 8 in a first configuration, a second configuration, a third
Petition 870200011939, of 01/24/2020, p. 10/198
7/184 configuration and a fourth configuration, respectively.
[00025] Figure 19 is a side cross-sectional view of the cartridge insulation module shown in Figure 8.
[00026] Figure 20 is a cross-sectional view of a portion of an isolation module valve assembly shown in Figure 19, taken along line X1-X1 in Figure 19.
[00027] Figure 21 is a perspective view of a portion of an isolation module valve assembly shown in Figure 19.
[00028] Figure 22 is a cross-sectional perspective view of the cartridge shown in Figure 8.
[00029] Figure 23 is a perspective view of a cartridge PCR module shown in Figure 8
[00030] Figure 24 is a cross-sectional perspective view of the cartridge shown in Figure 8.
[00031] Figure 25 is a side perspective view of a cartridge according to an embodiment.
[00032] Figure 26 is a side perspective view of a cartridge isolation module shown in Figure 25, in a first configuration.
[00033] Figure 27 is a side cross-sectional view of the insulation module shown in Figure 26, in the first configuration. [00034] Figure 28 is a side cross-sectional view of the insulation module shown in Figure 26, in a second configuration.
[00035] Figure 29 is a side perspective view of the cartridge PCR module shown in Figure 25, in a first configuration. [00036] Figure 30 is a side cross-sectional view of the PCR module shown in Figure 29, in the first configuration.
[00037] Figure 31 is a lateral cross-sectional view of the
Petition 870200011939, of 01/24/2020, p. 11/198
8/184 PCR module shown in Figure 29, in a second configuration. [00038] Figures 32 and 33 are seen in lateral cross-section of the cartridge shown in Figure 25, in a first configuration and in a second configuration, respectively.
[00039] Figure 34 is a schematic illustration of a portion of an instrument according to a modality.
[00040] Figure 35 is a schematic illustration in cross-section in perspective according to a modality.
[00041] Figure 36 is a perspective view of an instrument according to a modality.
[00042] Figure 37 is a perspective view of a first actuator assembly of the instrument shown in Figure 36.
[00043] Figure 38 is an exploded perspective view of the first actuator assembly shown in Figure 37.
[00044] Figure 39 is a rear perspective view of the first actuator assembly shown in Figure 37.
[00045] Figure 40 is a perspective view of a portion of the first actuator assembly shown in Figure 37.
[00046] Figure 41 is a top perspective view of an instrument transfer actuator assembly shown in Figure 36.
[00047] Figure 42 is a bottom perspective view of the transfer actuator assembly shown in Figure 41.
[00048] Figure 43 is a rear perspective view of the transfer actuator assembly shown in Figure 41.
[00049] Figure 44 is a perspective view of a portion of the transfer actuator assembly shown in Figure 41.
[00050] Figure 45 is a perspective view of a portion of the transfer actuator assembly shown in Figure 41.
[00051] Figure 46 is a perspective view of an axis of
Petition 870200011939, of 01/24/2020, p. 12/198
9/184 helical transmission of the transfer actuator assembly shown in Figure 41.
[00052] Figure 47 is a top perspective view of a second actuator assembly of the instrument shown in Figure 36.
[00053] Figure 48 is a side perspective view of the second actuator assembly shown in Figure 47.
[00054] Figures 49 to 51 are seen in perspective of the portions of the second actuator assembly shown in Figure 47.
[00055] Figure 52 is a side perspective view of an instrument heater assembly shown in Figure 36.
[00056] Figure 53 is a perspective view of a heater assembly receiving block shown in Figure 52.
[00057] Figures 54 and 55 are a front view and a top view, respectively, of the heater assembly receiving block shown in Figure 52.
[00058] Figure 56 is a cross-sectional view of the heater assembly receiving block shown in Figure 52 taken along line X2-X2 shown in Figure 54.
[00059] Figure 57 is a perspective view of a heater assembly clamp shown in Figure 52.
[00060] Figure 58 is a perspective view of a heater assembly mounting block shown in Figure 52.
[00061] Figure 59 is a perspective view of a heatsink assembly heatsink shown in Figure 52.
[00062] Figure 60 is a perspective view of a heater assembly mounting plate shown in Figure 52.
[00063] Figures 61 and 62 are seen in perspective of a first insulating member and a second insulating member, respectively, of the heater assembly shown in Figure 52.
[00064] Figure 63 is a perspective view of a block of
Petition 870200011939, of 01/24/2020, p. 13/198
10/184 heating the heater assembly shown in Figure 52. [00065] Figures 64 and 66 are a front perspective view and a rear perspective view, respectively, of an optical assembly of the instrument shown in Figure 36.
[00066] Figure 65 is an exploded perspective view of the optical assembly shown in Figures 64 and 66.
[00067] Figure 67 is a perspective view of an optical assembly mounting member shown in Figures 64 and 66.
[00068] Figure 68 is a perspective view of a shoe of the optical assembly shown in Figures 64 and 66.
[00069] Figure 69 is a perspective view of a movable needle of the optical assembly shown in Figures 64 and 66.
[00070] Figure 70 is a perspective view of a portion of a fiber optic module of the optical assembly shown in Figures 64 and 66.
[00071] Figures 71 to 73 are block diagrams of the electronic control system of the instrument shown in Figure 36.
[00072] Figures 74 to 76 are schematic illustrations of an optical assembly according to a modality, in a first configuration, in a second configuration and in a third configuration, respectively.
[00073] Figures 77 to 80 are flowcharts of methods for detecting target analytes in a sample containing a nucleic acid according to the modalities.
[00074] Figure 81 is a molecular signature produced using systems and methods according to a modality.
[00075] Figure 82 is a perspective view in cross section of a portion of an insulation module according to a modality that is configured to receive acoustic energy.
[00076] Figure 83 is a cross-sectional perspective view
Petition 870200011939, of 01/24/2020, p. 14/198
11/184 of a portion of an insulation module according to a modality that is configured to receive acoustic energy.
[00077] Figure 84 is a perspective cross-sectional view of a portion of the cartridge shown in Figure 26 and an acoustic transducer.
[00078] Figure 85 is a perspective view of a cartridge according to an embodiment.
[00079] Figure 86 is a perspective view of the cartridge shown in Figure 85 without the liner.
[00080] Figure 87 is a perspective view of a cartridge PCR module shown in Figure 85.
[00081] Figure 88 is a cross-sectional view of a PCR module according to an embodiment.
[00082] Figure 89 is a perspective view of a cartridge according to an embodiment.
[00083] Figure 90 is a perspective view of a cartridge according to an embodiment.
[00084] Figure 91 is a perspective view of a cartridge according to an embodiment.
[00085] Figure 92 is a perspective view of a cartridge according to an embodiment.
[00086] Figure 93 is an exploded perspective view of the cartridge shown in Figure 92.
[00087] Figure 94 is a perspective view of a cartridge having multiple PCR bottles according to one embodiment. DETAILED DESCRIPTION
[00088] Cartridges and instruments for isolating and reacting samples are described. In some embodiments, an apparatus includes an isolation module, which can be used, for example, to isolate a sample of nucleic acid or a sample of
Petition 870200011939, of 01/24/2020, p. 15/198
12/184 analyte, and a reaction module, which can be used, for example, to amplify the nucleic acid sample, or to test the binding of the analyte to other compounds. The isolation module includes a first compartment and a second compartment. The first compartment defines a first chamber and a second chamber. At least the first chamber is configured to contain a sample, such as, for example, a sample containing a nucleic acid. The second compartment includes a side wall and a piercable member that collectively defines a first volume configured to contain a first substance. The first substance can, for example, be a reagent, a wash buffer solution, a mineral oil and / or any other substance to be added to the sample. At least a portion of the second compartment is configured so that it is arranged within the first compartment in such a way that the first volume is in fluid communication with the first chamber when a portion of the piercable member is pierced. The reaction module defines a reaction chamber and a second volume configured to contain a second substance. The reaction module is configured so that it is coupled to the isolation module in such a way that the reaction chamber and the second volume are in fluid communication with the second chamber of the first compartment.
[00089] In some embodiments, an apparatus includes a first module, a second module, and a third module. The first module defines a first chamber and a second chamber. At least the first chamber is configured to contain a sample. The second module defines a first volume configured to contain a first substance. The first substance can, for example, be a reagent, a wash buffer solution, a mineral oil and / or any other substance to be added to the sample. A portion of the
Petition 870200011939, of 01/24/2020, p. 16/198
13/184 the second module is configured so that it is arranged inside the first chamber of the first module when the second module is coupled to the first module in such a way that the first volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber and a second volume. The second volume is configured to contain a second substance. A portion of the third module is disposed within the second chamber of the first module when the third module is coupled to the first module in such a way that the reaction chamber and the second volume are in fluid communication with the second chamber of the first module.
[00090] In some embodiments, an apparatus includes a first module, a second module, and a third module. The first module defines a first chamber and a second chamber. The first module includes a first transfer mechanism configured to transfer a sample between the first chamber and the second chamber while maintaining fluidic isolation between the first chamber and the second chamber. The second module defines a volume configured to contain a substance, such as, for example, a reagent, or the like. A portion of the second module is configured so that it is disposed within the first chamber of the first module when the second module is coupled to the first module in such a way that the volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber. The third module is configured so that it is coupled to the first module in such a way that the reaction chamber is in fluid communication with the second chamber. The third module includes a second transfer mechanism configured to transfer a portion of the sample between the second chamber and the reaction chamber.
Petition 870200011939, of 01/24/2020, p. 17/198
14/184
[00091] In some embodiments, an apparatus includes a first module and a second module. The first module includes a reaction flask, a substrate and a first transfer mechanism. The reaction flask defines a reaction chamber, and can, for example, be a PCR flask. The first transfer mechanism includes a plunger movably arranged within a compartment such that the compartment and the plunger define a first volume containing a first substance. The plunger can be moved between a first position and a second position. The first substance can, for example, be a reagent, mineral oil or the like. The substrate defines at least a portion of a first flow path and a second flow path. The first flow path is configured so that it is in fluid communication with the reaction chamber, with the first volume and with an isolation chamber of an isolation module. The second flow path configured so that it is in fluid communication with the isolation chamber. A portion of the plunger is arranged within the first flow path such that the first volume is fluidly isolated from the reaction chamber when the plunger is in the first position. The plunger portion is arranged separate from the first flow path in such a way that the first volume is in fluid communication with the reaction chamber when the plunger is in the second position. The plunger is configured to produce a vacuum inside the reaction chamber to transfer a sample from the isolation chamber to the reaction chamber when the plunger is moved from the first position to the second position. The second module includes a second transfer mechanism, and defines a second volume configured to contain a second substance. The second module is configured to be coupled to the first module in such a way that the second volume can be selectively placed in fluid communication with
Petition 870200011939, of 01/24/2020, p. 18/198
15/184 the isolation chamber through the second flow path. The second transfer mechanism is configured to transfer the second substance from the second volume to the isolation chamber when the second transfer mechanism is activated.
[00092] In some embodiments, an instrument to manipulate and / or drive a cartridge containing a sample may include a block, a first optical member, a second optical member and an optical assembly. The block defines a reaction volume configured to receive at least a portion of a reaction vessel. The block may include and / or be attached to a mechanism to facilitate, produce, support and / or promote a reaction associated with the sample. In some embodiments, for example, the block can be coupled to a heating element configured to thermally cycle the sample. The first optical member is arranged at least partially within the block such that the first optical member is in optical communication with the reaction volume. The second optical member is arranged at least partially within the block such that the second optical member is in optical communication with the reaction volume. The optical assembly includes an excitation module configured to produce a plurality of excitation light beams and a detection module configured to receive a plurality of emission light beams. The optical assembly is coupled to the first optical member and the second optical member in such a way that each between the plurality of excitation light beams can be transported to the reaction volume and each between the plurality of emission light beams can be received from the reaction volume.
[00093] In some embodiments, an instrument for manipulating and / or activating a cartridge includes a chassis, an acoustic transducer and an actuation mechanism. The chassis is configured to contain a cartridge
Petition 870200011939, of 01/24/2020, p. 19/198
16/184 having a compartment that defines a volume. The volume can receive a portion of a sample, such as, for example, a sample containing nucleic acids. The acoustic transducer is configured to produce acoustic energy. The actuation mechanism is configured to move at least a portion of the acoustic transducer in contact with a portion of the cartridge. The actuation mechanism is further configured to adjust a force exerted by the acoustic transducer portion against the cartridge portion.
[00094] The term light beam is used in this document to describe any projection of electromagnetic energy, whether in the visible spectrum or not. For example, a beam of light may include a collimated projection of electromagnetic radiation into the visible spectrum that is produced by a laser, a light-emitting diode (LED), a flash lamp, or the like. A beam of light can be continuous within a desired period of time or discontinuous (for example, pulsed or intermittent) within the desired period of time. In certain situations, a beam of light may include and / or be associated with information (that is, the beam of light may be an optical signal), such as an amount of an analyte present in a sample.
[00095] The term parallel is used in this document to describe a relationship between two geometric constructions (for example, two lines, two planes, a line and a plane, or the like) in which two geometric constructions do not substantially intersect since they are extend substantially to infinity. For example, depending on the usage in question, a first line is said to be parallel to a second line when the first line and the second line do not cross as they extend to infinity. Similarly, when a flat surface (i.e., a two-dimensional surface) is said to be parallel to a line, each point along the line is separated from the portion closest to the surface by a substantially equal distance. Two
Petition 870200011939, of 01/24/2020, p. 20/198
17/184 geometric constructions are described as being parallel or substantially parallel to each other when they are nominally parallel to each other, such as, for example, when they are parallel to each other within a tolerance. These tolerances may include, for example, manufacturing tolerances, measurement tolerances or the like.
[00096] The term normal is used in this document to describe a relationship between two geometric constructions (for example, two lines, two planes, a line and a plane, or the like) where the two geometric constructions intersect at an angle of approximately 90 degrees within at least one plane. For example, depending on the usage in question, a first line is said to be normal to a plane when the line and plane intersect at an angle of approximately 90 degrees within a plane. Two geometric constructions will be described in this document as being normal or substantially normal to each other when they are nominally normal to each other, such as, for example, when they are normal to each other within a tolerance. These tolerances may include, for example, manufacturing tolerances, measurement tolerances or the like
[00097] Figures 1 and 2 are schematic illustrations of a cartridge 1001 according to a modality, in a first configuration and in a second configuration, respectively, which includes an 1100 isolation module and a 1200 reaction module. insulation 1100 and reaction module 1200 are coupled together in such a way that insulation module 1100 and reaction module 1200 can be placed in fluid communication with each other. As described in this document, insulation module 1100 and reaction module 1200 can be coupled together in a suitable manner. In some modalities, for example, the module
Petition 870200011939, of 01/24/2020, p. 21/198
18/184 of insulation 1100 and reaction module 1200 can be constructed and coupled separately to form cartridge 1001. This arrangement between insulation module 1100 and reaction module 1200 allows several different configurations of insulation module 1100 to be used with several different configurations of reaction module 1200. Different configurations of isolation module 1100 and / or reaction module 1200 may include different reagents and / or different structures within isolation module 1100 and / or reaction module 1200.
[00098] The cartridge 1001 can be manipulated and / or activated by any of the instruments described in this document. In some embodiments, cartridge 1001 can be used to perform sample preparation, nucleic acid isolation and / or polymerase chain reactions (PCRs) in the sample. In such embodiments, the isolation module 1110 can isolate a target nucleic acid from the sample contained therein. The isolated nucleic acid can then be amplified (for example, using PCR) in reaction module 1200, as described below. The modular arrangement of cartridge 1001 allows any number of different reaction modules 1200 that contain, for example, different reagents and / or that are configured to amplify a different type of sample, to be used with an 1100 isolation module, and vice versa.
[00099] Isolation module 1100 includes a first compartment 1110 and a second compartment 1160. As described in more detail in this document, the second compartment 1160 is coupled to the first compartment 1110 in such a way that the second compartment 1160 can be placed in fluid communication with the first compartment 1110. In some embodiments, the first compartment 1110 and the second compartment 1160 are modularly arranged, in such a way that
Petition 870200011939, of 01/24/2020, p. 22/198
19/184 different configurations of the first compartment 1110 and the second compartment 1160 can be used with each other. Different configurations of the first compartment 1110 and the second compartment 1160 can include, for example, different chemicals, reagents, samples and / or different internal structures. [000100] The first compartment 1110 defines a first chamber 1114 and a second chamber 1190. At least one between the first chamber 1114 and the second chamber 1190 may contain an S sample. The S sample can be any biological sample, for example, a biological sample containing one or more target nucleic acids, such as, for example, urine, blood, other materials containing tissue samples or the like. Sample S can be introduced into the first chamber 1114 or the second chamber 1190 by any suitable mechanism, including, for example, pipetting or injecting sample S into the first chamber 1114 and / or the second chamber 1190 through a opening or a piercing member in the first compartment 1110 (not shown). Although the first chamber 1114 is shown to be in fluid communication with the second chamber 1190, in other embodiments, the first chamber 1114 can be selectively placed in fluid communication with the second chamber 1190. Otherwise, in some embodiments, the first compartment 1110 can include any suitable mechanism, such as a valve (not shown in Figures 1 and 2), that can selectively place the first chamber 1114 in fluid communication with the second chamber 1190. Furthermore, in other embodiments, the first compartment 1110 may have any suitable flow control and / or transfer mechanism (not shown in Figures 1 and 2) to facilitate the transfer and / or transfer control of a substance between the first chamber 1114 and the second chamber 1190, including, for example, valves,
Petition 870200011939, of 01/24/2020, p. 23/198
20/184 capillary flow control, pumps, or the like. In still other embodiments, the first chamber 1114 can be fluidly isolated from the second chamber 1190.
[000101] The second compartment 1160 includes a side wall 1147 and a piercing member 1170. The side wall 1147 and the piercing member 1170 define a first volume 1163. The first volume 1163 can be completely or partially filled with a substance R1. The R1 substance can be any biological or chemical substance such as, for example, a mineral oil, a wash buffer, a flourishing dye, a reagent, or the like. As shown in Figures 1 and 2, the second compartment 1160 portion is disposed within the first compartment 1110 such that when the piercing member 1170 is punctured, broken, cut and / or broken, the first volume 1163 is in fluid communication with the first chamber 1114 as shown in Figure 2. As similarly stated, the insulation module 1110 can be moved from a first configuration (Figure 1) to a second configuration (Figure 2) when the piercable member 1170 is drilled. When the first volume 1163 is in fluid communication with the first chamber 1114 as shown in Figure 2 (that is, when the isolation module is in the second configuration), substance R1 can be transferred from the first volume 1163 in the first chamber 1114 Substance R1 can be transferred from the first volume 1163 in the first chamber 1114 through any suitable mechanism, for example, through gravitational forces, capillary forces or some other actuation mechanism (not shown in Figures 1 and 2) acting on the first volume 1163.
[000102] The piercable member 1170 can be constructed from a material that is substantially impermeable and / or chemically
Petition 870200011939, of 01/24/2020, p. 24/198
21/184 inert to substance R1. In this way, substance R1 can be stored within the first volume 1163 for an extended period of time without compromising the ability to use the second compartment 1160 and any desired application, such as any of the modalities described in this document. In addition, in some embodiments, the piercable member 1170 can be constructed from a material having certain temperature characteristics such that the desired properties and integrity of the piercable member 1170 are maintained above a certain temperature range. For example, in some embodiments, it may be desirable to store the second compartment 1160 containing substance R1 in a refrigerated condition, or it may be desirable to manufacture the second compartment 1160 by thermally laminating the piercing member 1170. In such embodiments, the piercing member 1170 it can be selected in such a way that the cooling condition and / or the thermal lamination condition do not substantially degrade the desired properties and the integrity of the piercable member 1170 for the intended application. In some embodiments, the piercable member 1170 can be constructed from a polymer film, such as any form of polypropylene. In some embodiments, the 1170 drillable member can be constructed from biaxially oriented polypropylene (BOP).
[000103] Although Figures 1 and 2 show at least a portion of the second compartment 1160 being disposed within the first compartment 1110, in other embodiments, the first compartment 1110 and the second compartment 1160 can be coupled together having at least a portion of the first compartment 1110 disposed within the second compartment 1160, or having the first compartment 1110 and the second compartment 1160 coupled together through an interface or fitting without being disposed within
Petition 870200011939, of 01/24/2020, p. 25/198
22/184 of these. The second compartment 1160 can be coupled to the first compartment 1110 by any suitable mechanism, such as, for example, by an adhesive joint; a welded joint; a pressure fitting (for example, an arrangement in which fitting protrusions arranged in the first compartment are received and / or retained by the corresponding openings defined by the compartment, or vice versa); an interference fit, in which two parts that are fixed by friction after being pressed together (for example, such as a Luer-Slip®); a threaded coupling, which includes a removable coupling, such as Luer-Lok®; or a flange connection. The coupling between the first compartment 1110 and the second compartment 1160 can be fluid impermeable, such that when the piercing member 1170 is broken or ruptured as shown in Figure 2, the transfer of fluids between the first volume 1163 and the first chamber 1114 does not result in leakage and / or contamination. The fluid impermeable coupling between the first compartment 1110 and the second compartment 1160 can be obtained through the use of a tapered fit of fitting components, o-rings, gaskets or the like.
[000104] Reaction module 1200 defines a reaction chamber 1262 and a second volume 1213. The second volume 1213 contains a substance R2. Substance R2 can be any biological or chemical substance, such as a mineral oil, a wash buffer, a reagent, or the like, that participates in or otherwise supports a reaction within the 1262 reaction chamber and / or any other cartridge portion 1001. Reaction module 1200 is coupled to isolation module 1100 in such a way that reaction chamber 1262 and second volume 1213 can be placed in fluid communication with the second chamber 1190 of isolation module 1100. The module reaction 1200 can be coupled to the 1100 isolation module via
Petition 870200011939, of 01/24/2020, p. 26/198
23/184 of any suitable mechanism, such as, for example, by an adhesive joint; a welded joint; a pressure fitting (for example, an arrangement in which the fitting protrusions in the first compartment are received inside and / or retained by the corresponding openings defined by the second compartment or vice versa); an interference fit, in which two parts are rubbed together after being pressed together (for example, such as a Luer-Slip®); a threaded coupling, which includes a removable coupling, such as Luer-Lok®; or a flange connection. The coupling between the first compartment 1110 and the reaction module 1200 may be impermeable to fluids in such a way that the transfer of fluids between the isolation module 1100 and the reaction module 1200 does not result in leakage and / or contamination. The fluid impermeable coupling between reaction module 1200 and isolation module 1100 can be achieved using a tapered fit of plug-in components, O-rings, gaskets or the like. In some embodiments, the coupling between isolation module 1100 and reaction module 1200 is removable.
[000105] This provision allows substances to be transferred from reaction chamber 1262 and / or the second volume 1213 to the second chamber 1190, or vice versa. For example, in use, samples, reagents, and / or other support materials, such as one or more between sample S, substance R1 or substance R2 can be transferred into or out of reaction chamber 1262 in connection with the desired reaction. The transfer of fluids between the second chamber 1190, the reaction chamber 1262 and / or the second volume 1213 can be carried out through gravitational forces, capillary forces, hydraulic pressure or the like. In some embodiments, hydraulic pressure can be applied through a piston pump, a deflector pump or any other suitable
Petition 870200011939, of 01/24/2020, p. 27/198
24/184 transfer. In some embodiments, such a fluid transfer mechanism may be external to cartridge 1001 or internal to cartridge 1001 (for example, arranged at least partially within insulation module 1100 and / or reaction module 1200). [000106] In some embodiments, substance R1 and sample S, or a portion thereof, can be transferred from the first volume 1163 and the first chamber 1114, through the second chamber 1190, and up to the reaction chamber 1262 in connection with a reverse transcription process, creating a complementary single-stranded deoxyribonucleic acid (cDNA) from a ribonucleic acid (RNA) model using a reverse transcriptase enzyme. After the reverse transcription process has ended, substance R2 can be transferred from second volume 1213 through second chamber 1190 to reaction chamber 1262 to perform a PCR process on the newly synthesized cDNA, or on the DNA present in sample S In such embodiments, substance R2 may include one or more PCR reagents, including Taq polymerase. In some embodiments, substance R1 and / or substance R2 may include DNA-binding dyes (for example, minor groove ligand (MGB), MGB and fluorophore coupled to the 5 'end of a DNA probe, where the DNA hybridizes specifically to a target sequence), so the progress of the PCR process can be monitored in real time by detecting the fluorescence of the fluorescent reporter molecule in reaction chamber 1262 using any of the instruments and / or methods described in this document.
[000107] In some embodiments, cartridge 1001 (Figures 1 and 2) is used to both isolate and amplify a sample of nucleic acid. For example, isolation can occur in the first chamber 1114 or in the second chamber 1190. Substance R1, in one embodiment, includes a reagent for isolating nucleic acid. DNA, RNA and a
Petition 870200011939, of 01/24/2020, p. 28/198
25/184 combination of these can be isolated by the cartridges provided herein. For example, the substance R1, in one embodiment, comprises magnetic microspheres derivatized with a reagent to isolate DNA or RNA.
[000108] Both individual nucleic acids and total nucleic acids can be isolated in the cartridges provided herein. For example, substance R1 includes, in one embodiment, microspheres derivatized with a polyA sequence, designed to isolate the total pool of messenger RNA present in a sample. In another embodiment, substance R1 includes microspheres derivatized with specific nucleic sequences, designed to isolate only a portion of the nucleic acid in the sample.
[000109] Once the nucleic acid is isolated, it can be amplified. In one embodiment, amplification occurs by PCR. For the purposes of this invention, reference to PCR in a nucleic acid sample includes reverse transcription PCR (RT-PCR). Specifically, when the nucleic acid sample is one or more target RNAs, or a population of RNAs (for example, total mRNA), RT-PCR will be performed on the target RNAs. The PCR master mix provided here may therefore include reagents for reverse transcription. The reverse transcription step can take place in the same chamber or PCR module, or in a different camera or module. In one embodiment, reverse transcription and PCR are performed in the same chamber, providing a master mix for RT-PCR. An individual of ordinary skill in the art will readily know whether RT-PCR or PCR is required, based on the nucleic acid sample that is originally isolated. Any of the cartridges provided herein can be used to isolate DNA and / or RNA, and perform RT-PCR and / or PCR.
[000110] For example, in one modality, if the RNA is isolated
Petition 870200011939, of 01/24/2020, p. 29/198
26/184 first, a reverse transcriptase reaction is performed on the isolated sample, for example, in the second chamber 1190 or in the reaction chamber 1262. If the DNA is isolated, it can be amplified by PCR, for example, in the reaction 1262. Similarly, if the RNA is first isolated from sample S, it is subjected to a reverse transcription reaction, for example, in the 1262 reaction chamber, and the product of this reaction is used in a PCR reaction at downstream, for example, in reaction chamber 1262. In some embodiments, multiple target nucleic acids are amplified in the PCR, and the PCR reaction is monitored in real time. The amplification of multiple targets is monitored, in one modality, using individual DNA hybridization probes, specific to each target, where each probe includes a fluorophore that emits light at a different wavelength, or that can be excited in a unique wavelength. The DNA hybridization probe, in one embodiment, is provided in second volume 1213 as substance R2 (or a portion thereof).
[000111] The probe used to monitor the PCR, in one embodiment, is a DNA oligonucleotide that specifically hybridizes to a DNA target of interest, and includes a non-fluorescent suppressor at the 3 'termination and a fluorophore at the 5' termination . Additionally, in this embodiment, the DNA oligonucleotide includes a MGB at the 5 'end, directly linked to the oligonucleotide, or linked to the fluorophore (see Lukhtanov et al. (2007). Nucleic Acids Research 35, p. E30). The DNA oligonucleotide probe fluoresces when attached to a target, but not while in solution. Therefore, by synthesizing the product in the PCR, more hybridization will occur, and more fluorescence is generated. Therefore, the amount of fluorescence is proportional to the amount of target generated.
[000112] Real-time monitoring of a PCR reaction does not
Petition 870200011939, of 01/24/2020, p. 30/198
27/184 is limited to the cartridges shown in Figures 1 and 2. Instead, any of the cartridges provided in this document can employ a real-time PCR, for example, with the DNA hybridization probes described above.
[000113] In some embodiments, cartridge 1001 can be manipulated by one of the instruments and / or methods described herein to facilitate the occurrence of a PCR process inside reaction chamber 1262. In such embodiments, reaction module 1200 can be coupled and / or placed in contact with a heat transfer device to allow the contents of reaction chamber 1262 to be thermally cycled in connection with the PCR process. In such modalities, the reaction module 1200 can be operationally coupled to an optical device to allow real-time monitoring of the PCR process. In other embodiments, reaction module 1200 and / or isolation module 1100 can be operationally coupled with other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy or the like, to facilitate a reaction and / or an isolation process that occurs in it.
[000114] Although Figures 1 and 2 show the reaction chamber 1262 and the second volume 1213 being in fluid communication with the second chamber 1190, in other embodiments, the fluid communication between the reaction chamber 1262, the second volume 1213 and / or the second chamber 1190 of the isolation module can be selective. Otherwise, in some embodiments, reaction module 1200 and / or isolation module 1100 may include a mechanism, such as a valve, or a pierceable membrane, which can selectively place the second chamber 1190 in fluid communication with the second volume 1213 and / or reaction chamber 1262. Although isolation module 1100 is shown by defining a first volume 1163 in
Petition 870200011939, of 01/24/2020, p. 31/198
28/184 some modalities, the 1100 isolation module can define any number of volumes and / or can contain any number of different substances. Similarly, although reaction module 1200 is shown by defining a second volume 1213, in some embodiments, reaction module 1200 can define any number of volumes and can contain any number of different substances. [000115] Figure 3 is a schematic illustration of a 2001 cartridge according to an embodiment that includes a first module 2110, a second module 2160 and a third module 2200. The first module 2110 defines a first chamber 2114 and a second chamber 2190 The first chamber 2114 and / or the second chamber 2190 may contain any biological sample containing a target nucleic acid, such as, for example, urine, blood, other materials containing tissue samples or the like. Although the first chamber 2114 is shown to be in fluid communication with the second chamber 2190, in other embodiments, the first chamber 2114 can be selectively placed in fluid communication with the second chamber 2190. Otherwise, in some embodiments, the first module 2110 can include any suitable mechanism, such as a valve (not shown in Figure 3), which can selectively place the first chamber 2114 in fluid communication with the second chamber 2190. Furthermore, in other embodiments, the first 2110 module may have any control suitable flow and / or transfer mechanism (not shown in Figure 3) to facilitate the transfer and / or control of the transfer of a substance between the first chamber 2114 and the second chamber 2190, including, for example, valves, a device capillary flow control, pumps, or the like.
[000116] The second module 2160 defines a first volume 2163 that can completely or partially contain any biological or chemical substance. The substance can, for example, be an oil
Petition 870200011939, of 01/24/2020, p. 32/198
29/184 mineral, a wash buffer, a reagent, or the like, that can participate and / or otherwise withstand a reaction within the first chamber 2114 and / or any other portion of the cartridge 2001. In one embodiment, the reaction in the first chamber 2114 is an isolation reaction, for example, a nucleic acid or peptide isolation. The second module 2160 can be coupled to the first module 2110 in any suitable manner as described in this document. In some embodiments, for example, the first module 2110 and the second module 2160 can be constructed separately and coupled together in such a way that the first module 2110 and the second module 2160 are modularly arranged. In such a modular arrangement, several different configurations of the first module 2110 and the second module 2160 can be used. The different configurations of the first module 2110 and / or the second module 2160 can include different reagents and / or different structures within the first module 2110 and / or the second module 2160. As shown in Figure 3, a portion of the second module 2160 is arranged within the first chamber 2114 of the first module 2110 such that the first volume 2163 can be placed in fluid communication with the first chamber 2114. In other embodiments, the first volume 2163 can be selectively placed in fluid communication with the first chamber 2114. In some embodiments, for example, the first 2110 module and / or the second 2160 module may include any suitable mechanism, such as a valve and / or any suitable fluid control and / or transfer mechanism as described in this document, which may selectively place the first volume 2163 in fluid communication with the first chamber 2114 when the second module 2160 is coupled to the first m ode 2110. In some embodiments, substances and / or samples can be transferred between the first volume 2163 and the first
Petition 870200011939, of 01/24/2020, p. 33/198
30/184 chamber 2114 using any suitable fluid transfer mechanism as described in this document. For example, in use, a sample, isolated sample (for example, isolated DNA, isolated RNA, isolated peptides, isolated proteins), a reagent (for example, an isolating reagent), and / or other supporting substances can be transferred into and / or out of the first chamber 2114 in connection with a desired reaction. In still other embodiments, the first volume 2163 can be fluidly isolated from the first chamber 2114, for example, through a valve, piercable member, or a selective transfer mechanism as described in the present document (not shown in Figure 3).
[000117] The third module 2200 defines a reaction chamber 2262 and a second volume 2213. The reaction chamber 2262 and / or the second volume 2213 may contain completely or partially one or more biological or chemical substances, such as a mineral oil, a wash buffer, one or more PCR reagents, a reagent, or the like, that participate in or otherwise support a reaction inside the 2262 reaction chamber and / or any other portion of the 2001 cartridge. The third module 2200 can be coupled to the first 2110 module in any suitable manner as described in this document. In some embodiments, the first module 2110 is an isolation module 2110, for example, for isolating one or more target nucleic acids from a biological sample. In some embodiments, the first 2110 module is used for RNA isolation and first-strand cDNA synthesis. In this embodiment, the first volume 2163 includes an isolation reagent and reagents for a reverse transcription (RT) reaction. In some embodiments, for example, the first module 2110 and the third module 2200 can be separately constructed and coupled together in such a way
Petition 870200011939, of 01/24/2020, p. 34/198
31/184 so that the first module 2110 and the third module 2200 are modularly arranged. In such a modular arrangement, different configurations of the first module 2110 and the third module 2200 can be used. Different configurations of the first module 2110 and / or the third module 2200 may include different reagents and / or different structures within the first module 2110 and / or the third module 2200. As shown in Figure 3, a portion of the third module 2200 is arranged within the second chamber 2190 of the first module 2110 such that the reaction chamber 2262 and the second volume 2213 are in fluid communication with the second chamber 2190. In other embodiments, reaction chamber 2262 and / or second volume 2213 can be selectively placed in fluid communication with the second chamber 2190. Otherwise, in some embodiments, the first module 2110 and / or the third module 2200 may include any suitable mechanism, such as a valve and / or any suitable fluid control and / or transfer mechanism as described in this document , which can place reaction chamber 2262 and / or second volume 2213 in selective fluid communication with the second chamber 2190. In some embodiments, substances and / or samples can be transferred between the second chamber 2190, and the reaction 2262 and / or second volume 2213 using any suitable fluid transfer mechanism as described in this document. For example, in use, samples, reagents, and / or other support materials can be transferred into or out of the 2262 reaction chamber in connection with a desired reaction. In yet other embodiments, the reaction chamber 2262 and / or the second volume 2213 can be fluidly isolated from the second chamber 2190, for example, through a pierceable member or a selective transfer mechanism as described in the present document (not shown) .
Petition 870200011939, of 01/24/2020, p. 35/198
32/184
[000118] In some embodiments, the 2001 cartridge can be used to perform sample preparation, nucleic acid isolation and / or polymerase chain reactions (PCRs) in the sample. In such embodiments, a target nucleic acid can be isolated from the sample within the first 2110 module. The isolated nucleic acid can be RNA, DNA, or a combination of these. As previously described, if the RNA is isolated, before the PCR, a reverse transcription reaction is performed on the 2001 cartridge, for example, in the first chamber 2114 or in the second chamber 2190. The isolated nucleic acid (or newly synthesized cDNA if the RNA was isolated) can then be amplified (for example, using PCR) in the third module 2200, as described in this document, for example, a real-time PCR with a DNA oligonucleotide probe comprising a fluorophore and MGB at the termination 5 'and a non-fluorescent suppressor at the 3' termination. The modular arrangement of the 2001 cartridge allows any number of different third party modules 2200 containing, for example, different reagents and / or configured to amplify a different type of sample, to be used with the first module 2110, or vice versa. In some embodiments, the 2001 cartridge can be manipulated using any of the instruments and / or methods described herein to facilitate the occurrence of a PCR process inside the 2262 reaction chamber. In such embodiments, the third module 2200 can be coupled and / or placed in contact with a heat transfer device to allow the contents of the 2262 reaction chamber to be thermally cycled in connection with the PCR process. In such embodiments, the third module 2200 can be operationally coupled to an optical device to monitor the PCR process. In other embodiments, the third module 2200 and / or the first module 2110 can be operationally coupled to other energy sources, such as an optical energy source, energy
Petition 870200011939, of 01/24/2020, p. 36/198
33/184 ultrasonic, magnetic energy, hydraulic energy or similar, to facilitate a reaction and / or an isolation process that occurs in it.
[000119] Although Figure 3 shows the integrated cartridge 2001 defining a first volume 2163 and a second volume 2213, in some embodiments, the integrated cartridge 2001 can define any number of first volumes 2163 and / or second volumes 2213 to contain any number of different substances and / or perform additional functionality. For example, the first volumes 2163 and / or the second volumes 2213 may contain separate wash buffers, elution buffers, reagents for reverse transcription reaction, PCR reagents, lysis buffer.
[000120] As previously described, in some embodiments, any of the cartridges described herein may include one or more transfer mechanisms configured to transfer a sample between several defined chambers within the cartridge. For example, Figure 4 is a schematic illustration of a cartridge 3001 according to an embodiment that includes a first module 3110, a second module 3160 and a third module 3200. The first module 3110 defines a first chamber 3114 and a second chamber 3190 In some embodiments, the first module 3110 serves as an isolation module, for example, to isolate one or more target nucleic acids, a population of nucleic acids (for example, total RNA, total DNA, mRNA), or target peptides or proteins from a biological sample. The first chamber 3114 and / or the second chamber 3190 can contain any biological sample, for example, a biological sample containing a target nucleic acid, such as, for example, urine, blood, other materials containing tissue samples or the like. A first transfer mechanism 3140 is arranged between the first chamber 3114 and the second chamber 3190.
Petition 870200011939, of 01/24/2020, p. 37/198
34/184
[000121] In some embodiments, the first transfer mechanism 3140 may be a selective transfer mechanism to selectively transfer samples and / or substances between the first chamber 3114 and the second chamber 3190. In such embodiments, for example, the first transfer mechanism transfer 3140 can transfer samples and / or substances with particular properties between the first chamber 3114 and the second chamber 3190, while limiting and / or preventing the transfer of samples and / or substances having different properties between the first chamber 3114 and / or the second chamber 3190. In some embodiments, the first transfer mechanism 3140 may be an apparatus that uses magnetic components to transfer samples and / or substances based on the magnetic properties of the samples and / or substances. In other embodiments, the first transfer mechanism 3140 can transfer samples and / or substances based on the electrical surface charge of the samples and / or substances, such as, for example, through the use of electrophoresis. In yet other embodiments, the first 3140 transfer mechanism can transfer samples and / or substances based on the sizes of the molecules or ions within the samples and / or the substances. In such embodiments, the first transfer mechanism 3140 may include a reverse osmosis mechanism that serves to selectively transfer samples and / or substances. Otherwise, in some embodiments, the first transfer mechanism 3140 may depend on and / or produce a force, including, for example, a magnetic force, an electrostatic form, a pressure, or the like, to act on the target samples and / or substances and / or molecules and / or ions. The first transfer mechanism 3140 may also include any suitable structures and / or may combine multiple selective transfer mechanisms (for example, to confer physical movements
Petition 870200011939, of 01/24/2020, p. 38/198
35/184 and / or provide additional selectivity). In some embodiments, the first transfer mechanism 3140 can selectively transfer certain molecules or ions between the first chamber 3114 and the second chamber 3190, while maintaining substantial fluidic isolation between the first chamber 3114 and the second chamber 3190. In some embodiments, the first transfer mechanism 3140 may be a magnetic valve as described in US Patent No. 7,727,473, entitled CASSETTE FOR SAMPLE PREPARATION, deposited on October 17, 2006, hereby incorporated in its entirety for reference. In yet other embodiments, the first transfer mechanism 3140 can non-selectively transfer substances and / or samples between the first chamber 3114 and the second chamber 3190. [000122] The second module 3160 defines a first volume 3163 which may contain completely or partially any biological or chemical substance such as, for example, a mineral oil, a nucleic acid isolating reagent, a reverse transcription reagent, an elution buffer, a lysis buffer, a wash buffer, a reagent, or similar, which can participate and / or otherwise withstand the reaction inside the first chamber 3114 and / or any other portion of the cartridge 3001. The second module 3160 can be coupled to the first module 3110 in any suitable manner as described in this document . In some embodiments, for example, the first module 3110 and the second module 3160 can be constructed separately and coupled together in such a way that the first module 3110 and the second module 3160 are modularly arranged. In such a modular arrangement, different configurations of the first module 3110 and the second module 3160 can be used. Different configurations of the first module 3110 and / or the second module 3160 may include different reagents and / or different
Petition 870200011939, of 01/24/2020, p. 39/198
36/184 structures within the modules. As shown in Figure 4, the portion of the second module 3160 is arranged within the first chamber 3114 of the first module 3110 in such a way that the first volume 3163 is in fluid communication with the first chamber 3114. In other embodiments, the first volume 3163 can be selectively placed in fluid communication with the first chamber 3114. Otherwise, in some embodiments, the first module 3110 and / or the second module 3160 may include any suitable mechanism, such as a valve and / or any suitable fluid control and / or transfer mechanism as described in this document, which can selectively place the first volume 3163 in fluid communication with the first chamber 3114. In some embodiments, substances and / or samples can be transferred using any transfer mechanism. suitable fluids as described in this document between the first volume 3163 and the first chamber 3114. For example, in use, oysters, reagents, and / or other support materials can be transferred into or out of the first 3114 chamber in connection with a desired reaction. In yet other embodiments, the first volume 3163 can be fluidly isolated from the first chamber 3114, for example, via a pierceable member or a selective transfer mechanism as described in the present document (not shown not shown).
[000123] The third module 3200 defines a reaction chamber 3262. The reaction chamber 3262 can completely or partially contain any biological or chemical substance, such as a mineral oil, a reverse transcription reagent, an elution buffer, a buffer of lysis, a PCR reagent (for example, Taq polymerase, primers, DNA oligonucleotide probe to monitor the reaction, Mg2 +), a wash buffer, a reagent, or the like, that participate in or otherwise support the reaction reaction chamber 3262 and / or
Petition 870200011939, of 01/24/2020, p. 40/198
37/184 any other portion of the cartridge 3001. The third module 3200 may be coupled to the first module 3110 in any suitable manner as described herein. In some embodiments, for example, the first module 3110 and the third module 3200 can be built separately and coupled together in such a way that the first module 3110 and the third module 3200 are modularly arranged. In this modular arrangement, different configurations of the first 3110 module and the third 3200 module can be used. Different configurations of the first 3110 module and / or the third 3200 module can include different reagents and / or different structures within the modules. As shown in Figure 4, a portion of the third module 3200 is disposed within the second chamber 3190 of the first module 3110 such that the reaction chamber 3262 can be in fluid communication with the second chamber 3190 subjected to the control of the second transfer mechanism. 3240. [000124] The second transfer mechanism 3240 can transfer the substance and / or the reagent from the second chamber 3190 to the reaction chamber 3262 or vice versa. In some embodiments, for example, the second transfer mechanism can transfer a predetermined volume of the substance and / or the reagent between the second chamber 3190 and the reaction chamber 3262. As similarly stated, in some embodiments, the second transfer mechanism transfer 3240 can transfer the substance and / or the reagent between the second chamber 3190 and the reaction chamber 3262 at a predetermined volumetric flow rate. In some embodiments, for example, the second transfer mechanism 3240 can be a pump configured to apply positive pressure or vacuum to the second chamber 3190 and / or the reaction chamber 3262. In such embodiments, the second transfer mechanism 3240 can be a pump driven by a plunger using any of the
Petition 870200011939, of 01/24/2020, p. 41/198
38/184 instruments and / or methods described in this document. In some embodiments, the second transfer mechanism 3240 may have a piercable member as described herein, such that the second transfer mechanism 3240 can pierce, break, tear and / or rupture the piercable member to transfer the substance and / or the sample contained in the reaction chamber 3262 in the second chamber 3190 or vice versa. In other embodiments, for example, the second 3240 transfer mechanism can be a capillary flow control device. In yet other embodiments, the second 3240 transfer mechanism can be another selective or non-selective transfer mechanism as described herein.
[000125] In some embodiments, cartridge 3001 can be used to perform sample preparation, nucleic acid isolation, reverse transcription (if RNA is isolated first), and / or polymerase chain reactions (PCRs) in the sample . In such embodiments, a target nucleic acid can be isolated from the sample within the first module 3110. The isolated nucleic acid can then be amplified (for example, using PCR) in the third module 3200, as described below. As described in this document, PCRs on multiple targets can be monitored in real time with a cartridge of the invention, for example, cartridge 3001. In one embodiment, amplification of multiple targets occurs with the DNA oligonucleotide probes described by Lukhtanov et al. (Nucleic Acids Research 35, p. E30, 2007). The modular arrangement of the cartridge 3001 allows any number of different third party 3200 modules containing, for example, different reagents and / or configured to amplify a different type of sample, to be used with a first module 3110, and vice versa. In some embodiments, the 3001 cartridge can be handled by anyone
Petition 870200011939, of 01/24/2020, p. 42/198
39/184 of the instruments and / or methods described in this document to facilitate the occurrence of a PCR process inside the 3262 reaction chamber. In such modalities, the third 3200 module can be coupled and / or placed in contact with a heat transfer to allow the contents of the 3262 reaction chamber to be thermally cycled in connection with the PCR process. In such embodiments, the third 3200 module can be operationally coupled to an optical device to monitor the PCR process. In other embodiments, the third 3200 module and / or the first 3110 module can be operationally coupled to other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy or the like, to facilitate a reaction and / or an isolation process that occurs in it.
[000126] Although in one embodiment, the cartridge 3001 shown and described in relation to Figure 4 includes a first module, a second module and a third module, in other embodiments, a cartridge can include two modules coupled together. For example, Figure 5 is a schematic illustration of a portion of a cartridge 4001 according to an embodiment that includes a first module 4200 and a second module 4160. The portion of cartridge 4001 can be coupled to an insulation module 4110, as shown in Figure 5. The first 4200 module includes a 4260 reaction flask, a 4220 substrate, and a 4140 first transfer mechanism. The 4260 reaction flask defines a 4262 reaction chamber that can contain any or all samples and / or biological or chemical substance containing a target nucleic acid, such as, for example, urine, blood, other materials containing tissue samples, or the like, and / or mineral oil, wash buffer, lysis buffer, reverse transcription reagent, reagent PCR, a reagent, or the like, that participate in or otherwise support a reaction
Petition 870200011939, of 01/24/2020, p. 43/198
40/184 inside reaction chamber 4262 and / or any other portion of cartridge 4001.
[000127] The 4260 reaction flask can be any suitable container that serves to hold a sample, for example, a nucleic acid sample, isolated or otherwise, in a way that allows a reaction associated with the sample to occur. In some embodiments, the 4260 reaction flask may have a thin wall configured so that it is received inside and / or arranged against a heating element and / or a block (see, for example, block 1710 described below). The 4260 reaction flask can be constructed from any suitable materials with certain properties compatible with a desired reaction and / or process. In some embodiments, the 4260 reaction flask can be substantially constructed from a thermally conductive material to allow thermal cycling of the substances and / or samples within the 4260 reaction flask. In some embodiments, the 4260 reaction flask can be substantially constructed from a mechanically robust material such that the side wall of the 4260 reaction flask substantially retains its shape and / or size when a positive pressure or vacuum acts on the volume inside the 4260 reaction flask. In some embodiments, the 4260 reaction flask can be substantially constructed from a material chemically inert to the reaction inside the 4260 reaction flask in such a way that the material forming the 4260 reaction flask does not contaminate or otherwise affect the reaction inside the flask reaction 4260.
[000128] The 4260 reaction flask can also be any suitable container that serves to contain the sample in a way that allows the monitoring of such a reaction (for example, the detection of an analyte within the sample that results or is associated with the reaction). In
Petition 870200011939, of 01/24/2020, p. 44/198
41/184 some embodiments, for example, the 4260 reaction flask may be a PCR flask, a test tube, a microcentrifuge tube, or the like. In addition, in some embodiments, at least a portion of the 4260 reaction flask may be substantially transparent to allow optical monitoring of a reaction occurring in it. [000129] In some embodiments, the 4260 reaction flask can be integrally constructed by the 4220 substrate. In other embodiments, the 4260 reaction flask can be coupled to the 4220 substrate through any suitable mechanism as described in this document.
[000130] Substrate 4220 defines at least a portion of a first flow path 4221 and a second flow path 4222. The first flow path 4221 is configured so that it is in fluid communication with the reaction chamber 4262 and a flow chamber. isolation 4114 from a 4110 isolation module. The first 4140 transfer mechanism is configured to transfer an S sample (or portion thereof) from isolation chamber 4114 to reaction chamber 4262 (as shown by the arrow AA) when the first 4140 transfer mechanism is triggered. Substrate 4220 can define the portion of the first flow path 4221 and the second flow path 4222 using any suitable structure, material and / or manufacturing process. In some embodiments, substrate 4220 may be a single layer. In other embodiments, the substrate 4220 can be constructed from multiple separate layers of material manufactured and coupled together to define the structure and flow paths. In some embodiments, substrate 4220 can be constructed using processes, including, for example, chemical engraving, mechanical and / or ionic grinding, embossing, lamination, and / or silicon bonding. In some embodiments, at least a portion of substrate 4220 can be configured,
Petition 870200011939, of 01/24/2020, p. 45/198
42/184 arranged and / or in contact with a heating element such that in use, the portion of the substrate that defines the first flow path and / or the second flow path can be heated. For example, in some embodiments, substrate 4220 can be disposed within any of the instruments described herein, and can heat the first flow path 4221 and a second flow path 4222 in such a way that the substance contained therein (for example) , the portion of a sample being transferred between isolation chamber 4114 and reaction chamber 4262) can be heated up to and / or maintained at a temperature approximately greater than 50 ° C. As described in greater detail in this document, this arrangement facilitates a hot start transfer of substances and / or reagents associated with a PCR process.
[000131] The first transfer mechanism 4140 is at least partially contained within the first module 4200 and configured to facilitate the transfer of sample S, from the isolation chamber 4114 to the reaction chamber. In some embodiments, the first 4140 transfer mechanism can facilitate the transfer of the S sample, while maintaining fluid isolation between the first 4221 flow path and regions outside the first 4200 module. For example, in some embodiments, the first transfer mechanism transfer 4140 can be any mechanism that produces a force and / or facilitates the transfer of sample S without the addition of a substance from a region outside the first 4200 module (for example, without the addition of a compressed gas, or the like) . This arrangement reduces potential contamination, improves process automation and / or otherwise improves the speed and / or accuracy of sample transfer S. For example, sample transfer S can be programmed to proceed at different time steps , where each time step transfers different amounts of
Petition 870200011939, of 01/24/2020, p. 46/198
43/184 sample S. Improving the transfer precision of sample S can also improve the quality of PCR analysis. The first transfer mechanism can be any suitable mechanism as described in this document. For example, in some embodiments, the first transfer mechanism 4140 may be a selective transfer mechanism for selectively transferring sample S between isolation chamber 4114 and reaction chamber 4262. In some embodiments, the first transfer mechanism 4140 may apply magnetic, electrostatic and / or pressure forces to effect sample transfer S.
[000132] The first 4200 module can be coupled to the 4110 isolation module in any suitable manner as described in this document to allow fluid communication between the first 4200 module and the 4110 isolation module. In some embodiments, for example, the first 4200 module and 4110 isolation module can be separately constructed and coupled together in such a way that the first 4200 module and 4110 isolation module are modularly arranged. In such a modular arrangement, different configurations of the first 4200 module and the 4110 isolation module can be used. Different configurations of the first 4200 module and / or the 4110 isolation module can include different reagents and / or different structures within the modules.
[000133] The second module 4160 includes a second transfer mechanism 4240 and defines a volume 4163 configured to contain substance R1. Depending on the use in question, substance R1 and substance R2 may refer to one or more reagents. The R1 substance can be any biological or chemical substance such as, for example, a mineral oil, a wash buffer, a fluorescent dye, a lysis buffer, a wash buffer, an elution buffer, a reverse transcription reagent, a PCR reagent (for example
Petition 870200011939, of 01/24/2020, p. 47/198
44/184 example, one or more among a Taq polymerase, primers, DNA hybridization probes, such as the probes described by Lukhtanov et al. (2007). Nucleic Acids Research 35, p. e30), a reagent or the like. Although Figure 5 shows the second module 4160 including a volume 4163, in other embodiments, the second module 4160 can include any number of volumes 4163 and / or containers within which various substances (including substance R1 and / or different substances) can be stored. The second module 4160 is configured so that it is coupled to the first module 4200 in such a way that volume 4163 can be selectively placed in fluid communication with the reaction chamber 4262 through the second flow path 4222. The second transfer mechanism 4240 is configured to transfer at least a portion of substance R1 from volume 4163 to reaction chamber 4262 (as shown by arrow BB) when the second transfer mechanism 4240 is activated.
[000134] The second transfer mechanism 4240 can transfer the substance R1 from the second volume 4163 to the reaction chamber 4262 or vice versa. In some embodiments, for example, the second transfer mechanism can transfer a predetermined volume of substance R1 between the second volume 4163 and reaction chamber 4262. In some embodiments, for example, the second transfer mechanism can transfer substance R1 in a predetermined volumetric flow rate between the second volume 4163 and the reaction chamber 4262. In some embodiments, for example, the second transfer mechanism 4240 can be a pump configured to apply positive pressure or a vacuum to the second volume 4163 and / or the reaction chamber 4262. In such embodiments, the second transfer mechanism 4240 can be a pump driven by a plunger using any of the
Petition 870200011939, of 01/24/2020, p. 48/198
45/184 instruments and / or methods described in this document. In some embodiments, the second transfer mechanism 4240 may have a piercable member as described in this document, such that while in use, the second transfer mechanism 4240 may pierce, break, tear and / or rupture the piercable member and transfer the substance and / or the sample contained in the reaction chamber 4262 in the second volume 4163 or vice versa. In some other embodiments, for example, the second 4240 transfer mechanism may be a capillary flow control device. In yet other embodiments, the second transfer mechanism 4240 can be any other transfer mechanism as described herein.
[000135] In some embodiments, cartridge 4001 can be used to perform sample preparation, nucleic acid isolation and / or polymerase chain reactions (PCRs) in the sample, or an isolated portion of the sample (for example, a isolated nucleic acid sample). In such embodiments, the 4110 isolation module can isolate a target nucleic acid from the sample contained therein. The isolated nucleic acid can then be amplified (for example, using PCR) in reaction chamber 4262, as described below. Alternatively or additionally, if the RNA is isolated, a reverse transcription reaction can be performed in the 4262 reaction chamber. In another embodiment, if the RNA is isolated, an integrated reverse transcription PCR reaction is performed in one or more reaction, for example, reaction chamber 4262. The modular arrangement of cartridge 4001 allows any number of different second 4160 modules containing, for example, different reagents and / or configured to amplify a different type of sample, or isolate a different type sample, be used with the first 4200 module, and vice versa. In some modalities, the
Petition 870200011939, of 01/24/2020, p. 49/198
46/184 cartridge 4001 can be manipulated by any of the instruments and / or methods described in this document to facilitate the occurrence of an amplification process, for example, a PCR process, inside the 4262 reaction chamber. the 4260 reaction flask can be coupled and / or placed in contact with a heat transfer device to allow the contents of the 4262 reaction chamber to be thermally cycled in connection with the PCR process. In such embodiments, the 4260 reaction flask can be operationally coupled to an optical device to monitor the PCR process. In other embodiments, the 4260 reaction flask and / or the 4110 isolation module can be operationally coupled to other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy or the like to facilitate a reaction and / or an isolation process that occurs in it.
[000136] Figures 6 and 7 are schematic illustrations of a portion of the 5001 cartridge according to an embodiment in a first configuration and a second configuration, respectively. The cartridge portion 5001 includes a first module 5200 and a second module 5100. The first module 5200 includes a 5260 reaction flask, a 5220 substrate and a first 5235 transfer mechanism. The 5260 reaction flask defines a 5262 reaction chamber that it can contain a sample in order to allow a reaction associated with sample S. to occur. The 5260 reaction flask can be of any suitable shape and / or size, and can be constructed using any suitable materials, as described in this document. In some embodiments, for example, the 5260 reaction flask may be a PCR flask, a test tube or the like.
[000137] The first 5235 transfer mechanism includes a
Petition 870200011939, of 01/24/2020, p. 50/198
47/184 plunger 5240 movably arranged within a 5230 compartment such that the 5230 compartment and the 5235 plunger define a first 5213 volume. The first 5213 volume contains a first substance R1. The first substance R1 can, for example, be a reagent (for example, a PCR reagent, such as Taq polymerase, primers, DNA hybridization probes, such as those described above, or a combination of these), a reverse transcription reagent , a mineral oil or the like. The 5240 plunger can be driven by any suitable mechanism, such as, for example, any of the instruments described in this document.
[000138] The substrate 5220 defines at least a portion of a first flow path 5221 and a second flow path 5222. The first flow path 5221 is configured so that it is in fluid communication with the reaction chamber 5262, the first volume 5213 and a 5114 isolation chamber from a 5110 isolation module (shown in Figure 6 in a dotted line format). The second flow path 5222 is configured so that it is in fluid communication with the insulation chamber 5114. The insulation chamber 5114 can be any suitable insulation chamber and / or insulation module of the types shown and described in this document. In addition, the isolation chamber 5114 can be coupled to the first module 5200 in any suitable manner as described in this document. In some embodiments, the isolation chamber 5114 can be attached to the first module 5200 and modularly arranged as described in this document. The removable coupling between the isolation chamber 5114 and the first module 5200 can be impermeable to fluids using any suitable mechanism as described in this document.
[000139] The second 5100 module includes a second transfer mechanism 5150 and defines a second volume 5163 configured for
Petition 870200011939, of 01/24/2020, p. 51/198
48/184 contain the second substance R2. The second module 5100 is configured to be coupled to the first module 5200 in such a way that the second volume 5163 can be selectively placed in fluid communication with the isolation chamber 5114 through the second flow path 5222. The second module 5100 can include any mechanism and / or device configured to selectively place the second volume 5163 in fluid communication with the isolation chamber 5114 and / or the second flow path 5222. For example, in some embodiments, the second module 5100 may include a piercing member that defines a portion of a boundary of the second volume 5163 and which fluidly isolates the second volume 5163 from the isolation chamber 5114 and / or the second flow path 5222. In other embodiments, the second module 5100 may include a valve configured to selectively place the second volume 5163 in fluid communication with the isolation chamber 5114 and / or the second flow path 5222.
[000140] The second transfer mechanism 5150 is configured to transfer at least a portion of the second substance R2 from the second volume 5163 in the isolation chamber 5114 when the second transfer mechanism 5150 is activated. The second 5150 transfer mechanism can be any suitable transfer mechanism as described herein. For example, in some embodiments, the second transfer mechanism 5150 can apply magnetic, electrostatic and / or pressure forces to effect the transfer of substance R2 from the second volume 5163 to the isolation chamber 5114. In some embodiments, for example , the second 5250 transfer mechanism can be a piston driven pump using any of the instruments and / or methods described in this document. In some other modalities, for example, the second
Petition 870200011939, of 01/24/2020, p. 52/198
49/184 5250 transfer mechanism can be a capillary flow control device.
[000141] The 5001 cartridge can be moved between at least a first configuration (Figure 6) and a second configuration (Figure 7) to facilitate a reaction and / or test involving a sample S, which is initially disposed in the isolation chamber 5114 When cartridge 5001 is in the first configuration, plunger 5240 is in a first position within housing 5230 such that portion 5246 of plunger 5240 is arranged within the first flow path 5221. Therefore, when cartridge 5001 is in the first configuration, the first volume 5213 is fluidly isolated from reaction chamber 5262. In this way, when cartridge 5001 is in the first configuration, the first substance R1 is kept within the first volume 5213 and prevents it from being transported in the reaction chamber 5262 (for example, by leak, gravity feed, capillary action or similar). In addition, when the cartridge 5001 is in the first configuration, the second volume 5163 is fluidly isolated from the second flow path 5222 and the isolation chamber 5114. In this way, when the cartridge 5001 is in the first configuration, the second substance R2 is kept inside second volume 5163 and prevents it from being transported in the isolation chamber 5114 (for example, by leakage, gravity feed, capillary action or similar).
[000142] The cartridge 5001 is moved to the second configuration (Figure 7) placing the second volume 5163 in fluid communication with the isolation chamber 5114 through the second flow path 5222, activating the second transfer mechanism 5150 to transporting at least a portion of the second substance R2 in the isolation chamber 5114 (as shown by the arrow CC in Figure 7), and activating the first 5235 transfer mechanism.
Petition 870200011939, of 01/24/2020, p. 53/198
50/184
More particularly, the second volume 5163 can be placed in fluid communication with the isolation chamber 5114 through the second flow path 5222 through any suitable mechanism, such as, for example, by drilling a piercable member, activating a valve or similar. In some embodiments, the second volume 5163 can be placed in fluid communication with the isolation chamber 5114 by activating the second transfer mechanism 5150. In this way, the second volume 5163 can be placed in fluid communication with the isolation chamber 5114 and a portion of the second substance R2 can be transported in the isolation chamber 5114 in one operation and / or in response to a single trigger event.
[000143] The first 5235 transfer mechanism is actuated by moving the 5240 plunger inside the 5230 housing as shown by the DD arrow in Figure 7. As similarly stated, when the first 5235 transfer mechanism is activated, the 5240 plunger is moved in. 5230 bay from a first position (as shown in Figure 6) to a second position (as shown in Figure 7). Therefore, when the first transfer mechanism 5235 is activated, the portion 5246 of the plunger 5240 is at least partially removed from the first flow path 5221, thus placing the first volume 5213 in fluid communication with the reaction chamber 5262 through the first flow path 5221. In this way, a portion of the first substance R1 can be transported from the first volume 5213 in reaction chamber 5262, as shown by the arrow EE in Figure
7.
[000144] Furthermore, when the plunger 5240 is moved from the first position to the second position, a vacuum is produced inside the 5262 reaction chamber. This pressure differential inside the cartridge
Petition 870200011939, of 01/24/2020, p. 54/198
51/184
5001 (i.e., between reaction chamber 5262 and isolation chamber 5114) results in at least a portion of the contents of isolation chamber 5114 (i.e., sample S and / or the second substance R2) to be transported in the reaction chamber 5262 through the first flow path 5221, as shown by arrows FF and GG in Figure 7. In this way, substances and / or samples can be added, mixed and / or transported between the isolation chamber 5114 and the reaction chamber 5262 by activating the first transfer mechanism 5235 and / or the second transfer mechanism 5150. Mixing the sample S and the substance R2 inside the isolation chamber 5114 instead of transferring the sample S and the substance R2 separately within reaction chamber 5262, an additional transfer step can be eliminated. Furthermore, this arrangement and / or method can improve the mixture of sample S and substance R2, thus improving the precision and efficiency of the reaction in the 5262 reaction chamber.
[000145] Although described occurring in a particular order, in other modalities, the operations associated with the movement of the 5001 cartridge from the first configuration to the second configuration can occur in any order. In addition, in other embodiments, the 5001 cartridge can be placed in any number of different configurations involving any desired combination of operations.
[000146] In some embodiments, the 5001 cartridge can be used to perform polymerase chain reactions (PCRs) on at least a portion of the S sample (which can, for example, be one or more isolated target nucleic acids). In such embodiments, the isolated nucleic acids can be amplified (for example, using PCR) in the 5262 reaction chamber, as described herein. In some embodiments, the 5001 cartridge can be
Petition 870200011939, of 01/24/2020, p. 55/198
52/184 manipulated by any of the instruments and / or methods described in this document to facilitate the occurrence of a PCR process inside the 5262 reaction chamber. In such embodiments, the 5260 reaction flask can be attached and / or placed in contact with a heat transfer device to allow the contents of the 5262 reaction chamber to be thermally cycled in connection with the PCR process. In such embodiments, the 5260 reaction flask can be operationally coupled to an optical device to allow real-time monitoring of the PCR process. In other embodiments, the 5260 reaction flask and / or the second 5100 module can be operationally coupled to other energy sources, such as optical energy, ultrasonic energy, magnetic energy, hydraulic energy, or the like, to facilitate a reaction and / or an isolation process that occurs in it.
[000147] In some embodiments, the first substance R1 may include a mineral oil, wax, or the like, such that after the first substance R1 is transferred in reaction chamber 5262, the first substance R1 can form a layer on the surface of the fluid mixture (i.e., sample S and the second substance R1) in reaction chamber 5262. The surface layer of the first substance R1 can reduce evaporation of the fluid mixture in reaction chamber 5262 during the reaction process ( for example, during thermal cycling), thus improving efficiency, precision and / or reaction control. More particularly, by reducing the evaporation of the fluid mixture in the reaction chamber 5262, the relative concentrations or the proportion of the different constituents in the reaction mixture can be controlled more precisely. In addition, reducing evaporation of the fluid mixture in the 5262 reaction chamber can also minimize condensation on the walls of the 5260 reaction flask, thus improving the accuracy of optical monitoring or analysis
Petition 870200011939, of 01/24/2020, p. 56/198
53/184 of the reaction.
[000148] Mineral oil can have any mineral oil with suitable properties, such as, for example, the desired physical properties, including, for example, density and / or surface tension. Mineral oil, or the like, can also be selected in such a way that it is chemically inert and physically stable when exposed to conditions inside the 5262 reaction chamber.
[000149] Figures 8 to 24 are several views of a 6001 cartridge according to an embodiment. In certain views, such as, for example, Figures 8 and 9, portions of cartridge 6001 are shown to be semitransparent in such a way that the components and / or features within cartridge 6001 can be shown more clearly. The 6001 cartridge includes a 6100 sample preparation (or isolation) module and a 6200 amplification (or PCR) module that are coupled together to form an integrated 6001 cartridge. One or more 6001 cartridges can be arranged within any instrument of the types described here (see, for example, the instrument 3002 described below) that is configured to manipulate, activate and / or interact with the 6001 cartridge to perform isolation, transcription and / or nucleic acid amplification in a test sample contained inside cartridge 6001. The cartridge 6001 allows for efficient and accurate diagnostic testing of samples by limiting the degree of sample handling during and between the isolation, transcription and / or PCR amplification processes. In addition, the modular layout of the 6100 isolation module and the amplification (or PCR) module 6200 allows any number of different PCR modules 6200, each containing different reagents and / or configured to amplify a different type of nucleic acid, either used with any number of different 6100 isolation modules, each containing different reagents and / or configured to isolate a different type of
Petition 870200011939, of 01/24/2020, p. 57/198
54/184 nucleic acid, and vice versa. This arrangement also allows the 6100 isolation module and the 6200 amplification module to be stored separately. Separate storage can be useful, for example, if the reagents included within the 6100 isolation module have different storage requirements (for example, expiration dates, lyophilization requirements, storage temperature limits, etc.) in relation to the reagents included within the 6200 amplification module.
[000150] As shown in Figure 11, the 6100 isolation module includes a first compartment (or isolation) 6110 and a second compartment (or reagent) 6160 that is coupled and / or is at least partially within the first compartment 6110. The second compartment 6160 is not shown in Figures 10 and 22 for the sake of clarity. Figures 11 to 14 show the second compartment 6160 and certain components contained therein, and Figures 15 to 18 show the second compartment 6160 in several different stages of activation. The second compartment 6160 includes a first end portion 6161 and a second end portion 6162, and defines a series of holding chambers 6163a, 6163b, 6163c and 6163d that contain the reagents and / or other substances used in the isolation process. As described in greater detail in this document, the retention chambers may contain a protease (for example, Proteinase K), a lysis solution to solubilize the bulky material, a binding solution to magnetically charge the resident nucleic acid sample within the lysis chamber 6114, and a solution of magnetic microspheres that bind to the magnetically charged nucleic acid to aid in the transport of the nucleic acid within the 6100 isolation module and / or the first compartment 6110.
[000151] Each of the retention chambers 6163a, 6163b, 6163c and
Petition 870200011939, of 01/24/2020, p. 58/198
55/184
6163d includes an actuator 6166 (see, for example, Figure 14) arranged movably in these. More particularly, as shown in Figure 18, an actuator 6166a is disposed within the holding chamber 6163a, an actuator 6166b is disposed within the holding chamber 6163b, an actuator 6166c is disposed within the holding chamber 6163c, and an actuator 6166d is inside the 6163d holding chamber. As shown in Figure 15, a pierceable member 6170 is arranged around the second end portion 6162 of the second compartment 6160 such that the inner portions of the second compartment 6160, the pierced member 6170 and the actuators 6166a, 6166b, 6166c and 6166d collectively encircle and / or define the retention chambers 6163a, 6163b, 6163c and 6163d. As stated similarly, the internal portions of the second compartment 6160, the pierceable member 6170 and actuators 6166a, 6166b, 6166c and 6166d collectively define fluidically isolated chambers 6163a, 6163b, 6163c and 6163d within which the reagents and / or substances can be stored. The piercable member 6170 can be constructed from any suitable material of the types described herein, such as any form of polypropylene. In some embodiments, the 6170 drillable member can be constructed from biaxially oriented polypropylene (BOP).
[000152] As shown in Figure 14, each of the 6166 actuators includes a 6167 plunger portion, a drilling portion
6168 and one or more 6169 actuator openings. Actuator openings
6169 are configured to receive a portion of an actuator assembly to facilitate movement of the 6166 actuator within the chamber (for example, chamber 6163a), as described in this document. In particular, the 6169 actuator openings can receive a protrusion, such as a protrusion 3446a from a
Petition 870200011939, of 01/24/2020, p. 59/198
56/184 3400 actuator assembly, as described below in relation to Figures 37 to 40. This arrangement allows the plunger 6166 to be actuated from the first end portion 6161 of the second housing 6160. In some embodiments, the 6166 actuator may include a retaining mechanism (for example, a protrusion, a snap ring or the like) configured to retain a protrusion of an actuator assembly (for example, the 3400 actuator assembly) to facilitate the reciprocal movement of the 6166 actuator by the actuator assembly .
[000153] The plunger portion 6167 of the actuator 6166 is configured to engage the portion of the second compartment 6160 that defines the chamber (e.g. the chamber 6163a) within which the 6166 actuator is arranged in such a way that the plunger portion 6167 and the second compartment portion 6160 forms a substantially impermeable and / or fluid-tight seal. Therefore, when actuator 6166 is disposed within the chamber (for example, chamber 6163a), leaks and / or transport of the substance contained within the chamber are minimized and / or eliminated. In this way, the end face of the plunger portion 6167 defines a portion of the chamber boundary (for example, the chamber 6163a). The plunger portion 6167 is also configured such that when a force is exerted on the 6166 actuator (for example, by the 3400 actuator assembly shown and described below), the 6166 actuator will move inside the chamber (for example, the chamber 6163a) to transport the substance contained within the chamber in the 6114 lysis chamber, as described below. In this way, the 6166 actuator can act as a transfer mechanism for transporting the substances from the chamber (for example, chamber 6163a) in another portion of the isolation module 6100.
[000154] The 6168 drilling portion of the 6166 actuator is configured
Petition 870200011939, of 01/24/2020, p. 60/198
57/184 to puncture, break, tear and / or rupture a portion of piercable member 6170 when actuator 6166 is moved into the chamber (e.g., chamber 6163a) to place the chamber in fluid communication with a region outside the chamber. In this way, each of the chambers 6163a, 6163b, 6163c and 6163d can be selectively placed in fluid communication with another portion of the isolation module 6100 (for example, the lysis chamber 6114) to allow the transfer of the substance contained within each one chambers 6163a, 6163b, 6163c and 6163d when each of the actuators 6166a, 6166b, 6166c and 6166d is actuated, as described below.
[000155] The second compartment 6160 includes a mixing pump 6181, which can be activated (for example, by mounting actuator 3400 of the instrument 3002) to agitate, mix and / or produce a turbulent movement within the sample, reagents and / or other substances contained with a portion (for example, lysis chamber 6114) of isolation module 6100. As shown in Figure 12, pump 6181 includes a nozzle 6186 that can direct flow, increase flow pressure and / or increase turbulence within the portion of the insulation module 6100 to accentuate the mixture within it. Although the 6181 mixing pump is shown as a bellows pump, in other embodiments, the 6181 mixing pump can be any suitable mechanism for transferring energy in a solution within the 6114 lysis chamber. Such mechanisms may include, for example, a piston pump, a rotating member, or the like. In some embodiments, the second compartment 6160 may include any other suitable mechanism for mixing the substances within the isolation chamber 6114 to promote cell lysis of the contained sample and / or the isolation of the nucleic acids contained therein. In some embodiments, the second compartment 6160 may include an ultrasonic mixing mechanism, an
Petition 870200011939, of 01/24/2020, p. 61/198
58/184 thermal mix or similar.
[000156] As shown in Figure 11, the second compartment 6160 is disposed within an opening 6115 defined by the first end portion 6111 of the first compartment 6110. Therefore, when the second compartment 6160 is disposed within the first compartment 6110, a portion of the second compartment 6160 defines at least a portion of a boundary of the lysis chamber 6114. More particularly, when the second compartment 6160 is arranged within the first compartment 6110, the pierceable member 6170 defines a portion of the boundary of the lysis chamber 6114. This arrangement allows substances contained within the second compartment 6160 to be transported in the lysis chamber 6114 when the pierced member portion 6170 is punctured, punctured, torn and / or broken (see, for example, Figure 15). Although at least a portion of the second compartment 6160 is shown to be disposed within the first compartment 6110 and / or the lysis chamber 6114, in other embodiments, the second compartment 6160 can be coupled to the first compartment 6110 without any portion of the second compartment being inside the first compartment. In still other embodiments, the portion of the first compartment can be arranged within the second compartment when the first compartment and the second compartment are coupled together.
[000157] As shown in Figures 12 and 13, the second compartment 6160 includes a seal 6172 arranged around the second end portion 6162 such that when the second compartment 6160 is coupled to the first compartment 6110, the seal 6172 and a portion the side wall of the first 6110 compartment collectively form a substantially impermeable and / or fluid-tight seal between the first 6110 compartment and the
Petition 870200011939, of 01/24/2020, p. 62/198
59/184 second compartment 6160. Otherwise, seal 6172 fluidly insulates lysis chamber 6114 from a region outside cartridge 6001. In some embodiments, seal 6172 can also acoustically isolate second compartment 6160 from the first 6110 compartment.
[000158] The first end portion 6161 of the second compartment 6160 includes protrusions 6171 configured to be received within the corresponding openings 6119 (see, for example, Figure 10) defined by the first compartment 6110. Therefore when the second compartment 6160 is arranged inside of the first compartment 6110, protrusions 6171 and openings 6119 collectively retain the second compartment 6160 within the first compartment 6110. As similarly stated, protrusions 6171 and openings 6119 collectively limit the movement of the second compartment 6160 in relation to the first compartment 6110.
[000159] The modular arrangement of the first compartment 6110 and the second compartment 6160 allows any number of second compartments 6160 (or reagent compartments), containing different reagents and / or substances to promote nucleic acid isolation, to be used with the first compartment 6110 to form the isolation module 6100. This arrangement also allows the first compartment 6110 and the second compartment 6160 to be stored separately. Separate storage can be useful, for example, if the reagents included within the second 6160 compartment have different storage requirements (for example, expiration dates, lyophilization requirements, storage temperature limits, etc.) from the substances contained inside the first 6110 compartment.
Petition 870200011939, of 01/24/2020, p. 63/198
60/184
[000160] In use, substances contained within the second compartment 6160 can be transported in the first compartment 6110 to facilitate the isolation process. Figures 15 to 18 show a cross-sectional view of a portion of the insulation module 6100 in various stages of activation. For example, Proteinase K can be stored in chamber 6163d, and transferred in lysis chamber 6114 as shown in Figure 15. More particularly, actuator 6166d can be moved within chamber 6163d as shown by the arrow HH when driven by any external force , such as, for example, a force applied by the drive assembly 3400 of the instrument 3002 described in this document. When the actuator 6166d moves towards the lysis chamber 6114, the drilling portion 6168d comes into contact and pierces a portion of the piercing member 6170. In some embodiments, the piercing member 6170 may include a piercing, a stress contraction elevator or other structural discontinuity to ensure that the piercing member 6170 easily pierces the desired portion of the piercing member 6170. In this way, the movement of the 6166d actuator places the 6163d chamber in fluid communication with the 6114 lysis chamber. A continued movement of the 6166d actuator transfers the contents of chamber 6163d (e.g. Proteinase K) in lysis chamber 6114. In this way, actuator 6166d functions as both a valve and a transfer mechanism.
[000161] In another embodiment, the contents of chamber 6163d may include proteinase K (for example, 10 mg / mL, 15 mg / mL or 20 mg / mL, mannitol, water and bovine serum albumin. In an additional embodiment, microspheres are coated or derivatized by proteinase K. In another embodiment, the contents of chamber 6163d may include a proteinase K, mannitol, water and gelatin.
Petition 870200011939, of 01/24/2020, p. 64/198
61/184 In an additional embodiment, the microspheres are coated or derivatized by proteinase K. In another embodiment, the contents of chamber 6163d are lyophilized, for example, as a 50 pL pellet.
[000162] In another embodiment, chamber 6163d also provides a positive control reagent. The positive control reagent, in one embodiment, consists of a plurality of microspheres derivatized by an internal control nucleic acid sequence. In an additional embodiment, the microspheres are provided in a solution of mannitol, BSA and water. In yet another additional embodiment, the microspheres and the solution are provided as a lyophilized pellet, for example, as a 50 pL pellet.
[000163] Although specifically described for chamber 6163d, proteinase K, a solution comprising proteinase K and / or the positive control reagent, in other embodiments, is present as an R1 or R2 substance.
[000164] Similarly, a lysis solution can be stored in chamber 6163c, and transferred to lysis chamber 6114 as shown in Figure 16. More particularly, actuator 6166c can be moved into chamber 6163c as shown by arrow II when actuated by any suitable external force, such as, for example, a force applied by drive assembly 3400 of instrument 3002 described in this document. When the actuator 6166c moves towards the lysis chamber 6114, the drilling portion 6168c contacts and pierces a portion of the piercable member 6170. In this way, the movement of the actuator 6166c places the chamber 6163c in fluid communication with the lysis 6114. A continuous movement of the 6166c actuator transfers the contents of the 6163c chamber (for example, the lysis solution) into the 6114 lysis chamber. In this way, the 6166c actuator functions as both a valve and a transfer mechanism. In one embodiment, the lysis solution
Petition 870200011939, of 01/24/2020, p. 65/198
62/184 stored in chamber 6163c, or another chamber, comprises a filtered solution of, guanidine HCl (for example, 3 M, 4 M, 5 M, 6 M, 7 M or 8 M), Tris HCl (for example, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM or 30 mM), triton-X-100 (e.g. 1.5%, 2%, 2.5%, 3%, 3.5%, 4% , 4.5% or 5%), NP-40 (e.g. 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5% or 5%), Tween-20 (for example, 5%, 10%, 15%, or 20%), CaCl2 (for example, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM or, 5 mM), molecular grade water. Although specifically described for chamber 6163c, the lysis solution, in other embodiments, is present as an R1 or R2 substance.
[000165] Similarly, a connection solution can be stored in chamber 6163b, and transferred in lysis chamber 6114 as shown in Figure 17. More particularly, actuator 6166b can be moved within chamber 6163b as shown by the arrow JJ when actuated by any suitable external force, such as, for example, a force applied by drive assembly 3400 of instrument 3002 described in this document. When actuator 6166b moves towards lysis chamber 6114, drilling portion 6168b comes into contact and pierces a portion of piercable member 6170. In this way, the movement of actuator 6166b places chamber 6163b in fluid communication with the lysis 6114. A continued movement of actuator 6166b transfers the contents of chamber 6163b (for example, the connection solution) into lysis chamber 6114. In this way, actuator 6166b functions as both a valve and a transfer mechanism. In one embodiment, the binding solution comprises isopropanol, for example, 100% isopropanol, 90% isopropanol, 80% isopropanol, 70% isopropanol, in a volume of about 50 pL, about 100 pL, about 125 pL, about 150 pL, about 175 pL or about 200 pL. Although specifically described for camera 6163b, the connection solution, in other
Petition 870200011939, of 01/24/2020, p. 66/198
63/184 modalities, is present as a substance R1 or R2.
[000166] Similarly, a set of magnetic microspheres can be stored in chamber 6163a, and transferred in lysis chamber 6114 as shown in Figure 18. More particularly, actuator 6166a can be moved within chamber 6163a as shown by the arrow KK when driven by any suitable external force, such as, for example, a force applied by drive assembly 3400 of instrument 3002 described in this document. When the actuator 6166a moves towards the lysis chamber 6114, the drilling portion 6168a comes into contact and pierces a portion of the piercable member 6170. In this way, the movement of the actuator 6166a places the chamber 6163a in fluid communication with the lysis 6114. A continuous movement of the actuator 6166a transfers the contents of the chamber 6163a (for example, the magnetic microspheres) in the lysis chamber 6114. In this way, the actuator 6166a functions as both a valve and a transfer mechanism. Microspheres in one mode are paramagnetic. In one embodiment, the microspheres are magnetic silica microspheres, and are provided in a concentration of 1.0 mg / mL, or 1.5 mg / mL, 2.0 mg / mL, 2.5 mg / mL, 3, 0 mg / ml or 3.5 mg / ml. In an additional embodiment, the magnetic silica microspheres stored in isopropanol, for example, about 50% isopropanol, about 55% isopropanol, about 60%
isopropanol, fence in 61% in isopropanol, fence in 62% in isopropanol, fence in 63% in isopropanol, fence in 64% in isopropanol, fence in 65% in isopropanol, fence in 66% in isopropanol, fence in 67% in isopropanol, fence in 68% in isopropanol, fence in 69% in isopropanol, fence in 70% in isopropanol, fence in 75% in isopropanol, fence in 80% in isopropanol, or about 85% isopropanol. In one mode, at
microspheres are provided as a volume of about 50 pL,
Petition 870200011939, of 01/24/2020, p. 67/198
64/184 about 100 pL, about 125 pL, about 150 pL, about 175 pL or about 200 pL. Although specifically described for chamber 6163a, microspheres, in other embodiments, are present as an R1 or R2 substance.
[000167] As shown in Figure 10, the first compartment 6110 includes a first end portion 6111 and a second end portion 6112, and defines the lysis chamber 6114, two wash chambers 6121 and 6122, three transfer mounting lumens 6123, 6124 and 6125, and an elution chamber 6190. The first compartment 6110 also defines an opening 6115 adjacent to the isolation chamber 6114. As shown in Figure 11 and described earlier, the second compartment 6160 is arranged within the opening 6115 in such a way. such that a portion of the second compartment 6160 (e.g., the piercable member 6170) defines at least a portion of an insulation chamber boundary 6114.
[000168] The first end portion 6111 also defines a filling opening 6116 through which the lysis chamber 6114 can be placed in fluid communication with a region outside the insulation module 6100. As shown in Figures 8 to 10, the module insulating material 6100 includes a cap 6118 that is removably attached around the fill opening 6116. In use, a sample containing a target nucleic acid, such as, for example, urine, blood and / or other materials containing tissue samples can be transported in the lysis chamber 6114 through the filling opening 6116. The sample can be introduced into the lysis chamber 6114 by any suitable mechanism, including, for example, pipetting or injecting the sample into the first chamber 6114 through the fill opening 6116. In some embodiments, a sample filter can be arranged inside the fill opening 6116 and / or the fill cap 6118. The filter
Petition 870200011939, of 01/24/2020, p. 68/198
65/184 can, for example, be a hydrophobic filter.
[000169] After the sample is disposed in the 6114 lysis chamber, reagents and / or substances can be added to facilitate cell lysis to the 6114 lysis chamber, as previously described. In addition, the sample can be agitated and / or mixed through the 6181 pump to facilitate the lysis process, as previously described. In some embodiments, the contents of the lysis chamber 6144 can be heated (for example, by the third heating module 3780, as shown and described below with reference to instrument 3002).
[000170] The 6100 insulation module includes a series of transfer assemblies (also referred to as transfer mechanisms), shown in Figures 15 to 19 as a transfer assembly 6140a, a transfer assembly 6140b and a transfer assembly 6140c. As described in this document, transfer assemblies are configured to transfer substances (for example, portions of the sample including the magnetically charged particles and the isolated nucleic acid attached to them) between the lysis chamber 6114, the washing chamber 6121, the washing chamber 6122, and the elution chamber 6190. More particularly, the transfer assemblies 6140 are configured to transfer substances between the lysis chamber 6114, the washing chamber 6121, the washing chamber 6122, and the elution chamber 6190 while substantially maintaining insulation chamber 6114, washing chamber 6121, washing chamber 6122, and elution chamber 6190 fluidly isolated from other chambers (e.g., the adjacent washing chamber) defined by first compartment 6110.
[000171] The transfer assembly 6140a is arranged inside the transfer assembly lumen 6123, in such a way that the
Petition 870200011939, of 01/24/2020, p. 69/198
66/184 transfer assembly 6140a is between the lysis chamber 6114 and the washing chamber 6121. Consequently, the transfer assembly 6140a is configured to transfer substances between the lysis chamber 6114 and the washing chamber 6121.
[000172] The transfer assembly 6140b is arranged inside the transfer assembly lumen 6124, such that the transfer assembly 6140b is between the wash chamber
6121 and the washing chamber 6122. Consequently, the transfer assembly 6140b is configured to transfer substances between the washing chamber 6121 and the washing chamber 6122.
[000173] The transfer assembly 6140c is arranged inside the transfer assembly lumen 6125, such that the transfer assembly 6140c is between the wash chamber
6122 and the elution chamber 6190. Consequently, the transfer assembly 6140c is configured to transfer substances between the washing chamber 6122 and the elution chamber 6190.
[000174] Each of the transfer assemblies is described with reference to Figures 20 and 21, which shows a representative transfer assembly 6140. The transfer assembly 6140 includes a compartment 6141 and a movable member 6146 which is rotatable disposed within the compartment 6141. The compartment 6141 defines a first opening 6142 and a second opening 6143. When the transfer assembly 6140 is disposed within the transfer assembly lumen (for example, the transfer assembly lumen 6123), the compartment 6141 is aligned such that the first opening 6142 is aligned and / or is in fluid communication with the first chamber (for example, the lysis chamber 6114) and the second opening 6143 is aligned and / or is in fluid communication with the second chamber ( for example, the washing chamber 6121). The 6141 compartment can be fastened inside the
Petition 870200011939, of 01/24/2020, p. 70/198
67/184 transfer assembly (for example, the 6123 transfer assembly lumen) by any suitable mechanism, such as, for example, through a mechanical fastener or retainer, a chemical or adhesive bond, an interference fit , a welded joint or the like. In addition, compartment 6141 may include one or more seals (not shown in Figures 20 and 21) such that the first chamber (for example, the lysis chamber 6114) and the second chamber (for example, the washing chamber 6121 ) are kept in fluidic isolation from each other. As similarly stated, compartment 6141 and first compartment 6110 can collectively form a substantially impermeable and / or fluid-tight seal to eliminate and / or reduce leakage of substances between the first chamber (for example, the 6114 lysis chamber) and the second chamber (for example, the washing chamber 6121). [000175] The movable member 6146 includes an outer surface 6147 that defines a recess or cavity 6148. The movable member 6146 is arranged within the compartment 6141 such that the movable member 6146 can rotate as shown by the arrow MM in Figures 20 and 21 The outer surface 6147 of the movable member 6146 is shown to be separate from the inner surface 6145 of compartment 6141 in Figure 20 for the sake of clarity. The outer surface 6147 is in sliding contact with the inner surface 6145 of compartment 6141 in such a way that the outer surface 6147 and the inner surface 6145 produce a substantially impermeable and / or fluid-tight seal. In this way, leaks of substances between the first chamber (for example, the lysis chamber 6114) and the second chamber (for example, the washing chamber 6121) through the interface between compartments 6141 and the movable member 6146 are eliminated and / or reduced.
[000176] The movable member 6146 also defines a 6149 lumen
Petition 870200011939, of 01/24/2020, p. 71/198
68/184 configured to receive a portion of an actuator 510. The actuator 510 can be any suitable actuator, such as an axis 3510 of the transfer actuator assembly 3500 of the instrument 3002 shown and described below with reference to Figures 41 to 46. As shown in Figure 20, a shape of the actuator 510 can correspond to a shape of the lumen 6149 defined by the movable member 6146 such that the rotation of the actuator 510 results in the rotation of the movable member 6146. As similarly stated, the actuator 510 it can be arranged in a compatible manner within the lumen 6149 such that a relative rotational movement between the actuator 510 and the movable member 6146 is limited. In some embodiments, actuator 510 and lumen 6149 may have a substantially similar hexagonal and / or octagonal shape.
[000177] In use, the movable member 6146 can be moved between the first position (not shown) and the second position (Figure 20) by rotating the movable member 6146 as shown by the MM arrow. When the movable member 6146 is in the first position, the recess or cavity 6148 is aligned and / or is in fluid communication with the first chamber (for example, the lysis chamber 6114). When the movable member 6146 is in the second position, the recess or cavity 6148 is aligned and / or is in fluid communication with the second chamber (for example, the washing chamber 6121). Consequently, one or more substances contained in the first chamber (for example, the lysis chamber 6114) can be transferred to the second chamber (for example, the washing chamber 6121) by capturing or disposing a portion of the substance within the cavity 6148 when the movable member 6146 is in the first position, rotating the movable member in the second position and removing the substance from the cavity 6148.
[000178] In some modalities, the substance can be captured,
Petition 870200011939, of 01/24/2020, p. 72/198
69/184 disposed and / or maintained within cavity 6148 by a magnetic force. For example, in some embodiments, actuator 510 may include a magnetic portion. In use, actuator 510 is aligned to the desired transfer assembly 6140 and moved in lumen 6149, as shown by the arrow LL in Figure 19. Because the shape of actuator 510 can match the shape of lumen 6149, as described above, an alignment operation can be performed in some embodiments to ensure that the actuator 510 fits within the lumen 6149. When the magnetic portion of the actuator 510 is within the lumen 6149, and when the movable member 6146 is in the first position, a magnetic portion (for example, the magnetic microspheres and the nucleic acid attached to them) of the sample is moved from the first chamber (for example, the lysis chamber 6114) in cavity 6148. Actuator 510 is then rotated, as shown by MM arrow in Figures 20 and 21. When the movable member 6146 is in the second position, the actuator 510 can be removed from the lumen 6149, thus removing the magnetic force that is holding the magnetic portion of the sample into cavity 6148. Consequently, the sample portion can then be moved from cavity 6148 and into the second chamber (for example, the washing chamber 6121). The sample portion can be moved from cavity 6148 and into the second chamber (e.g., the washing chamber 6121) through any suitable mechanism, such as, for example, gravity action, fluidic motion, or the like. For example, as described below, in some embodiments, the 6130a mixing mechanism may include a nozzle (for example, nozzle 6131a) to direct a pressure jet into and / or adjacent to cavity 6148 to move the sample portion from the cavity 6148 and the second chamber (for example, the washing chamber 6121).
[000179] The use of the 6140 transfer mechanism as
Petition 870200011939, of 01/24/2020, p. 73/198
70/184 described in this document can eliminate the need for a separate refuse chamber within the first compartment 6110 and / or flow paths to transport the refuse. Instead, as previously described, the target portion of the sample is moved between the various chambers (for example, from the washing chamber 6121 to the washing chamber 6122) while other portions of the sample are maintained in the anterior chamber (for example , the washing chamber 6122). Furthermore, due to the fact that the transfer mechanism 6140 maintains fluidic isolation between the two chambers (for example, the washing chamber 6121 and the washing chamber 6122), the waste solution is prevented from entering the chamber (for example, the washing chamber 6122) next to the target portion of the sample. Therefore, this arrangement also eliminates the need to filter the mechanisms within the first compartment 6110, between the described chambers and / or within the flow paths defined by the 6100 insulation module.
[000180] The use of the 6140 transfer mechanism as described in this document also allows the target portion of the sample to be transported within the 6100 insulation module while maintaining the pressure within the insulation module at or near ambient pressure. As stated similarly, the 6140 transfer mechanism as described in this document transfers the target portion of the sample without producing a substantial pressure differential within the 6100 isolation module. Therefore, this arrangement can reduce sample leakage from the sample module. isolation.
[000181] The 6100 isolation module includes two mixing mechanisms 6130a and 6130b (also referred to as washing pumps). As described in this document, the mixing mechanisms 6130a and 6130b are configured to produce a fluid flow
Petition 870200011939, of 01/24/2020, p. 74/198
71/184 inside the washing chamber 6121 and the washing chamber 6122, respectively, in order to promote the washing or mixing of the sample portion contained therein. As stated similarly, the mixing mechanisms 6130a and 6130b are configured to transfer energy in the washing chamber 6121 and washing chamber 6122, respectively.
[000182] The 6130a mixing mechanism includes an actuator 6132a and a nozzle 6131a. The mixing mechanism 6130a is coupled to the first compartment 6110 in such a way that at least a portion of the nozzle 6131a is disposed within the washing chamber 6121. In particular, the mixing mechanism 6130a includes a coupling portion 6133a that is configured so that is coupled to a corresponding coupling portion 6134a of the first compartment 6110. Although the coupling portions 6133a and 6134a are shown defining a threaded coupling, in other embodiments, the mixing mechanism 6130a can be coupled to the first compartment 6110 by any method, such as, for example, through a mechanical fastener or retainer, a chemical or adhesive bond, an interference fit, a welded joint or the like.
[000183] Similarly, the mixing mechanism 6130b includes an actuator 6132b and a nozzle 6131b. The mixing mechanism 6130b is coupled to the first compartment 6110 in such a way that at least a portion of the nozzle 6131b is arranged within the washing chamber 6122. In particular, the mixing mechanism 6130b includes a coupling portion 6133b that is configured so that is coupled to a corresponding coupling portion 6134b of the first compartment 6110. Although the coupling portions 6133b and 6134b are shown defining a threaded coupling, in other embodiments, the 6130b mixing mechanism can be coupled
Petition 870200011939, of 01/24/2020, p. 75/198
72/184 to the first compartment 6110 by any suitable method, such as, for example, through a mechanical fastener or retainer, a chemical or adhesive bond, an interference fit, a welded joint or the like.
[000184] Actuators 6132a and 6132b include a top surface 6136a and 6136b, respectively, which is configured to be brought into contact and / or actuated by an instrument drive assembly, such as, for example, the drive 3600 of instrument 3002 described in this document. In use, the drive assembly can press and / or move the top surface 6136a and 6136b of each actuator 6132a and 6132b to produce pressure within each mixing mechanism 6130a and 6130b. The pressure is transported in the washing chambers 6121 and 6122 to promote washing, mixing and / or other interactions between the samples arranged in it. As previously described, in some embodiments, at least one of the nozzles (for example, the nozzle 6131a) may include a tip portion that is angled, bent and / or otherwise shaped to direct the pressure energy and / or flow produced by the actuator (for example, the 6132a actuator) towards a particular region within the wash chamber (for example, the wash chamber 6121). For example, in some embodiments, the nozzle 6131a can be shaped to direct the pressure and / or flow energy produced by the actuator 6132a towards the cavity 6148 of the transfer mechanism 6140.
[000185] Although actuators 6132a and 6132b are shown as a bellows pump, in other embodiments, the 6130a mixing mechanism and / or the 6130b mixing mechanism can include any mechanism suitable for producing and / or transferring energy in the wash chambers 6121 and 6122. These mechanisms may include, for example, a piston pump, a rotating member, or the like. In
Petition 870200011939, of 01/24/2020, p. 76/198
73/184 In some embodiments, a mixing mechanism may include an ultrasonic energy source, a thermal energy source or the like.
[000186] Although the mixing mechanisms 6130a and 6130b are shown and described producing and / or transferring energy in the washing chambers 6121 and 6122, respectively, in other embodiments, a mixing mechanism can also define a volume within which a substance ( for example, a wash buffer solution) can be stored in fluidic isolation from the wash chamber. Therefore, when the mixing mechanism is activated, the substance can be transferred in the washing chamber. Thus, in some embodiments, a mixing mechanism can also function as a transfer mechanism.
[000187] The amplification module (or PCR) includes a 6210 compartment (having a first 6211 end portion and a second 6212 end portion), a 6260 PCR bottle and a 6250 transfer tube. The 6260 PCR bottle is coupled to the first end portion 6211 of compartment 6210 and defines a volume 6262 within which a sample can be arranged to facilitate a reaction associated with the sample. The 6260 PCR flask can be any container suitable for containing a sample to allow a reaction associated with the sample to occur. The 6260 PCR flask can also be any suitable container for containing the sample in a way that allows monitoring of such a reaction (for example, the detection of an analyte within the sample that results or is associated with the reaction). In some embodiments, at least a portion of the 6260 PCR flask may be substantially transparent to allow an optical monitoring of a reaction occurring within it to be an optical system (for example, a 3800 optical assembly of the 3002 instrument described in this document. ).
[000188] As shown in Figures 8, 9, 10 and 22, the module of
Petition 870200011939, of 01/24/2020, p. 77/198
74/184 amplification 6200 is coupled to the second end portion 6112 of the first compartment 6110 of the isolation module 6100 in such a way that at least a portion of the transfer tube 6250 is disposed within the elution chamber 6190 of the isolation module 6100. Thus, as described herein, the isolated nucleic acid, any substances and / or any PCR reagents disposed within the elution chamber 6190 can be transported from the elution chamber 6190 to the PCR vial 6260 through transfer tube 6250 .
[000189] Compartment 6210 defines a series of reagent chambers 6213a, 6213b, 6213c (see, for example, Figure 22) and a pump well 6241. Reagent chambers 6213a, 6213b, 6213c can contain any suitable substances associated to a reaction and / or process taking place in the 6260 PCR vial. Reagent chambers 6213a, 6213b, 6213c may include, for example, an elution fluid, a master mix, probes and / or primers to facilitate the PCR process . As shown in Figure 24, compartment 6210 defines a series of passages 6221a, 6221b, 6221c configured to place each of the reagent chambers 6213a, 6213b, 6213c in fluid communication with the 6190 elution chamber of the 6100 isolation module. shown in Figure 22, in some embodiments, a piercable member can be disposed within any of the reagent chambers 6213a, 6213b, 6213c and / or within any of passages 6221a, 6221b, 6221c to fluidly isolate the respective reagent chamber from the 6190 elution chamber. Similar to that previously described with reference to the piercable member 6170, in such embodiments, the piercable member can be pierced by the reagent plunger to selectively place the reagent chamber in fluid communication with the elution chamber .
Petition 870200011939, of 01/24/2020, p. 78/198
75/184
[000190] A reagent plunger 6214a is movably disposed within reagent chamber 6213a, a reagent plunger 6214b is movably disposed within reagent chamber 6213b, and a reagent plunger 6214c is movably disposed within the 6213c reagent chamber. In this way, when the reagent plunger (for example, reagent plunger 6214a) is moved, as shown by the arrow NN in Figure 22, the reagent plunger transfers the contents of the reagent chamber (for example, the reagent chamber 6213a) in the elution chamber 6190 through the associated passage (for example, passage 6221a). In this way, the reagent plunger acts as a transfer mechanism.
[000191] Reagent plungers 6214a, 6214b, 6214c can be brought into contact and / or actuated by an instrument mounting assembly, such as, for example, the drive mounting 3600 of instrument 3002 described in this document. In some embodiments, reagent plungers 6214a, 6214b, 6214c may include a retaining mechanism (for example, a protrusion, a snap ring or the like) configured to retain a portion of an actuator assembly (for example, the assembly of actuator 3400) to facilitate the reciprocal movement of reagent plungers 6214a, 6214b, 6214c by the actuator assembly. [000192] The PCR module includes a transfer mechanism 6235 configured to transfer substances from and / or between the elution chamber 6190 of the isolation module 6100 and the PCR bottle 6260 of the PCR module 6200. The transfer mechanism 6235 includes a transfer piston 6240 disposed within the pump cavity 6241. When the transfer piston 6240 is moved within the pump cavity 6241, as shown by the arrow OO in Figure 22, a vacuum and / or positive pressure is produced within of the PCR 6262 volume. This pressure differential between the
Petition 870200011939, of 01/24/2020, p. 79/198
76/184
PCR 6262 and elution chamber 6190 results in at least a portion of the contents of elution chamber 6190 being transferred in (or from) PCR volume 6262 through transfer tube 6250 and passage 6222 (see, for example, Figure 24). In this way, substances and / or samples can be added, mixed and / or transported between the elution chamber 6190 and the PCR volume 6262 by activating the transfer mechanism 6235. The transfer mechanism 6235 can be activated by any suitable mechanism, such as, for example, the drive assembly 3600 of the instrument 3002 described in this document.
[000193] The transfer piston 6240 and the pump cavity 6241 can be in any suitable location within the PCR module 6200. For example, although the transfer piston 6240 is shown to be disposed substantially above the PCR bottle 6260, in in other embodiments, the transfer piston 6240 can be disposed substantially above the elution chamber 6190.
[000194] In some embodiments, compartment 6210 defines one or more ventilation passages for fluidly coupling the 6190 elution chamber and / or the 6260 PCR flask to the atmosphere. In some embodiments, any of these ventilation passages may include a frit to minimize and / or prevent loss of sample and / or reagents from the 6190 elution chamber and / or the 6260 PCR flask.
[000195] In use, after the nucleic acid is isolated and processed within the 6100 isolation module, as previously described, it is transferred in the 6190 elution chamber via the 6140c transfer assembly. Then, the magnetic microspheres are removed (or washed) from the nucleic acid by an elution buffer, and removed from the 6190 elution chamber. Therefore, the 6190 elution chamber contains the isolated 2 / or purified nucleic acid. In some
Petition 870200011939, of 01/24/2020, p. 80/198
77/184 embodiments, the elution buffer is contained within the 6190 elution chamber. In other embodiments, the elution buffer is contained in one of the reagent chambers (for example, reagent chamber 6213c) of the PCR module 6200, and is transferred to the 6190 elution chamber, as described above. In one embodiment, the elution buffer comprises a filtered solution of molecular grade water, tris HCl (for example, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM), magnesium chloride (for example, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM or about 20 mM), glycerol (for example, about 2%, about 3%, about 4%, about 5%, about of 6%, about 7%, about 8%, about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% or about 25%). In one embodiment, the pH of the elution buffer is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about of 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8 , 9 or about 9.0). In another embodiment, the elution buffer comprises a bactericide, for example, the elution buffer provided above which further comprises a bactericide. In one embodiment, the elution buffer also serves as a wash buffer. Although specifically described for the 6190 elution chamber, the aforementioned elution buffer, in other embodiments, is present as an R1 or R2 substance.
[000196] In some embodiments, the PCR reagents are then transported from the PCR module 6200 in the 6190 elution chamber. More particularly, the reagent plungers 6214a, 6214b and / or 6214c are driven (for example, by instrument 3002) for
Petition 870200011939, of 01/24/2020, p. 81/198
78/184 introduce the reagents into the elution chamber 6190 through passages 6221a, 6221b, 6221c. The PCR sample is then transported from elution chamber 6190 in PCR vial 6260 through transfer tube 6250 and passage 6222. In particular, transfer piston 6240 can be driven to produce a pressure differential within of the PCR module 6200 to transport the PCR sample from the 6190 elution chamber in the PCR 6260 flask, as previously described. In this way, the PCR sample (the isolated nucleic acid and the PCR reagents) is prepared in the 6190 elution chamber. Mixing the reagents and the nucleic acid sample inside the 642 elution chamber (instead of transporting the nucleic acid isolated in the PCR flask 6260 and mixing it), further transfer of the nucleic acid is avoided. This provision can result in an improved accuracy of post-PCR analysis, such that, in some cases, the analysis can be semi-quantitative in nature.
[000197] In other embodiments, however, the PCR sample (the isolated nucleic acid and the PCR reagents) can be prepared in the PCR 6260 vial. In such embodiments, for example, the PCR reagents can be stored in the vial PCR 6260, for example, in a lyophilized form. The isolated nucleic acid can be transported in the 6260 PCR vial and mixed with lyophilized PCR reagents to reconstitute the reagents inside the 6260 PCR vial.
[000198] After the PCR sample is in the 6260 PCR flask, the PCR sample can be thermally cycled (for example, through the heating assembly 3700 of the instrument 3002) to perform the desired amplification. Upon termination and / or during thermal cycling, the PCR sample can be optically analyzed (for example, through the 3800 optical assembly of the 3002 instrument)
Petition 870200011939, of 01/24/2020, p. 82/198
79/184 to analyze the sample. A description of instrument 3002 is provided below.
Figures 25 to 33 are several views of a cartridge 7001 according to an embodiment. Certain features of the 7001 cartridge are similar to the corresponding features of the 6001 cartridge, and therefore will not be described below. Where applicable, the discussion presented above for cartridge 6001 is incorporated into the discussion of cartridge 7001. For example, although actuators (for example, actuator 7163a) within second compartment 7160 have a size and / or shape that is different from the size and / or formed of the actuators (for example, the 6163a actuator) within the second compartment 6160, many aspects of the structure and function of the actuators within the second compartment 6160 are similar to those of the actuators within the compartment 7160. Consequently, the description presented above for the actuators (for example, the 6160a actuator) are applicable to the actuators (for example, the 7160a actuator) described below.
[000199] The 7001 cartridge includes a 7100 sample preparation (or isolation) module and a 7200 amplification (or PCR) module that are coupled together to form an integrated 7001 cartridge. A 7005 coating is arranged around a portion of the 7100 isolation module and the 7200 PCR module. One or more 7001 cartridges can be disposed within any suitable instrument of the types described here (see, for example, instrument 3002 described below) that is configured to manipulate, drive and / or interact with cartridge 7001 to perform isolation, transcription and / or nucleic acid amplification in a test sample contained within cartridge 7001.
[000200] As shown in Figures 26 to 28, the 7100 insulation module includes a first (or insulation) compartment
Petition 870200011939, of 01/24/2020, p. 83/198
80/184
7110 and a second (or reagent) compartment 7160 that is coupled to and / or at least partially within the first compartment 7110. The second compartment 7160 defines a series of holding chambers 7163a, 7163b, 7163c and 7163d containing the reagents and / or other substances used in the isolation process. As described in this document, the holding chambers can contain a protease (for example, Proteinase K), a lysis solution to solubilize the bulky material, a binding solution to magnetically load the resident nucleic acid sample into the lysis chamber 7114, and a solution of magnetic microspheres that bind to the magnetically charged nucleic acid to aid in the transport of the nucleic acid within the 7100 isolation module and / or the first compartment 7110. In one embodiment, the aforementioned solutions provided above are used in the cartridge provided in Figures 26 to 28.
[000201] Each of the holding chambers 7163a, 7163b, 7163c and 7163d includes an actuator arranged in a movable manner. More particularly, as shown in Figures 27 and 28, an actuator 7166a is disposed within the holding chamber 7163a, an actuator 7166b is disposed within the holding chamber 7163b, an actuator 7166c is disposed within the holding chamber 7163c, and an actuator 7166d is disposed within the holding chamber 7163d. Each of the 7166a actuators,
7166b, 7166c and 7166d is similar to the 6166 actuator shown and described previously (see, for example, Figure 14). In particular, each of the actuators 7166a, 7166b, 7166c and 7166d can function as a transfer mechanism for transporting substances from the chamber (for example, chamber 7163a) in another portion of the 7100 isolation module when moved in the indicated direction by the PP arrow in Figure 28.
[000202] As shown in Figure 27, a piercable member
Petition 870200011939, of 01/24/2020, p. 84/198
81/184
7170 is arranged around a portion of the second compartment 7160 in such a way that the internal portions of the second compartment 7160, the piercing member 7170 and the actuators 7166a, 7166b, 7166c and 7166d collectively surround and / or define the holding chambers 7163a, 7163b, 7163c and 7163d. As stated similarly, the internal portions of the second compartment 7160, the pierceable member 7170 and actuators 7166a, 7166b, 7166c and 7166d collectively define fluidically isolated chambers 7163a, 7163b, 7163c and 7163d within which reagents and / or substances can be stored. The piercable member 7170 can be constructed from any suitable material of the types described herein, such as any form of polypropylene. In some embodiments, the 7170 drillable member can be constructed from biaxially oriented polypropylene (BOP).
[000203] The second compartment 7160 includes a mixing pump 7181, which can be activated (for example, by mounting actuator 3400 of the instrument 3002) to agitate, mix and / or produce a turbulent movement within the sample, reagents and / or other substances contained with a portion (for example, lysis chamber 7114) of insulation module 7100.
[000204] As shown in Figures 26 to 28, the second compartment 7160 is disposed within an opening defined by the first compartment 7110. Therefore, when the second compartment 7160 is disposed within the first compartment 7110, the portion of the second compartment 7160 defines at least minus a portion of a boundary of the lysis chamber 7114. More particularly, when the second compartment 7160 is disposed within the first compartment 7110, the pierceable member 7170 defines a portion of the boundary of the lysis chamber 7114. This arrangement allows the substances contained inside the second compartment 7160 are
Petition 870200011939, of 01/24/2020, p. 85/198
82/184 carried in the lysis chamber 7114 when the portion of the piercable member 7170 is punctured, punctured, torn and / or broken. In a similar manner as previously described with reference, the insulation module 6100, the substances contained within the second compartment 7160 can be transported in the first compartment 7110 when actuators 7166a, 7166b, 7166c and 7166d are actuated.
[000205] As shown in Figures 27 and 28, the first compartment 7110 includes a first portion (or top) 7112 and a second portion (or bottom) 7111. In some embodiments, the top portion 7112 can be constructed separately from the bottom portion 7111, and can then be coupled to bottom portion 7111 to form the first compartment 7110. The first compartment defines the lysis chamber 7114, two wash chambers 7121 and 7122, three transfer mounting lumens (no shown in Figures 27 and 28), and an elution chamber 7190. The first compartment 7110 also defines an opening adjacent to the isolation chamber 7114 within which a portion of the second compartment 7160 is provided. [000206] As shown in Figures 26- 28, the insulation module 7100 includes a cap 7118 that is removably attached to compartment 7110. In use, a sample containing a target nucleic acid, such as, for example, urine, blood and / or other materials containing hand strips of tissue can be transported in the lysis chamber 7114 through a filling opening 7116 by removing the cap 7118. The sample can be introduced into the lysis chamber 7114 by any suitable mechanism, including, for example, pipetting or injecting the sample in the first chamber 7114 through the filling opening 7116.
[000207] After the sample is placed in the 7114 lysis chamber, reagents and / or substances can be added to facilitate cell lysis to the
Petition 870200011939, of 01/24/2020, p. 86/198
83/184 lysis chamber 7114, as previously described. In addition, the sample can be agitated and / or mixed through the 7181 pump to facilitate the lysis process, as previously described. In some embodiments, the contents of the lysis chamber 7144 can be heated (for example, by the third heating module 3780, as shown and described below with reference to the instrument 3002). In addition, the second portion 7111 of the first compartment 7110 includes an acoustic coupling portion 7182. Consequently, in some embodiments, at least a portion of an acoustic transducer (not shown) can be arranged in contact with the acoustic coupling portion 7182. In this way, the acoustic and / or ultrasonic energy produced by the transducer can be transported through the acoustic coupling portion 7182 and the side wall of the first compartment 7110, and in the solution inside the lysis chamber 7114.
[000208] The 7100 isolation module includes a series of transfer assemblies (also referred to as transfer mechanisms), shown in Figures 26 to 28 as a transfer assembly 7140a, a transfer assembly 7140b and a transfer assembly 7140c. As described in this document, transfer assemblies are configured to transfer substances (for example, portions of the sample including the magnetically charged particles and the isolated nucleic acid attached to them) between the lysis chamber 7114, the wash chamber 7121, the wash chamber 7122, and elution chamber 7190. More particularly, transfer assemblies 7140 are configured to transfer substances between lysis chamber 7114, wash chamber 7121, wash chamber 7122, and elution chamber 7190 while maintaining the isolation chamber 7114, the wash chamber 7121, the wash chamber 7122, and the elution chamber 7190
Petition 870200011939, of 01/24/2020, p. 87/198
84/184 substantially fluidly isolated from other chambers (for example, the adjacent washing chamber) defined by the first compartment 7110. Transfer assemblies 7140a, 7140b and 7140c are similar in structure and function to the transfer assemblies 6140 shown and described previously in isolation module 6100, and therefore will not be described in detail below.
[000209] The 7100 insulation module includes two wash buffer modules 7130a and 7130b that are coupled to the upper portion 7112 of the first compartment 7110. As described in this document, each wash buffer module 7130a and 7130b contains a substance (for example, example, a reagent, a wash buffer solution, a mineral oil and / or any other substance to be added to the sample), and is configured to transfer the substance into wash chamber 7121 and wash chamber 7122, respectively, when triggered . In addition, each wash buffer module 7130a and 7130b is configured to produce a flow of fluids within the wash chamber 7121 and wash chamber 7122, respectively, to promote washing and / or mixing of the sample portion contained therein. . As similarly stated, each wash buffer module 7130a and 7130b is configured to transfer energy in wash chamber 7121 and wash chamber 7122, respectively. In one embodiment, the wash buffer module 7130a and / or 7130b comprises a wash buffer comprising a molecular grade filtered water solution, tris HCl (for example, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM), magnesium chloride (e.g., about 1 mM, about 2 mM, about 3 mM, about 4 mM , about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10
Petition 870200011939, of 01/24/2020, p. 88/198
85/184 mM or about 20 mM), glycerol (for example, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8% , about 9%, about 10%, about 12%, about 14%, about 16%, about 18%, about 20% or about 25%). In one embodiment, the pH of the wash buffer is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about of 8.1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8 , 9 or about 9.0). In another embodiment, the wash buffer comprises a bactericide, for example, the wash buffer provided above which further comprises a bactericide.
[000210] Although specifically described for chambers 7130a and / or 7130b, the wash buffer described immediately above, in other embodiments, is present as a substance R1 and / or R2.
[000211] In another embodiment, the wash buffer module 7130a and / or 7130b comprises a wash buffer comprising a filtered solution of molecular grade water, guanidine HCl (e.g., about 0.7 mM, about 0 .8 mM, about 0.81 mM, about 0.82 mM, about 0.83 mM, about 0.84 mM, about 0.85 mM, about 0.9 mM, about 1, 0 mM), tris HCl (for example, about 10 mM, about 15 mM, about 20 mM, about 25 mM, about 30 mM, about 35 mM, or about 40 mM, and can have a pH of about 7.5, about 8 or about 8.5), triton-X-100 (for example, about 0.25%, about 0.5%, about 0.75%, about 1%), Tween-20 (for example, about 0.25%, about 0.5%, about 0.75%, about 1%), EDTA (for example, about 0.1 mM , about 0.2 mM, about 0.3 mM, about 0.5 mM, about 0.75 mM, about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM or about 20 mM), isopropanol (for example, about
Petition 870200011939, of 01/24/2020, p. 89/198
86/184 of 10%, about 20%, about 30%, about 40%, about 50%, about 60%). In one embodiment, the pH of the elution buffer is about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8.0, about 8 , 1, about 8.2, about 8.3, about 8.4, about 8.5, about 8.6, about 8.7, about 8.8, about 8.9 or about 9.0). Although described specifically for chambers 7130a and / or 7130b, the wash buffer described immediately above, in other embodiments, is present as a substance R1 and / or R2.
[000212] The wash buffer module 7130a includes an actuator 7150a which is movably disposed within a compartment 7137a. compartment 7137a is coupled to the upper portion 7112 of the first compartment 7110 in such a way that the wash buffer module 7130a is substantially aligned with the wash chamber 7121. In particular, compartment 7137a includes a pair of protrusions 7133a which is configured so that it is arranged within a corresponding opening defined by a coupling portion 7134a of the upper portion 7112 of the first compartment 7110. Although the wash buffer module 7130a is shown to be coupled to the first compartment 7110 by a pressure fitting, in other embodiments, the wash buffer module 7130a can be coupled to the first compartment 7110 by any suitable method, such as, for example, by a threaded coupling, a mechanical fastener or retainer, a chemical or adhesive connection, an interference fit, a welded joint or the like.
[000213] Actuator 7150a includes a plunger portion 7151a, a drilling portion 7152a and an engagement portion 7153a. The engagement portion 7153a is configured to engage, be removably coupled and / or to be received within a portion of an actuator assembly to facilitate movement of the 7150a actuator within the
Petition 870200011939, of 01/24/2020, p. 90/198
87/184 compartment 7137a, as described in this document. The 7150a actuator can be manipulated and / or actuated by any suitable instrument, such as the 3600 actuator assembly described below in relation to Figures 47 to 51.
[000214] The plunger portion 7151a of actuator 7150a is disposed within housing 7137a. A piercable member 7135a is arranged around the end portion of compartment 7137a such that the end face of the plunger portion 7151a, compartment 7137a and piercable member 7135a collectively define a volume within which a substance is disposed. The plunger portion 7151a and the inner surface of housing 7137a are configured to form a substantially impermeable and / or fluid-tight seal. In some embodiments, the plunger portion 7151a may include a sealing member, an O-ring or the like. [000215] The drilling portion 7152a of the 7150a actuator is configured to drill, break, tear and / or break the piercing member portion 7135a when the 7150a actuator is moved into compartment 7137a in the direction indicated by the arrow QQ in Figure 28. From this In this way, the movement of the 7150 actuator places the chamber in fluid communication with the washing chamber 7121. As similarly stated, the wash buffer module 7130a can be selectively placed in fluid communication with the washing chamber 7121 when the 7150a actuator is triggered. After the substance inside the wash buffer module 7130a is transported in the wash chamber 7121, the actuator 7150a can be reciprocated within compartment 7137a to produce a pressure that is carried inside the wash chamber 7121 to promote washing, mixing and / or other interaction between the samples arranged in it. The upper portion 7112 of the first compartment 7110 includes a nozzle 7131a configured to direct
Petition 870200011939, of 01/24/2020, p. 91/198
88/184 the pressure energy and / or the flow produced by the 7150a actuator towards a particular region within the washing chamber 7121. [000216] The 7130b wash buffer module includes an 7150b actuator that is movably disposed within of a 7137b compartment. The compartment 7137b is coupled to the upper portion 7112 of the first compartment 7110 in such a way that the wash buffer module 7130b is substantially aligned with the wash chamber 7122. In particular, the compartment 7137b includes a pair of protrusions 7133b which is configured so that it is disposed within a corresponding opening defined by a coupling portion 7134b of the upper portion 7112 of the first compartment 7110. Although the wash buffer module 7130b is shown to be coupled to the first compartment 7110 through a press fit, in other embodiments , the wash buffer module 7130b can be attached to the first compartment 7110 by any suitable method, such as, for example, through a threaded coupling, a mechanical fastener or retainer, a chemical or adhesive connection, an adjustment of interference, a welded joint or the like.
[000217] Actuator 7150b includes a plunger portion 7151b, a drilling portion 7152b and an engagement portion 7153b. The engagement portion 7153b is configured to engage, be removably coupled and / or to be received within a portion of an actuator assembly to facilitate movement of the 7150b actuator within compartment 7137b, as described in this document. The 7150b actuator can be manipulated and / or actuated by any suitable instrument, such as the 3600 actuator assembly described below in relation to Figures 47 to 51.
[000218] The plunger portion 7151b of actuator 7150b is disposed within housing 7137b. A 7135b drillable member is
Petition 870200011939, of 01/24/2020, p. 92/198
89/184 disposed around the end portion of the compartment 7137b such that the end face of the plunger portion 7151b, the compartment 7137b and the pierceable member 7135b collectively define a volume within which a substance is disposed. The plunger portion 7151b and the inner surface of housing 7137b are configured to form a substantially impermeable and / or fluid-tight seal. In some embodiments, the plunger portion 7151b may include a sealing member, an O-ring or the like. [000219] Drilling portion 7152b of actuator 7150b is configured to drill, break, tear and / or break the portion of piercable member 7135b when actuator 7150b is moved into compartment 7137b in the direction indicated by the arrow QQ in Figure 28. From this In this way, the movement of the 7150b actuator places the chamber in fluid communication with the washing chamber 7122. As similarly stated, the wash buffer module 7130b can be selectively placed in fluid communication with the washing chamber 7122 when the 7150b actuator is triggered. After the substance inside the wash buffer module 7130b is transported in the wash chamber 7122, the actuator 7150b can be reciprocated within the compartment 7137b to produce a pressure that is carried in the wash chamber 7122 to promote washing, mixing and / or other interaction between the samples arranged in it. The upper portion 7112 of the first compartment 7110 includes a nozzle 7131b configured to direct the pressure energy and / or the flow produced by the actuator 7150b towards a particular region within the washing chamber 7122.
[000220] As shown in Figures 29 to 31, the amplification module (or PCR) 7200 includes a substrate 7220 that is constructed from a first layer (or higher) 7227 and a second layer (or lower) 7228. The module PCR 7200 includes a bottle of
Petition 870200011939, of 01/24/2020, p. 93/198
90/184
PCR 7260 coupled to second layer 7228, a transfer mechanism 7235, a first reagent module 7270a and a second reagent module 7270b. The 7260 PCR bottle is coupled to the first end portion 7211 of compartment 7210 and defines a volume 7262 within which a sample can be arranged to facilitate a reaction associated with the sample. The 7260 PCR flask can be any container suitable for containing a sample to allow a reaction associated with the sample to occur. The 7260 PCR flask can also be any suitable container for containing the sample to allow monitoring of such a reaction (for example, the detection of an analyte within the sample that results in or is associated with the reaction). In some embodiments, at least a portion of the 7260 PCR vial may be substantially transparent to allow an optical monitoring of a reaction occurring therein to be an optical system (for example, the 3800 optical assembly of the 3002 instrument described in this document) .
[000221] As shown in Figures 32 and 33, the amplification module 7200 is coupled to the first compartment 7110 of the isolation module 7100 in such a way that at least a portion of a transfer tube 7250 is arranged inside the elution chamber 7190 of the isolation module 7100. In this way, as described in this document, the isolated nucleic acid, any substances and / or any PCR reagents disposed within the 7190 elution chamber can be transported from the 7190 elution chamber to the 7260 PCR vial. through transfer tube 7250. More particularly, substrate 7220 defines a flow passage 7222 that places the PCR bottle 7260 in fluid communication with the elution chamber 7190 when the PCR module 7200 is coupled to the 7100 isolation module. shown in Figures 30 and 31, the flow passage portions 7222 are defined
Petition 870200011939, of 01/24/2020, p. 94/198
91/184 in transfer tube 7250 and a transfer port 7229 of second layer 7228 of substrate 7220. Although flow passage 7222 is shown to be defined primarily by second layer 7228 of substrate 7220, in other embodiments, flow 7222 can be defined by the first layer 7227 or in portions of both the first layer 7227 and the second layer
7228.
[000222] Substrate 7220 also defines a flow passage 7223, a flow passage 7221a and a flow passage 7221b. As described in greater detail in this document, flow passage 7223 is configured to place a volume 7237 defined within the transfer mechanism 7235 in fluid communication with the PCR bottle 7260 through the transfer port
7229. Flow passage 7221a is configured to place a volume defined by reagent module 7270a in fluid communication with the elution chamber 7190 through transfer tube 7250. Flow passage 7221b is configured to place a volume defined by the reagent 7270b in fluid communication with the PCR vial 7260 through transfer port 7229 and / or a portion of passage 7222. Either between flow passage 7223, flow passage 7221a and / or flow passage 7221b can be defined by the first layer 7227, the second layer 7228, or portions of both the first layer 7227 and the second layer 7228.
[000223] The PCR module 7200 includes two reagent modules 7270a and 7270b that are coupled to the top layer 7227 of the substrate 7220. As described in this document, each reagent module 7270a and 7270b contains a substance, R1 and R2, respectively. The reagent module 7270a is configured to transport substance R1 in the 7190 elution chamber via the
Petition 870200011939, of 01/24/2020, p. 95/198
92/184 flow passage 7221a, as described in this document. The 7270b reagent module is configured to transport substance R2 in the 7260 PCR vial through flow passage 7221b, as described in this document. In this way, each reagent module 7270a and 7270b functions as a reagent storage device and a transfer mechanism.
[000224] Substances R1 and R2 can, for example, be a reagent, an elution buffer solution, a wash buffer solution, a mineral oil and / or any other substance to be added to the sample, as described in this document. In some embodiments, substance R1 may include an elution buffer and mineral oil. In some embodiments, the substance R2 may include reaction reagents that facilitate a PCR process inside the 7260 PCR flask. In some embodiments, a PCR master mix can be arranged inside the 7260 PCR flask in such a lyophilized state. that the addition of substance R2 and / or a mixture of substance R1 and the target sample reconstitutes the lyophilized master mix to facilitate the PCR process.
[000225] For example, in a modality where HSV is amplified by PCR, the master mix is a lyophilized pellet comprising HSV1 and HSV2 primers specific for an HSV1 and / or HSV2 sequence, detection probe (for example, a probe hybridization oligonucleotide comprising a fluorophore and MGB at the 5 'termination and a non-fluorescent suppressor at the 3' termination), and internal control and probe primers, KCl (for example, about 40 mM, about 50 mM, about 60 mM, about 70 mM), mannitol (for example, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM), BSA (for example , about 0.1 mg / ml, about 0.5 mg / ml, about 1 mg / ml), dNTPs (e.g., about 0.2 mM, about 0.3 mM, about 0, 4 mM, about 0.5 mM, about
Petition 870200011939, of 01/24/2020, p. 96/198
93/184 mM), Taq polymerase (e.g., about 0.1 U / pL, about 0.2 U / pL, about 0.3 U / pL).
[000226] In another modality, a master mix comprises freeze-dried reagents to perform a multiplexing PCR on three targets and an internal control. In an additional embodiment, the target nucleic acids consist of an influenza A specific nucleic acid, an influenza B specific nucleic acid and an RSV specific nucleic acid. In an additional embodiment, the multiplexing reaction is monitored in real time, for example, by providing a hybridization oligonucleotide probe, specific for each target sequence, in which each probe comprises a fluorophore and MGB at the 5 'termination and a suppressor non-fluorescent at 3 'termination.
[000227] In another embodiment, the lyophilized master mix comprises reagents for both a PCR and a reverse transcriptase reaction. For example, in one embodiment, the lyophilized master mix includes reverse transcriptase enzymes and Taq polymerase enzymes, dNTPs, RNase inhibitor, KCl, BSA and primers to perform first-strand cDNA synthesis and PCR.
[000228] The master mix comprises different initiators and probes, depending on the target to be amplified. Each target will have a specific primer and probe set associated with it, and the primer and probe set can be lyophilized with the other PCR reagents mentioned above, to form a lyophilized master mix. Component concentrations will also vary depending on the particular target being amplified, and if multiple targets are amplified.
[000229] The reagent module 7270a includes an actuator 7280a that is movably disposed within a compartment 7277a. The compartment 7277a is coupled to the top layer 7227 of the substrate 7220 in such a way that the reagent module 7270a is substantially aligned with the passage 7221a, to the transfer tube
Petition 870200011939, of 01/24/2020, p. 97/198
94/184
7250 and / or to the elution chamber 7190. As shown in Figure 29, compartment 7277a includes a pair of protrusions 7273a that are configured to be arranged within a corresponding opening defined by a coupling portion 7234a of the top layer 7227 of the substrate 7220. While the 7270a reagent module is shown to be coupled to the 7220 substrate via a pressure fitting, in other embodiments, the 7270a reagent module can be coupled to the 7220 substrate by any suitable method, such as, for example, through a threaded coupling, a mechanical fastener or retainer, a chemical or adhesive bond, an interference fit, a welded joint or the like.
[000230] The actuator 7280a includes a plunger portion 7281a, a drilling portion 7282a and an engagement portion 7283a. The engagement portion 7283a is configured to engage, to be removably coupled and / or to be received within a portion of an actuator assembly to facilitate movement of the 7280a actuator within compartment 7277a, as described in this document. The 7280a actuator can be manipulated and / or actuated by any suitable instrument, such as the 3600 actuator assembly described below in relation to Figures 47 to 51.
[000231] The plunger portion 7281a of actuator 7280a is disposed within housing 7277a. A piercable member 7275a is arranged around the end portion of compartment 7277a such that the end face of plunger portion 7281a, compartment 7277a and pierced member 7275a collectively define a volume within which the substance R1 is disposed. The plunger portion 7281a and the inner surface of compartment 7277a are configured to form a substantially impermeable and / or fluid-tight seal. In some modalities, the
Petition 870200011939, of 01/24/2020, p. 98/198
95/184 plunger portion 7281a may include a sealing member, an O-ring or the like.
[000232] The drilling portion 7282a of the 7280a actuator is configured to drill, break, tear and / or break a portion of the piercable member 7275a when the 7280a actuator is moved into the 7277a housing in the direction indicated by the arrow SS in Figure 31. From this In this way, the movement of the actuator 7280a places the volume in fluid communication with passage 7221a, and therefore the elution chamber 7190. As similarly stated, reagent module 7270a can be selectively placed in fluid communication with elution chamber 7190 when actuator 7280a is actuated.
[000233] The reagent module 7270b includes an actuator 7280b that is movably disposed within a compartment 7277b. The compartment 7277b is coupled to the top layer 7227 of the substrate 7220 in such a way that the reagent module 7270b is substantially aligned with the passage 7221b. As shown in Figure 29, compartment 7277b includes a pair of protrusions 7273b that are configured so that they are arranged within a corresponding opening defined by a coupling portion 7234b of upper layer 7227 of substrate 7220. Although reagent module 7270b is shown as being coupled to the substrate 7220 via a pressure fitting, in other embodiments, the reagent module 7270b can be coupled to the substrate 7220 by any suitable method, such as, for example, through a threaded coupling, an mechanical fixation or retainer, a chemical or adhesive bond, an interference fit, a welded joint or the like.
[000234] The actuator 7280b includes a piston portion 7281b, a drilling portion 7282b and an engagement portion 7283b. The portion
Petition 870200011939, of 01/24/2020, p. 99/198
Coupling 96/184 7283b is configured to engage, be removably coupled and / or to be received within a portion of an actuator assembly to facilitate movement of the 7280b actuator within compartment 7277b, as described in this document. The 7280b actuator can be manipulated and / or actuated by any suitable instrument, such as the 3600 actuator assembly described below in relation to Figures 47 to 51.
[000235] The plunger portion 7281b of the actuator 7280b is disposed within the housing 7277b. A piercable member 7275b is arranged around the end portion of compartment 7277b such that the end face of plunger portion 7281b, compartment 7277b and pierced member 7275b collectively define a volume within which substance R2 is disposed. The plunger portion 7281b and the inner surface of compartment 7277b are configured to form a substantially impermeable and / or fluid-tight seal. In some embodiments, the plunger portion 7281a may include a sealing member, an O-ring or the like.
[000236] The drilling portion 7282b of the 7280b actuator is configured to drill, break, tear and / or break a portion of the piercable member 7275b when the 7280b actuator is moved into the 7277b compartment in the direction indicated by the arrow SS in Figure 31. From this In this way, the movement of the 7280b actuator places the volume in fluid communication with the passage 7221b, and therefore with the 7260 PCR chamber.
[000237] The PCR module 7200 includes a transfer mechanism 7235 configured to transfer substances from and / or between the elution chamber 7190 of the isolation module 7100 and the PCR bottle 7260 of the PCR module 7200. As described herein document, the 7235 transfer mechanism is also
Petition 870200011939, of 01/24/2020, p. 100/198
97/184 configured to define a volume 7237 within which a substance may be contained, and selectively place volume 7237 in fluid communication with the 7260 PCR vial. In this way, the transfer mechanism 7235 also acts as a control mechanism for flow.
[000238] The transfer mechanism 7235 includes an actuator 7240 disposed within a compartment 7236. The compartment 7236 is coupled and / or consists of a portion of the top layer 7227 of the substrate 7220. The compartment 7236 defines a volume 7237 within which a substance, such as, for example, mineral oil, can be stored. Although not shown including a piercable member, in other embodiments, a portion of volume 7237 can be surrounded and / or fluidly isolated by a piercable member, as described herein.
[000239] Actuator 7240 includes a plunger portion 7241, a valve portion 7242 and an engaging portion 7243. The engaging portion 7243 is configured to engage, be removably coupled and / or to be received within a portion of an actuator assembly to facilitate the movement of the 7240 actuator within the 7236 housing, as described in this document. The 7240 actuator can be manipulated and / or actuated using any suitable instrument, such as the 3600 actuator assembly described below in relation to Figures 47 to 51.
[000240] Plunger portion 7241 of actuator 7240 is disposed within compartment 7236. Plunger portion 7241 and the inner surface of compartment 7236 are configured to form a substantially impermeable and / or fluid-tight seal. In some embodiments, the plunger portion 7241 may include a sealing member, an O-ring or the like. In addition, a seal 7244 is disposed in the top portion of compartment 7236.
Petition 870200011939, of 01/24/2020, p. 101/198
98/184
[000241] The 7240 actuator is configured to be moved within compartment 7236 between a first position (Figure 30) and a second position (Figure 31). When the actuator 7240 is in the first position, the valve portion 7242 of the actuator 7240 is arranged at least partially within the flow passage 7223 such that the volume 7237 is substantially fluidly isolated from the flow passage 7223 and / or the 7260 PCR bottle. As stated similarly, when the 7240 actuator is in the first position, a portion of the valve portion 7242 is in contact with the top layer 7227 to produce a substantially impermeable and / or fluid-tight seal. When the actuator 7250 is moved into compartment 7236 in the direction indicated by the RR arrow in Figure 31, the valve portion 7242 is separated from the top layer 7227 and / or removed from flow passage 7223, thus placing the volume 7237 in communication fluidic with passage 7223, and therefore with the 7260 PCR chamber. In this way, when the 7240 actuator is moved, the substance within the 7237 volume can be transported in the 7262 PCR volume defined by the 7260 PCR bottle. [000242 ] In addition, when the 7240 actuator is moved into compartment 7236, as shown by the arrow RR in Figure 31, a vacuum is produced within the PCR volume 7262 of the PCR 7260 bottle. This pressure differential between the PCR volume 7262 and the elution chamber 7190 results in at least a portion of the contents of the elution chamber 7190 being transferred in the PCR volume 7262 through transfer tube 7250 and passage 7222 (see, for example, Figure 24). In this way, substances and / or samples can be added, mixed and / or transported between the elution chamber 7190 and the PCR volume 7262 by activating the transfer mechanism 7235. The transfer mechanism 7235 can be activated through any mechanism appropriate, such as,
Petition 870200011939, of 01/24/2020, p. 102/198
99/184 for example, the drive assembly 3600 of the instrument 3002 described in this document.
[000243] In use, after one or more target nucleic acids, or a population of nucleic acids is isolated and processed within the 7100 isolation module, as described above, this is transferred to the 7190 elution chamber via transfer assembly 7140c. The reagent module 7270a can then be triggered to carry the substance R1 in the 7190 elution chamber. For example, in some embodiments, the reagent module 7270a can be triggered to carry a solution containing an elution buffer and mineral oil. in the 7190 elution chamber. The magnetic microspheres are then removed (or washed) from the nucleic acid by the elution buffer, and removed from the 7190 elution chamber (for example, by transfer assembly 7140c). Therefore, the 7190 elution chamber contains isolated and / or purified nucleic acid.
[000244] The 7270b reagent module can be triggered to carry substance R2 in the PCR volume 7262. For example, in some embodiments, the 7270b reagent module can be triggered to carry a solution containing various reaction reagents in the PCR bottle. 7260. In some embodiments, the 7260 PCR bottle may contain additional reagents and / or substances, such as, for example, a PCR master mix, in a lyophilized state. Consequently, when the substance R2 is transported in the 7260 PCR vial, the lyophilized contents can be reconstituted in preparation for the reaction.
[000245] The target S sample can be transported (either before or after activating the reagent module 7270b described previously) from the elution chamber 7190 in the PCR vial 7260 through transfer tube 7250 and passage 7222. In particular , the 7240 actuator of the 7235 transfer mechanism can be actuated
Petition 870200011939, of 01/24/2020, p. 103/198
100/184 to produce a pressure differential within the PCR module 7200 to transport the PCR sample from the elution chamber 7190 in the PCR bottle 7260 through passage 7222, as previously described. In this way, the PCR sample (the isolated nucleic acid and the PCR reagents) can be partially prepared in the 7190 elution chamber. In addition, when the transfer mechanism 7235 is activated, the volume 7237 defined here is placed in fluid communication with PCR volume 7262 through passage 7223, as previously described. Therefore, in some embodiments, an additional substance (for example, a mineral oil) can be added to the PCR bottle through the same operation as the sample transfer operation.
[000246] After the PCR sample is in the 7260 PCR flask, at least a portion of the PCR S sample can be thermally cycled (for example, through the heating assembly 3700 of the instrument 3002) to perform the desired amplification. Upon termination and / or during thermal cycling, the PCR sample can be optically analyzed (for example, through the 3800 optical assembly of instrument 3002) to analyze the sample. Alternatively, as described in this document, the PCR sample can be optically analyzed during PCR, for example, with DNA hybridization probes, each conjugated to a MGB and fluorophore. A description of the instrument 3002 and other suitable instruments for handling the cartridge is provided below.
[000247] Any of the cartridges described here can be manipulated and / or activated by any suitable instrument to perform an isolation and / or reaction process on a sample contained within the cartridge. For example, in some embodiments, any of the cartridges described here can be manipulated and / or triggered by an instrument to perform isolation and amplification
Petition 870200011939, of 01/24/2020, p. 104/198
101/184 nucleic acid in real time in a test sample inside the cartridge. In this way, the system (for example, the cartridge or a series of cartridges and an instrument) can be used for many different assays, such as, for example, the rapid detection of influenza (influenza) A, influenza B, and syncytial virus (RSV) of nasopharyngeal samples.
[000248] In some modalities, an instrument can be configured to facilitate, produce, support and / or promote a reaction in a sample contained in a reaction chamber defined by a cartridge of the types shown and described in this document. This instrument may also include an optical assembly to detect one or more different substances and / or analytes within the sample before, during and / or after the reaction. For example, Figure 34 is a schematic illustration of an instrument 1002 according to an embodiment. Instrument 1002 includes a block 1710, a first optical member 1831, a second optical member 1832 and an optical assembly 1800. Block 1710 defines a reaction volume 1713 configured to receive at least a portion 261 of a reaction vessel 260 containing a sample S. Reaction vessel 260 can be any suitable container for containing sample S so as to allow a reaction associated with sample S. to occur. Reaction vessel 260 can also be any vessel suitable for containing sample S so to allow the monitoring of such a reaction (for example, the detection of an analyte within the sample S that results from, or is associated with, the reaction). In some embodiments, for example, reaction vessel 260 may be a PCR flask, test tube or the like. Furthermore, in some embodiments, at least portion 261 of reaction vessel 260 may be substantially transparent to allow optical monitoring of a reaction to occur.
Petition 870200011939, of 01/24/2020, p. 105/198
102/184
[000249] Block 1710 can be any suitable structure and / or can be coupled to any suitable mechanism to facilitate, produce, support and / or promote a reaction associated with sample S in reaction vessel 260. For example, in some embodiments, block 1710 can be coupled to and / or can include a mechanism to cyclically heat sample S in reaction vessel 260. In this way, block 1710 can produce a thermally induced reaction of sample S, such as, for example, a PCR. In other embodiments, block 1710 may be coupled to and / or may include a mechanism for introducing one or more substances into reaction vessel 260 to produce a chemical reaction associated with sample S. [000250] Reaction volume 1713 can be any size and / or shape suitable for containing portion 261 of reaction chamber 260. In some embodiments, for example, the shape of reaction volume 1713 may substantially match the shape of portion 261 of reaction chamber 260 (for example, as shown in Figure 34). In other embodiments, however, the shape of reaction volume 1713 can be dissimilar to the shape of portion 261 of reaction chamber 260. Although portion 261 of reaction chamber 260 is shown in Figure 34 as a separate sense from the side wall of the block 1710 which defines reaction volume 1713, in other embodiments, portion 261 of reaction chamber 260 may be in contact with a portion of block 1710. In yet other embodiments, reaction volume 1713 may contain a substance (for example, a salt water solution, a thermally conductive gel, or the like) disposed between the portion 261 of the reaction chamber 260 and the portion (e.g., a side wall) of the 1710 block.
[000251] Although block 1710 is shown in Figure 34 containing only portion 261 of reaction chamber 260 within reaction volume 1713, in other embodiments, block 1710 can be
Petition 870200011939, of 01/24/2020, p. 106/198
103/184 configured such that the entire reaction chamber 260 is received within reaction volume 1713. In some embodiments, for example, block 1710 may include a coating or other mechanism (not shown in Figure 34) that substantially retains the entire reaction chamber 260 within reaction volume 1713. Furthermore, in some embodiments, block 1710 may substantially surround the entire reaction chamber 260. In other embodiments, block 1710 may substantially surround portion 261 of reaction chamber 260 arranged within reaction volume 1713.
[000252] As shown in Figure 34, the first optical member 1831 is arranged at least partially within the block 1710 such that the first optical member 1831 is in optical communication with the reaction volume 1713. In this way, a beam of light (and / or an optical signal) can be transported between reaction volume 1713 and a region outside block 1710 through the first optical member 1831. The first optical member 1831 can be any suitable structure, device and / or mechanism through which a beam of light can be carried. In some embodiments, the first optical member 1831 can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber. In other embodiments, the first optical member 1831 may include a mechanism configured to modify and / or transform a beam of light, such as, for example, an optical amplifier, an optical signal converter, a lens, an optical filter or the like. In yet other embodiments, the second optical member 1832 may include a light-emitting diode (LED), a laser or other device configured to produce a beam of light.
[000253] The second optical member 1832 is arranged at least partially within the block 1710 such that the second member
Petition 870200011939, of 01/24/2020, p. 107/198
104/184 optical 1832 is in optical communication with reaction volume 1713. In this way, a beam of light (and / or an optical signal) can be transported between reaction volume 1713 and a region outside block 1710 through the second optical member 1832. The second optical member 1832 can be any suitable structure, device and / or mechanism through which a beam of light can be transported. In some embodiments, the second optical member 1832 can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber. In other embodiments, the second optical member 1832 may include a mechanism configured to modify and / or transform a beam of light, such as, for example, an optical amplifier, an optical signal converter, a lens, an optical filter or the like. In still other embodiments, the second optical member 1832 may include a photodiode or other device configured to receive and / or detect a beam of light.
[000254] Optical assembly 1800 includes an excitation module 1860 and a detection module 1850. The excitation module 1860 is configured to produce a series of excitation light beams (and / or optical signals, not shown in Figure 34) . Consequently, the excitation module 1860 can include any device and / or mechanism suitable for producing the series of excitation light beams, such as, for example, a laser, one or more light-emitting diodes (LEDs), a flash, or the like. In some embodiments, each beam of light produced by the 1860 excitation module may have substantially the same characteristics (for example, wavelength, amplitude and / or energy) as each of the other light beams produced by the 1860 excitation module. other modalities, however, a first beam of light produced by the 1860 excitation module may
Petition 870200011939, of 01/24/2020, p. 108/198
105/184 example, wavelength, amplitude and / or energy) different from one of the other beams of light produced by the excitation module 1860. In some embodiments, for example, the excitation module 1860 may include a series of LEDs, each one configured to produce a light beam having a different wavelength than the light beams produced by the other LEDs.
[000255] The 1850 detection module is configured to receive a series of emission light beams (and / or optical signals, not shown in Figure 34). Consequently, the detection module 1850 can include any suitable photodetector, such as, for example, an optical detector, a photoresistor, a photovoltaic cell, a photodiode, a phototube, a CCD camera or the like. The emission light beams can be produced by any suitable source, such as, for example, by exciting a constituent of sample S. In some embodiments, the 1850 detection module can be configured to selectively receive each light beam from emission independently if each light beam has the same characteristics (for example, wavelength, amplitude and / or energy) as each of the other emission light beams. In other embodiments, however, the 1850 detection module can be configured to selectively receive each beam of emission light based on the particular characteristics (for example, wavelength, amplitude and / or energy) of the light beam. In some embodiments, for example, the 1850 detection module may include a series of photodetectors, each configured to receive a beam of light having a different wavelength than the light beams received by the other photodetectors.
[000256] As shown in Figure 34, the first optical member 1831 and the second optical member 1832 are coupled to the optical assembly 1800. In this way, each one among the series of light beams of
Petition 870200011939, of 01/24/2020, p. 109/198
106/184 excitation can be carried in reaction volume 1713 and / or in portion 261 of reaction vessel 260, and each between the series of emission light beams can be received from reaction volume 1713 and / or the portion 261 of reaction vessel 260. More particularly, the first optical member 1831 is coupled to excitation module 1860 in such a way that the series of excitation light beams produced by excitation module 1860 can be transported in reaction volume 1713 and / or in the 261 portion of the reaction vessel 260. Similarly, the second optical member 1832 is coupled to the detection module 1850 in such a way that each of the plurality of emission light beams can be received from the volume of reaction 1713 and / or portion 261 of reaction vessel 260.
[000257] The series of light beams produced by excitation module 1860 is transported in reaction volume 1713 and / or in portion 261 of reaction vessel 260 by the first optical member 1831, and along a first light path 1806. Therefore, each of the series of light beams produced by the excitation module 1860 is transported in reaction volume 1713 and / or in portion 261 of reaction vessel 260 at a substantially constant location. Similarly, the series of light beams received by the detection module 1850 is received from reaction volume 1713 and / or portion 261 of reaction vessel 260 by the second optical member 1832, and along a second path of light 1807. Therefore, each of the series of light beams received by the detection module 1850 is received from reaction volume 1713 and / or portion 261 of reaction vessel 260 at a substantially constant location. By converting and receiving the excitation light beams and the emission light beams, respectively, at a constant location within the 1713 reaction volume, one can reduce the detection variability within an associated multi-channel analysis. to
Petition 870200011939, of 01/24/2020, p. 110/198
107/184 transporting excitation light beams from several different locations and / or receiving emission light beams from multiple different locations.
[000258] Furthermore, including the first optical member 1831 and the second optical member 1832 within the 1710 block, the position of the first optical member 1831 (and the first light path 1806) and / or the position of the second optical member 1832 (and the second light path 1807) in relation to reaction volume 1713 is constant. This arrangement can also reduce the test-test detection variability associated with light paths and / or optical members minimizing and / or eliminating the relative movement between the first optical member 1831, the second optical member 1832 and / or the volume of reaction 1713.
[000259] In some embodiments, the series of excitation light beams can be sequentially carried in reaction volume 1713, and the series of emission light beams can be received sequentially from reaction volume 1713. For example, in In some embodiments, the 1860 excitation module can produce a series of light beams, each having a different wavelength, sequentially (or cyclically). Each beam of light is transported in reaction volume 1713, where the beam of light can, for example, excite the sample S contained within the reaction vessel 260. Similarly, in such embodiments, the beam of emission light is produced (as a result of the excitation of certain analytes and / or targets within the S sample) sequentially (or cyclically). Therefore, the 1850 detection module can receive a series of light beams, each having a different wavelength, in a sequential (or cyclic) mode. In this way, the 1802 instrument can be used to detect multiple different analytes and / or targets within the sample S.
[000260] Although the portion of the first optical member 1831 arranged
Petition 870200011939, of 01/24/2020, p. 111/198
108/184 within block 1710 and the portion of the second optical member 1832 disposed within block 1710 are shown in Figure 34 as being substantially parallel and / or within the same plane, in other embodiments, a block may include a first optical member that is in any position and / or orientation relative to a second optical member. As stated similarly, although the first light path 1806 is shown in Figure 34 as being substantially parallel and / or within the same plane as the second light path 1807, in other embodiments, an instrument can be configured to produce a first path light that is in any position and / or orientation relative to a second light path.
[000261] For example, Figure 35 shows a schematic illustration in partial cross section of a portion of a 2002 instrument according to a modality. The 2002 instrument includes a block 2710, a first optical member 2831, a second optical member 2832 and an optical assembly (not shown in Figure 35). Block 2710 defines a reaction volume 2713 configured to receive at least a portion 261 of a reaction vessel 260 containing a sample S. The reaction vessel 260 can be any vessel suitable for containing sample S so as to allow it to occur a reaction associated with sample S, and allow the monitoring of such reaction, as described in this document. In some embodiments, for example, reaction vessel 260 may be a PCR flask, test tube or the like. In addition, in some embodiments, at least the portion 261 of the reaction vessel 260 may be substantially transparent to allow optical monitoring of a reaction occurring therein. [000262] Block 2710 can be any suitable structure and / or can be coupled to any suitable mechanism to facilitate,
Petition 870200011939, of 01/24/2020, p. 112/198
109/184 produce, support and / or promote a reaction associated with sample S in reaction vessel 260. For example, in some embodiments, block 2710 may be coupled and / or may include a mechanism for cyclically heating sample S in the vessel reaction 260. In this way, block 2710 can produce a thermally induced reaction of sample S, such as, for example, a PCR process. In other embodiments, block 2710 may be coupled and / or may include a mechanism for introducing one or more substances into reaction vessel 260 to produce a chemical reaction associated with sample S. [000263] Reaction volume 2713 can be of any size and / or suitable shape to contain portion 261 of reaction chamber 260. As shown in Figure 35, reaction volume 2713 defines a longitudinal geometric axis LA and substantially surrounds portion 261 of reaction chamber 260 when portion 261 is arranged within reaction volume 2713. In this way, any stimulus (for example, heating or cooling) provided to sample S by block 2710 or any mechanisms attached to it can be provided substantially and spatially uniformly.
[000264] As shown in Figure 35, the first optical member 2831 is arranged at least partially within the block 2710 in such a way that the first optical member 2831 defines a first light path 2806 and is in optical communication with the reaction volume 2713 In this way, a beam of light (and / or an optical signal) can be transported between reaction volume 2713 and a region outside block 2710 through the first optical member 2831. The first optical member 2831 can be any structure, device and / or suitable mechanism through which a beam of light can be transported, of the types shown and described in this document. In some embodiments, the first optical member 2831 can be any optical fiber suitable for carrying a beam of light, such as, for example,
Petition 870200011939, of 01/24/2020, p. 113/198
110/184 example, a multi-mode fiber or a single-mode fiber. [000265] The second optical member 2832 is arranged at least partially within block 2710 in such a way that the second optical member 2832 defines a second light path 2807 and is in optical communication with reaction volume 2713. In this way, a beam light (and / or an optical signal) can be transported between reaction volume 2713 and a region outside block 2710 through the second optical member 2832. The second optical member 2832 can be any suitable structure, device and / or mechanism through from which a beam of light can be carried, of the types shown and described in this document. In some embodiments, the second optical member 2832 can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber.
[000266] As previously described, the first optical member 2831 and the second optical member 2832 are coupled to the optical assembly (not shown in Figure 35). The optical assembly can produce one or more excitation light beams, and can detect one or more emission light beams. Therefore, one or more beams of excitation light can be carried in reaction volume 2713 and / or in reaction vessel 260, and one or more beams of emission light can be received from reaction volume 2713 and / or the portion 261 of reaction vessel 260. More particularly, the first optical member 2831t can carry a beam of excitation light from the optical assembly in reaction volume 2713 to excite a portion of sample S contained within reaction vessel 260. similarly, the second optical member 2832 can carry a beam of emission light produced by an analyte or other target within the sample S from reaction volume 2713 to the optical assembly. In this way, the optical assembly can monitor a reaction that
Petition 870200011939, of 01/24/2020, p. 114/198
111/184 occurs within reaction vessel 260.
[000267] As shown in Figure 35, the first optical member portion 2831 and the first light path 2806 are arranged substantially within a first PXY plane. The first PXY plane is substantially parallel and / or includes the longitudinal geometric axis LA of the reaction volume 2713. In other embodiments, however, the first PXY plane does not need to be substantially parallel and / or include the longitudinal geometric axis LA of the reaction volume. reaction 2713. The second optical member portion 2832 and the second light path 2807 are arranged substantially within a second PYZ plane. The second PYZ plane is substantially parallel and / or includes the longitudinal geometric axis LA of the reaction volume 2713. In other embodiments, however, the second PYZ plane does not need to be substantially parallel and / or include the longitudinal geometric axis LA of the reaction volume. reaction 2713. In addition, as shown in Figure 35, the first light path 2806 and the second light path 2807 define an angle of displacement é that is greater than approximately 75 degrees. More particularly, the first light path 2806 and the second light path 2807 define a displacement angle Θ when viewed in a direction substantially parallel to the longitudinal geometric axis LA of the reaction volume 2713 (i.e., which is within a plane substantially normal to the first PXY plane and the second PYZ plane) which is greater than approximately 75 degrees. Similarly, the first optical member 2831 and the second optical member 2832 define a displacement angle Θ that is greater than approximately 75 degrees. This arrangement minimizes the amount of the excitation beam that is received by the second optical member 2832 (i.e., the optical detection member), thereby improving the accuracy and / or sensitivity of optical detection and / or monitoring.
Petition 870200011939, of 01/24/2020, p. 115/198
112/184
[000268] In some embodiments, the 2002 portion of the instrument may produce the first light path 2806 and the second light path 2807 within reaction volume 2713 such that the displacement angle Θ is between approximately 75 degrees and approximately 105 degrees. In some embodiments, the instrument portion 2002 can produce the first light path 2806 and the second light path 2807 within reaction volume 2713 such that the displacement angle Θ is approximately 90 degrees.
[000269] Although the 2002 instrument portion is shown producing the first light path 2806 and the second light path 2807 which are substantially parallel and intersect at reaction volume 2713 at a PT point, in other modalities, block 2713, the first optical member 2831 and / or the second optical member 2832 can be configured in such a way that the first light path 2806 is not parallel and / or does not cross the second light path 2807. For example, in some embodiments, the first light path 2806 and / or the first optical member 2831 can be parallel and move away from (ie, deflect) the second light path 2807 and / or the second optical member 2831. As similarly stated, in some modalities, the the first optical member 2831 and the second optical member 1832 can be separated from a reference plane defined by block 2710 by a distance Y1 and Y2, respectively, where Y1 is different from Y2. Therefore, the position along the longitudinal axis LA in which the first optical member 2831 and / or the first light path 2806 cross reaction volume 2713 is different from the position along the longitudinal axis LA in which the second optical member 2832 and / or the second light path 2807 cross reaction volume 2713. In this way, the first light path 2806 and / or the first optical member 2831 can be deflected
Petition 870200011939, of 01/24/2020, p. 116/198
113/184 of the second light path 2807 and / or the second optical member 2831. [000270] In other embodiments, an angle γ1 defined by the longitudinal geometric axis LA and the first light path 2806 and / or the first optical member 2831 can be different from an angle γ2 defined by the longitudinal geometric axis LA and the second light path 2807 and / or the second optical member 2832 (i.e., the first light path 2806 may not be parallel to the second light path 2807). In still other embodiments, the block 2713, the first optical member 2831 and / or the second optical member 2832 can be configured in such a way that the first light path 2806 crosses the second light path 2807 at a location outside the reaction volume. 2713.
[000271] The distance Y1 and distance Y2 can be any suitable distance such that the first optical member 2831 and the second optical member 1832 are configured to produce and / or define the first light path 2806 and the second light path 2807, respectively, in the desired portion of the reaction vessel 260. For example, in some embodiments, the distance Y1 may be such that the first optical member 2831 and / or the first light path 2806 enter and / or cross the volume reaction 2713 at a location below the FL fill line location of sample S when reaction vessel 260 is disposed within block 2710. In this way, the excitation light beam carried by the first optical member 2831 will enter sample S below the fill line. This arrangement can improve the optical detection of analytes within the sample by reducing the attenuation of the excitation beam that can occur by transmitting the excitation beam through the empty space of the reaction vessel (that is, the portion of the reaction vessel 260 above the fill line LF which is substantially devoid of sample S). In other modalities, however, the Y1 distance can
Petition 870200011939, of 01/24/2020, p. 117/198
114/184 be such that the first optical member 2831 and / or the first light path 2806 enters reaction volume 2713 at a location above the location of the FL fill line of sample S when reaction vessel 260 is disposed inside block 2710.
[000272] Similarly, in some embodiments, the distance Y2 may be such that the second optical member 2832 and / or the second light path 2807 enter and / or cross reaction volume 2713 at a location below the location from the fill line FL of the sample S when the reaction vessel 260 is disposed within the block 2710. In this way, the beam of emission light received by the second optical member 2832 will leave the sample S below the fill line. This arrangement can improve the optical detection of analytes within the sample by reducing the attenuation of the beam of emission light that can occur by receiving the beam of emission light through the empty space of the reaction vessel. In other embodiments, however, the distance Y2 may be such that the second optical member 2832 and / or the second light path 2807 enter and / or cross reaction volume 2713 at a location above the location of the fill line. FL of sample S when reaction vessel 260 is disposed within block 2710.
[000273] Figures 36 to 70 show several views of an instrument 3002 and / or portions of an instrument configured to manipulate, drive and / or interact with a series of cartridges to perform a process of isolation and amplification of nucleic acid in samples of test inside the cartridges. Cartridges can include any of the cartridges shown and described in this document, such as, for example, cartridge 6001. This system can be used for many different assays, such as, for example, rapid detection of influenza (Flu) A , Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal samples. The 3002 instrument is shown without the
Petition 870200011939, of 01/24/2020, p. 118/198
115/184 housing 3002 and / or certain portions of the instrument 3002 to show more clearly the components contained therein. For example, Figure 47 shows the instrument 3002 without the optical mount 3800.
[000274] As shown in Figure 36, instrument 3002 includes a chassis and / or frame 3300, a first 3400 actuator assembly, a 3500 sample transfer assembly, a second 3600 actuator assembly, a 3700 heater assembly and a 3800 optical mount. The 3300 frame is configured to accommodate, contain and / or provide mounting for each of the components and / or assemblies of the 3002 instrument as described in this document. The first 3400 actuator assembly is configured to drive an actuator or transfer mechanism (for example, the 6166 actuator or transfer mechanism) of the isolation module (for example, the 6100 isolation module) of a cartridge to transport one or more reagents and / or substances in a lysis chamber inside the isolation module. The 3500 transfer actuator assembly is configured to drive a transfer assembly (for example, the 6140a transfer assembly) to transfer a sample portion between multiple chambers and / or volumes within an isolation module (for example, the isolation module insulation rating 7100). The second 3600 actuator assembly is configured to drive a mixing mechanism (for example, the 6130a mixing mechanism) and / or a wash buffer module (for example, the wash buffer module 7130a) of the insulation module ( eg the 6100 isolation module) and / or the PCR module (eg the PCR module 6200) to transport and / or mix one or more reagents and / or substances within a chamber in the isolation module and / or on the PCR module. The 3700 heater assembly is configured to heat one or more portions of a cartridge (for example,
Petition 870200011939, of 01/24/2020, p. 119/198
116/184 example, the PCR bottle 7260, substrate 7220 and / or a region of compartment 7110 adjacent to lysis chamber 7114) to promote and / or facilitate a process within the cartridge (for example, to promote, facilitate and / or produce a hot hot start process, a heated lysis process and / or a PCR process). The 3800 optical mount is configured to monitor a reaction that occurs with the cartridge. More specifically, the 3800 optical mount is configured to detect one or more different analytes and / or targets within a test sample in the cartridge. Each of these assemblies will be discussed separately below, followed by a description of the various methods that can be performed by the 3002 instrument.
[000275] As shown in Figure 36, frame 3300 includes a base frame 3310, a front member 3312, two side members 3314 and a rear member 3320. The base member 3310 supports the functional assemblies described in this document, and includes your mounting legs or bracket. In some embodiments, the support legs can be adjustable to allow the 3302 instrument to be horizontally level when mounted and / or installed on a laboratory bench. The rear member 3320 is coupled to the base member 3310 and configured to support and / or retain the power supply assembly 3361. The rear member 3320 can also provide a mounting bracket for any of the other components related to the operation of the 3302 instrument. , such as, for example, a processor, control elements (for example, motor controllers, heating system controllers or the like), a communication interface, a cooling system or the like. Figures 71 to 73 are block diagrams of a control and computational system of the instrument 3002.
[000276] Each side member 3314 includes an upper portion 3316
Petition 870200011939, of 01/24/2020, p. 120/198
117/184 and a lower portion 3315. The front member 3312 is coupled to each side member 3314 and defines an opening within which a tank 3350 containing multiple test cartridges can be arranged for processing. In some embodiments, the 3350 tank can be configured to contain six cartridges of the types shown and described in this document (shown in Figure 36, for example, as the 6001 cartridge). In use, the 3350 tank containing multiple cartridges is disposed within the instrument 3002 and is held in a fixed position in relation to the 3300 chassis during the isolation and / or amplification process. Therefore, the cartridges containing the samples are not moved between multiple stations to conduct the analysis. Instead, as described in this document, samples, reagents and / or other substances are transported, processed and / or manipulated within the various portions of the cartridge by instrument 3002, as described in this document. Although instrument 3002 is shown to be configured to receive a 3350 tank containing six cartridges, in other embodiments, an instrument can be configured to receive any number of 3350 tanks containing any number of cartridges.
[000277] Figures 37 to 40 show various views of the first 3400 actuator assembly of the instrument 3002. The first 3400 actuator assembly is configured to drive and / or manipulate a transfer mechanism and / or reagent actuator (for example, reagent actuators 6166a, 6166b, 6166c and 6166d) of an isolation module (for example, the 6100 isolation module) of a cartridge to transport one or more reagents and / or substances in a lysis chamber within the isolation module. In particular, the first 3400 actuator assembly can drive a first among the reagent actuators (for example, reagent actuator 6166d) from each of the cartridges disposed within the 3350 tank, and,
Petition 870200011939, of 01/24/2020, p. 121/198
118/184 then, at a different time, trigger one second between the reagent actuators (for example, reagent actuator 6166c) from each of the cartridges.
[000278] The first actuator assembly includes a 3445 drawbar, a first (or x-axis) 3440 and a second (or y-axis) 3441 supported by a frame assembly 3410. As shown in Figures 38 and 40, the drawbar 3445 includes a series of protrusions 3346a, 3346b, 3346c, 3346d, 3346e and 3346f. Each of the protrusions is configured to engage, be arranged and / or drive one or more reagent actuators (for example, reagent actuator 6166a) of an isolation module (for example, the 6100 isolation module) disposed within the instrument 3002. In some embodiments, the drawbar 3445 and / or protrusions (for example, protrusion 3346a) may include a retention mechanism (for example, a protrusion, a pressure ring or the like) configured to retain a protrusion and / or an opening of an actuator (for example, reagent actuator 6166a) to facilitate reciprocal movement of the reagent actuator within the isolation module.
[000279] The 3410 frame assembly includes a 3420 first geometry (or geometry axis) mounting frame that is movably coupled to a second 3430 geometry (or y axis) mounting frame. In particular, the 3420 first axis frame can be moved relative to the 3430 second axis frame along the y axis, as shown by the arrow AAA in Figure 37. As similarly stated, the 3420 first axis frame can be moved in relation to the second geometric axis frame 3430 in an alignment direction (that is, along the geometric axis y) to facilitate alignment of the drawbar 3445 and / or
Petition 870200011939, of 01/24/2020, p. 122/198
119/184 protrusions (for example, protrusion 3346a) with the desired series of actuators and / or transfer mechanisms.
[000280] The first geometric axis frame 3420 provides support for the first motor (or x-axis) 3440, which is configured to move the drawbar 3445 and / or the protrusions (for example, the protrusion 3346a) along the geometric axis x, as shown by the BBB arrow in Figure 37. As similarly stated, the 3440 first axis motor is coupled to the 3420 first axis frame, and configured to move the drawbar 3445 and / or the protrusions (for example, protrusion 3346a) in a driving direction (that is, along the geometric axis x) to drive the desired series of actuators and / or transfer mechanisms. The movement of the drawbar 3445 is guided by two guide axes of the geometric axis x 3421, in which each is movably arranged within a corresponding bearing 3422. The bearings 3422 are positioned in relation to the frame of the first geometric axis 3420 and / or the first 3440 engine by a 3423 bearing assembly member.
[000281] The second geometric frame 3430 is coupled between the two side frame members 3314 of the frame assembly 3300. The second geometric frame 3430 provides support to the second motor (or geometric axis y) 3441 and the frame first geometric axis 3420. The second motor 3441 is configured to move the frame of the first geometric axis 3420, and therefore the drawbar 3445 along the geometric axis y (or in an alignment direction), as shown by the BBB arrow in Figure 37. In this way, the drawbar 3445 and / or the protrusions (for example, the protrusion 3346a) can be aligned to the desired series of actuators and / or transfer mechanisms before actuating the actuators and / or
Petition 870200011939, of 01/24/2020, p. 123/198
120/184 transfer. The first geometry axis frame 3420 is coupled to the second geometry axis frame 3430 by a pair of bearing blocks 3432 that are slidably arranged around a corresponding pair of geometry axis guide y 3431.
[000282] In use, the first 3400 actuator assembly can sequentially drive a series of transfer mechanisms and / or reagent actuators (for example, the 6166a, 6166b, 6166c and 6166d actuators) of a set of cartridges (for example, cartridge 6001) arranged inside instrument 3001. First, the drawbar 3445 can be aligned with the desired transfer mechanism and / or reagent actuator (for example, actuator 6166d) by moving the first frame member 3420 in the alignment direction (that is, along the y-axis). The drawbar 3445 can then be moved in the driving direction (that is, along the geometric axis x) to drive the desired transfer mechanism and / or reagent actuator (for example, the 6166d actuator) from each cartridge. In this way, the first 3400 actuator assembly can drive and / or manipulate a reagent actuator from each of the cartridges disposed within the instrument 3002 in a parallel (or simultaneous) manner. In other embodiments, however, the 3400 actuator assembly and / or the 3445 drawbar can be configured to sequentially drive the corresponding reagent actuators for each of the cartridges arranged within the instrument 3002 in a sequential (or serial) manner.
[000283] The first 3400 actuator assembly can drive the desired transfer mechanism and / or reagent actuator by moving the drawbar 3445 in a first direction along the x axis. In other embodiments, however, the first 3400 actuator assembly can drive the desired transfer mechanism and / or reagent actuator by reciprocating the
Petition 870200011939, of 01/24/2020, p. 124/198
121/184 coupling 3445 (that is, alternatively moving the coupling bar 3445 in a first direction and in a second direction) along the geometric axis x. When the desired transfer mechanism and / or reagent actuator has been activated, the first 3400 actuator assembly can drive another transfer mechanism and / or reagent actuator (for example, 6166c actuators), in a similar manner as described previously .
[000284] Although the first 3400 actuator assembly is shown and described by driving a transfer mechanism and / or a reagent actuator, in other embodiments, the first 3400 actuator assembly can drive any suitable portion of any of the cartridges described herein document. For example, in some embodiments, the first 3400 actuator assembly can drive, manipulate and / or move an ultrasonic transducer to facilitate ultrasonic lysis.
[000285] Figures 41 to 46 show several views of the 3500 transfer actuator assembly of the instrument 3002. The 3500 transfer actuator assembly is configured to drive and / or manipulate an assembly or transfer mechanism, such as, for example, the transfer assembly 6140 shown and described previously with reference to Figures 20 and 21. In particular, the transfer actuator assembly 3500 can drive a first between transfer assemblies (for example, transfer assembly 6140a) from each one of the cartridges disposed within the 3350 tank, and then, at a different time, drive a second between the transfer assemblies (for example, the transfer assembly 6140b) from each of the cartridges.
[000286] The 3500 transfer actuator assembly includes a series of 3510 actuator shafts. Although the 3500 transfer actuator assembly includes six actuator shafts, only one is
Petition 870200011939, of 01/24/2020, p. 125/198
122/184 identified in Figures 41 to 46. Each of the 3510 actuator shafts is configured to engage, be arranged within and / or drive one or more transfer assemblies (for example, transfer assembly 6140a) of an isolation module (for example, insulation module 6100) disposed within instrument 3002. As shown in Figure 44, each actuator shaft 3510 has a first end portion 3511 and a second end portion 3512. The first end portion 3511 is coupled to a 3513 transmission gear (see Figures 41 and 42), which is successively driven by a 3541 helical drive shaft. As shown in Figures 41 and 42, a 3542 rotational position indicator is coupled to the first end portion 3511 of one of the 3510 actuator shafts. The 3542 rotational position indicator defines a 3543 slot and / or opening, the rotational position of which can be captured (for example, via an optimum pickup mechanism) ca) to provide feedback regarding the rotational position of the 3510 actuator shafts.
[000287] The second end portion 3512 of each axis 3510 includes an engagement portion 3514 configured to be received within and / or engaged with a transfer assembly (for example, transfer assembly 6140a) of a cartridge (for example, example, cartridge 6001) disposed within instrument 3002. In this way, engagement portion 3514 can manipulate and / or drive the transfer assembly to facilitate the transfer of portions of a sample within the cartridge, as described above. The engagement portion 3514 has a shape that corresponds to a shape of a portion of the transfer assembly (for example, the lumen 6149 defined by the movable member 6146) such that rotation of the actuator shaft 3510 results in the rotation of a portion transfer assembly. In particular, as shown in Figure 44, the portion
Petition 870200011939, of 01/24/2020, p. 126/198
123/184 hitch has an octagonal shape. In some embodiments, the engagement portion 3514 may include a retaining mechanism (for example, a protrusion, a snap ring or the like) configured to retain a protrusion and / or an opening of a transfer assembly to facilitate reciprocal movement of a portion of the transfer assembly within the isolation module.
[000288] The engagement portion 3514 defines a lumen 3515 within which a magnet (not shown) can be arranged. In this way, the 3510 actuator shaft can produce and / or exert a force (i.e., a magnetic force) on a portion of the contents (i.e., the magnetic microspheres) disposed within the cartridge (for example, the 6001 cartridge) to facilitate the transfer of a portion of the sample through the transfer assembly, as previously described.
[000289] The 3510 actuator shafts are driven by a first motor (or x-axis) 3580, a second motor (or y-axis) 3560, and a third motor (or rotational) 3540. As described in more detail below, the 3580 x axis motor is supported by the 3571 support frame, the 3560 y axis motor is supported by the 3550 hitch frame assembly, and the 3540 rotational motor is supported by the frame assembly rotation speed 3530.
[000290] The 3530 rotation frame assembly provides support for the 3540 rotational motor, which is configured to rotate the 3510 actuator shafts around the y-axis, as shown by the CCC arrow in Figure 41. As similarly stated, the 3540 rotational motor is coupled to the 3530 rotational frame assembly, and is configured to rotate the 3510 actuator shafts in a driving direction (ie around the y-axis) to drive the desired series of transfer assemblies. THE
Petition 870200011939, of 01/24/2020, p. 127/198
124/184 3530 pivot frame assembly includes a 3531 pivot plate, a pair of 3533 helical drive bearing blocks, and a 3541 helical drive shaft. The 3541 helical drive shaft is coupled to the 3540 rotary motor by mounting pulley, and is supported by the two 3533 helical drive bearing blocks. The 3541 helical drive shaft is engaged with the 3513 drive gear on each 3510 actuator shaft. Consequently, when the 3541 helical drive shaft is rotated in a first direction (that is, around the z axis), each 3510 actuator axis is rotated in a second direction (that is, around the y axis, as shown by the CCC arrow in Figure 41).
[000291] The 3530 pivot frame assembly also includes a 3534 y-axis position indicator that can be slidably arranged within a pair of corresponding sliding members 3553 in the 3550 hitch frame assembly. In this way, when the 3530 pivot frame assembly is moved along the y-axis (for example, in a hitch direction), as shown by the DDD arrow in Figure 41, the y-axis position indicator 3534 and the corresponding sliding members 3553 can guide linear movement and / or provide feedback regarding the position of the 3530 rotation frame assembly.
[000292] The 3550 hitch frame assembly supports the 3560 y-axis motor, which is configured to move the 3530 pivot frame assembly, and therefore the 3510 actuator shafts, along the y-axis, as shown by the DDD arrow in Figure 41. As similarly stated, the y-axis motor 3560 is coupled to the 3550 hitch frame assembly, and is configured to move the drive shafts
Petition 870200011939, of 01/24/2020, p. 128/198
125/184
3510 in the engaging direction (that is, along the y-axis) to drive the desired series of transfer mechanisms. The 3550 hitch frame assembly includes a 3551 support frame that provides support for the 3561 transmission link (which converts the rotational movement of the y-axis motor into a linear movement of the 3530 rotation frame assembly. rotation frame 3530 is guided by two guide axes of the y axis 3552, each of which is movably arranged within a corresponding 3554 bearing. The 3554 bearings are coupled to the rotation plate 3531, as shown in Figure 43.
[000293] The 3571 support frame is coupled between the lower end portion 3315 of the two side frame members s 3314 of the 3300 frame assembly. The 3571 support frame provides support for the 3580 x axis motor and the mounting of 3550 hitch frame. The 3580 x axis motor is configured to move the 3550 hitch frame assembly, and therefore the 3510 drive axes along the x axis (or in an alignment direction), as shown by the EEE arrow in Figure 41. In this way, the 3510 actuator shafts can be aligned to the desired series of transfer mechanisms before the transfer mechanisms are activated. The support frame 3571 is coupled to the coupling frame assembly 3550 by a pair of bearing blocks 3573 which are slidably arranged around a corresponding pair of guiding axes of the x 3572 axis.
[000294] In use, the transfer actuator assembly 3500 can sequentially drive a series of transfer mechanisms (for example, transfer assemblies 6140a, 6140b and 6166c) of a set of cartridges (for example, cartridge 6001) arranged
Petition 870200011939, of 01/24/2020, p. 129/198
126/184 inside the instrument 3001. First, the 3510 actuator shafts can be aligned to the desired transfer mechanism by moving the 3550 hitch frame assembly in the alignment direction (that is, along the x axis). The 3510 actuator shafts can then be moved in the engaging direction (i.e., along the y-axis) to engage the desired transfer mechanism (e.g., transfer assembly 6140a) from each cartridge. The 3510 actuator shafts can then be moved in the driving direction (i.e., rotation around the y-axis) to drive the desired transfer mechanism (for example, the 6140a transfer assembly) from each cartridge. In this way, the transfer actuator assembly 3500 can drive and / or manipulate a transfer mechanism from each of the cartridges disposed within the instrument 3002 in a parallel (or simultaneous) manner. In other embodiments, however, the mounting of the transfer actuator 3500 and / or the drive axes 3510 can be configured to sequentially drive the corresponding transfer mechanism of each of the cartridges arranged within the instrument 3002 in a sequential (or serial) manner .
[000295] Figures 47 to 51 show various views of the second actuator assembly 3600 of the instrument 3002. The second actuator assembly 3600 is configured to drive and / or manipulate a transfer mechanism (for example, the transfer mechanism 7235), the wash buffer module (for example, the wash buffer module 7130a), a mixing mechanism (for example, the 6130a mixing mechanism) and / or a reagent module (for example, the 7270a reagent module) any of the cartridges shown or described in this document. In particular, the second 3600 actuator assembly can drive a first among the transfer mechanisms, mixing mechanisms
Petition 870200011939, of 01/24/2020, p. 130/198
127/184 or similar (for example, the 6130a mixing mechanism) from each of the cartridges disposed within the 3350 tank, and then, at a different time, trigger one second between the transfer mechanisms, mixing mechanisms or (for example, the 6130b mixing mechanism) from each of the cartridges. [000296] The second actuator assembly 3600 includes a drawbar 3645, a first motor (or x-axis) 3640 and a second motor (or y-axis) 3641 supported by a frame assembly 3610. As shown in Figure 48, the drawbar 3645 includes a series of 3346 protrusions. Although the drawbar 3645 includes six protuberances (each corresponding to each cartridge within the 3350 tank), only one 3346 protrusion is labeled. Each of the protrusions is configured to engage, be arranged inside, manipulate and / or activate one or more transfer mechanisms (for example, the transfer mechanism 7235), wash buffer modules (for example, the wash buffer module 7130a), mixing mechanisms (for example, the 6130a mixing mechanism) and / or reagent modules (for example, reagent module 7270a) from a cartridge disposed within instrument 3002. In some embodiments, the 3645 drawbar and / or the 3346 protrusions may include a retaining mechanism (for example, a protrusion, a snap ring or the like) configured to retain a portion of an actuator (for example, the engagement portion 7153a of actuator 7150a, shown and described previously with reference to Figures 27 and 28) to facilitate the reciprocal movement of the actuator within a portion of the cartridge. [000297] Frame assembly 3610 includes a second geometric (or y-axis) mounting frame 3630 that is movably coupled to a first geometric (or x-axis) mounting frame 3620. In particular,
Petition 870200011939, of 01/24/2020, p. 131/198
128/184 the second geometry axis frame 3630 can be moved relative to the first geometric axis frame 3620 along the geometric axis x, as shown by the arrow GGG in Figure 47. As similarly stated, the second geometric axis frame 3630 can be moved relative to the first geometric axis frame 3620 in an alignment direction (ie along the x axis) to facilitate alignment of the drawbar 3645 and / or protrusions 3346 with the desired series of mechanisms transfer mechanisms, mixing mechanisms, reagent modules, or the like.
[000298] The second geometric axis frame 3620 provides support for the second motor (or geometric axis y) 3641, which is configured to move the drawbar 3645 and / or the protrusions 3346 along the geometric axis y, as shown by arrow FFF in Figure 47. As similarly stated, the second geometry axis motor 3641 is coupled to the second geometry axis frame 3620, and configured to move the drawbar 3645 and / or the protrusions 3346 in a driving direction ( that is, along the geometric axis y) to drive the desired series of transfer mechanisms, mixing mechanisms, reagent modules or the like. The movement of the coupling bar 3645 is guided by the two guide axes of the y axis 3631, each of which is movably arranged within a corresponding bearing coupled to the second geometric axis frame 3620.
[000299] The first geometric frame 3630 is coupled between the upper portion 3316 of the two side frame members 3314 of the frame assembly 3300. The first geometric frame 3630 provides support to the first motor (or x-axis) 3640 and the second geometric axis frame 3620. The first 3640 motor is configured to move the second geometric axis frame
Petition 870200011939, of 01/24/2020, p. 132/198
129/184
3620, and therefore the drawbar 3645 along the geometric axis x (or in an alignment direction), as shown by the arrow GGG in Figure 47. In this way, the drawbar 3645 and / or the protrusions 3346a can be aligned to the desired series of transfer mechanisms, mixing mechanisms, reagent modules, or the like, before activating such mechanisms. The second axis geometry frame 3620 is coupled to the first axis geometry frame 3630 by a pair of bearing blocks 3622 that are slidably arranged around a corresponding pair of axis axes x 3631. The first motor (or with geometric axis x) 3640 is coupled to the frame of the second geometric axis 3620 through the mounting member 3624 (see, for example, Figure 51).
[000300] In use, the second actuator assembly 3600 can sequentially drive a series of transfer mechanisms (for example, transfer mechanism 7235), wash buffer modules (for example, wash buffer module 7130a), mixing mechanisms (for example, the 6130a mixing mechanism) and / or reagent modules (for example, the 7270a reagent module) from a set of cartridges (for example, the 6001 cartridge) disposed within the 3001. Firstly, the coupling bar 3645 can be aligned with the desired mechanism (for example, the mixing mechanism 6130a) by moving the second frame member 3630 in the direction of alignment (i.e., along the x-axis). The drawbar 3645 can then be moved in the driving direction (i.e., along the y-axis) to drive the desired mechanism (e.g., the 6130a mixing mechanism) from each cartridge. In this way, the second actuator assembly 3600 can drive and / or manipulate a transfer mechanism, a wash buffer module, a
Petition 870200011939, of 01/24/2020, p. 133/198
130/184 mixture and / or a reagent module from each of the cartridges disposed within the instrument 3002 in a parallel (or simultaneous) manner. In other embodiments, however, the second actuator assembly 3600 and / or the drawbar 3645 can be configured to sequentially drive the corresponding mechanisms of each of the cartridges arranged within the instrument 3002 in a sequential (or serial) manner.
[000301] The second 3600 actuator assembly can drive the desired mechanism by moving the drawbar 3645 in a first direction along the y-axis. In other embodiments, however, the second actuator assembly 3600 can drive the desired transfer mechanism and / or reagent actuator by reciprocating the coupling bar 3645 (that is, alternatively moving the coupling bar 3645 in one direction and in a second direction) along the y-axis. When the desired mechanism has been actuated, the second 3600 actuator assembly can actuate another mechanism and / or actuator (for example, 6130b mixing mechanism), in a similar manner as described previously.
[000302] Although the second 3600 actuator assembly is shown and described by driving a transfer mechanism and / or a reagent actuator, in other embodiments, the second 3600 actuator assembly can drive any suitable portion of any of the cartridges described herein document. For example, in some embodiments, the second 3600 actuator assembly can drive, manipulate and / or move an ultrasonic transducer to facilitate the transmission of acoustic energy in a portion of the cartridge.
[000303] Figures 52 to 63 show various views of the 3700 heater assembly of the 3002 instrument. The 3700 heater assembly is configured to heat one or more portions of a cartridge (for example,
Petition 870200011939, of 01/24/2020, p. 134/198
131/184 example, the PCR bottle 7260, substrate 7220 and / or a region of compartment 7110 adjacent to lysis chamber 7114) to promote and / or facilitate a process within the cartridge (for example, to promote, facilitate and / or produce a hot start process, a heated lysis process and / or a thermal cycle process for PCR). In particular, the 3700 heater assembly can drive and / or heat a first portion (for example, the PCR bottle 6260) of each of the cartridges disposed within the 3350 tank, and then, at a different time, trigger and / or heating a second portion (e.g., the 6100 insulation module portion adjacent the lysis chamber 6114) from each of the cartridges.
[000304] The heater assembly 3700 includes a series of receiving blocks 3710 (each corresponding to each of the cartridges within the 3350 tank), a 3770 positioning assembly, a first 3730 heating module, a second 3750 heating module and a third heating module 3780. The receiving block 3710 is configured to receive at least a portion of a cartridge reaction chamber, such as the PCR bottle 6260 of cartridge 6001. As shown in Figures 53 to 56, the receiving block 3710 includes a mounting surface 3714 and defines a reaction volume 3713. Reaction volume 3713 has a size and / or shape that substantially corresponds to a size and / or shape of the PCR 6260 bottle in cartridge 6001. As shown in Figures 54 and 56, reaction volume 3713 defines a longitudinal geometric axis LA and substantially surrounds the portion of the PCR bottle 6260 when the PCR bottle 6260 is disposed within the reaction volume 3713 In this way, any stimulus (for example, heating or cooling) provided to the sample inside the 6260 PCR bottle by the 3700 heater assembly can be provided substantially and spatially
Petition 870200011939, of 01/24/2020, p. 135/198
132/184 uniform. Furthermore, as shown in Figure 56, the side wall of the receiving block portion 3710 that defines reaction volume 3713 has a substantially uniform wall thickness. This arrangement allows for a thermal transfer between reaction volume 3713 and the remaining portions of heater assembly 3700 to be substantially and spatially uniform.
[000305] The receiving block 3710 is coupled to a mounting block 3734 (see, for example, Figure 58) by a clamp block 3733 (see, for example, Figure 57) such that a thermoelectric device 3731 stay in contact with the mounting surface 3714. In this way, the reaction volume 3713 and the sample contained therein can be cyclically heated to produce a thermally induced reaction of the sample S, such as, for example, a PCR process.
[000306] Each receiving block 3710 defines a first lumen (or excitation) 3711, a second lumen (or emission) 3712 and a third lumen (or temperature monitoring) 3715. A thermocouple or other temperature measurement device suitable can be disposed adjacent to the PCR vial through the third lumen 3715. As shown in Figure 52, an excitation fiber 3831 is arranged at least partially within the first lumen 3711 such that the excitation fiber 3831 and / or the first lumen 3711 define a first light path 3806 and stay in optical communication with reaction volume 3713. In this way, a beam of light (and / or an optical signal) can be transported between reaction volume 3713 and a region outside the block 3710 through excitation fiber 3831 and / or first lumen 3711. Excitation fiber 3831 can be any suitable structure, device and / or mechanism through which a beam of light can be transported, of the types shown and described in this document. In some embodiments, the excitation fiber
Petition 870200011939, of 01/24/2020, p. 136/198
133/184
3831 can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber.
[000307] The detection fiber 3832 is arranged at least partially within the second lumen 3712 in such a way that the detection fiber 3832 and / or the second lumen 3712 define a second light path 3807 and are in optical communication with the volume of reaction 3713. In this way, a beam of light (and / or an optical signal) can be transported between reaction volume 3713 and a region outside block 3710 via the 3832 detection fiber and / or the second 3712 lumen. The fiber 3832 detection device can be any suitable structure, device and / or mechanism through which a beam of light can be transported, of the types shown and described in this document. In some embodiments, the 3832 detection fiber can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber.
[000308] As described below, excitation fiber 3831 and detection fiber 3832 are coupled to optical assembly 3800. Optical assembly 3800 can produce one or more excitation light beams, and can detect one or more light beams from issue. Therefore, excitation fiber 3831 can carry a beam of excitation light from the optical assembly in reaction volume 3713 to excite a portion of sample S contained within the 6260 PCR flask. Similarly, the 3832 detection fiber can transporting a beam of emission light produced by an analyte or other target within sample S from the PCR vial 6260 to the optical mount 3800.
[000309] As shown in Figure 55, the first lumen 3711 and the second lumen 3712 define an offset angle Θ that is approximately equal to 90 degrees. As stated so
Petition 870200011939, of 01/24/2020, p. 137/198
134/184 similarly, the first light path 3806 and the second light path 3807 define an offset angle Θ which is approximately equal to 90 degrees. More particularly, the first light path 3806 and the second light path 3807 define an offset angle Θ when viewed in a direction substantially parallel to the longitudinal geometric axis LA of reaction volume 3713 which is approximately equal to 90 degrees. Similarly, excitation fiber 3831 and detection fiber 3832, which are arranged within the first lumen 3711 and the second lumen 3712, respectively, define the displacement angle Θ which is approximately equal to 90 degrees. This arrangement minimizes the amount of the excitation beam that is received by the 3832 detection fiber, thereby improving the accuracy and / or sensitivity of optical detection and / or monitoring.
[000310] In some embodiments, the first lumen 3711 and the second lumen 3712 can be positioned such that the displacement angle Θ is greater than approximately 75 degrees. In other embodiments, the first lumen 3711 and the second lumen 3712 can be positioned such that the displacement angle Θ is between approximately 75 degrees and approximately 105 degrees. [000311] As shown in Figure 54, a center line of the first lumen 3711 is substantially parallel and displaced from (i.e., offset) from a center line of the second lumen 3712. As similarly stated, excitation fiber 3831 ( and, therefore, the first light path 3806) is deviated from the 3832 detection fiber (and therefore the second light path 3807). Otherwise, the first lumen 3711 (and / or the excitation fiber 3831) and the second lumen 3712 (and / or the detection fiber 3832) are separated from a reference plane defined by the receiving block 3710 by a distance Y1 and Y2, respectively, where Y1 is different from Y2.
Petition 870200011939, of 01/24/2020, p. 138/198
135/184
Therefore, the position along the longitudinal axis LA in which the excitation fiber 3831 and / or the first light path 3806 crosses the reaction volume 3713 is different from the position along the longitudinal axis LA n in which the sensing fiber 3832 and / or the second light path 3807 crosses reaction volume 3713. In this way, the first light path 3806 and / or the excitation fiber 3831 can be diverted from the second light path 3807 and / or the second optical member 3831.
[000312] Distance Y1 and distance Y2 can be any suitable distance such that excitation fiber 3831 and detection fiber 3832 are configured to produce and / or define the first light path 3806 and the second light path 3807, respectively, in the desired portion of the PCR vial 6260. For example, in some embodiments, the distance Y1 may be such that the first lumen 3711, excitation fiber 3831 and / or the first light path 3806 enter and / or cross reaction volume 3713 at a location below the location of a sample filling line inside the PCR vial 6260 disposed within the receiving block 3710. In this way, the excitation light beam carried by the excitation fiber 3831 will enter the sample below the fill line. In other embodiments, however, the distance Y1 can be such that the first lumen 3711, the excitation fiber
3831 and / or the first light path 3806 enter reaction volume 3713 at a location above the location of the sample fill line inside the 6260 PCR flask.
[000313] Similarly, in some embodiments, the distance Y2 can be such that the second lumen 3712, the detection fiber
3832 and / or the second light path 3807 enter and / or cross reaction volume 3713 at a location below the location of a sample fill line inside the 6260 PCR bottle disposed
Petition 870200011939, of 01/24/2020, p. 139/198
136/184 within the receiving block 3710. In other embodiments, however, the distance Y2 can be such that the second lumen 3712, the detection fiber 3832 and / or the second light path 3807 enter and / or cross reaction volume 3713 at a location above the sample fill line location inside the 6260 PCR flask.
[000314] The first 3730 heating module includes a series of 3731 thermoelectric devices (each corresponding to each of the cartridges and / or each of the 3710 receiving blocks), a 3734 mounting block, a series of clamp blocks 3733, and a 3732 heatsink. As shown in Figure 58, mounting block 3734 includes a first portion 3735 and a second portion 3737. The first portion 3735 includes an angled surface 3736 to which each of the 3731 thermoelectric devices is coupled. In this way, each receiving block 3710 is coupled to a mounting block 3734 by the corresponding clamp block 3733 in such a way that the thermoelectric device 3731 is in contact with the mounting surface 3714 of the receiving block 3710.
[000315] The second portion 3737 of mounting block 3734 is coupled to the heat sink 3732. The heat sink (see, for example, Figure 59) can be any device suitable for facilitating thermal transfer between the receiving blocks 3710 and a region outside the instrument 3002. In some embodiments, the 3732 heatsink may include a device and / or mechanism for actively cooling (i.e., removing heat) the 3734 mounting block.
[000316] Positioning assembly 3770 is coupled to heatsink 3732 and a portion of the frame assembly 3300, and is configured to move the 3700 heater assembly linearly along the y-axis. Therefore, when engaged, the 3770 positioning assembly can move the
Petition 870200011939, of 01/24/2020, p. 140/198
137/184 heater 3700 assembly in relation to the 3350 tank and / or the cartridges in such a way that the PCR bottle (for example, the PCR bottle 6260) is disposed within the receiving block 3710, as described previously. The 3770 positioning assembly includes a 3771 motor and a 3772 link assembly configured to convert the rotational motion of the 3771 motor into linear motion. The movement of the 3700 heater assembly is guided by a y-axis guide axis 3773.
[000317] In use, the first 3730 heating module can cyclically heat the PCR bottle of each of the cartridges disposed within the 3001 instrument to promote a PCR process and / or the mixing of the contents contained therein. Furthermore, due to the fact that each of the cartridges is heated by a separate thermoelectric device 3731 through the separate receiving block 3710, in some embodiments, the thermal cycling of a first cartridge can be conducted at a different time than the thermal cycling of a second cartridge. Furthermore, due to the fact that each cartridge can be thermally cycled independently of the other cartridges in the instrument, in some modalities, the thermal cycle protocol (for example, the times and temperatures of the thermal cycle events) for a first cartridge may be different from thermal cycle protocol for a second cartridge. In some embodiments, the first 3730 heating module is not used for thermal cycling, and is instead kept at a constant temperature, for example, a temperature to perform reverse transcription on an RNA sample.
[000318] The second heating module 3750 includes a series of 3751 resistance heaters (each corresponding to each of the cartridges and / or each of the 3710 receiving blocks), a mounting plate 3754, a first insulating member 3752 , it is a
Petition 870200011939, of 01/24/2020, p. 141/198
138/184 second insulating member 3753. As shown in Figure 60, mounting plate 3754 includes a first portion 3755 and a second portion 3760. The first portion 3755 provides a mounting bracket for each of the 3751 resistance heaters. similarly stated, each of the 3731 resistance heaters is coupled to the 3754 mounting plate.
[000319] The mounting plate 3754 is coupled to the mounting block 3734 of the first heating module 3730 in such a way that the first insulating member 3752 is disposed between the mounting block 3734 and the first portion 3755 of the mounting plate 3754, and the second insulating member 3753 is disposed between the mounting block 3734 and the second portion 3760 of the mounting plate 3754. In this way, the second heating module 3750 can function substantially independent of the first heating module 3730. As stated so similarly, this arrangement reduces and / or limits the thermal transfer between the mounting plate 3754 and the mounting block 3734.
[000320] The first portion 3755 of mounting plate 3754 includes a top surface 3758, and defines a recess 3756 and a series of lumens 3757 (each corresponding to each of the cartridges within the 3350 tank). In use, when the 3700 heater assembly is moved into position around each of the cartridges within the instrument 3002, each PCR vial is disposed through the corresponding lumen 3757 and the reaction volume 3713 defined by the corresponding receiving block 3710. Therefore, in some embodiments, when the 3700 heater assembly is positioned around each of the cartridges, a side wall of the 3754 mounting plate that defines the 3757 lumens is positioned around and / or substantially surrounds a portion of each vial of 6260 PCR. In other embodiments, however, the 6260 PCR bottle can be
Petition 870200011939, of 01/24/2020, p. 142/198
139/184 separate and / or non-resident within lumen 3757. For example, in some embodiments, only one transfer port (such as transfer port 7229 of PCR module 7200, shown and described previously with reference to Figures 30 and 31) can be arranged within lumen 3737 of mounting plate 3754 when heater assembly 3700 is positioned around each of the cartridges.
[000321] As shown in Figure 60, the second portion 3760 of mounting plate 3754 defines a series of recesses and / or cavities 3761 (each corresponding to each of the cartridges within the 3350 tank). In use, when the 3700 heater assembly is moved into position around each of the cartridges within the instrument 3002, a portion of the cartridge is disposed within the corresponding recess 3761 of the 3754 mounting plate. More particularly, as shown in Figure 52 , a portion of the insulation module (for example, the 6100 insulation module) that corresponds to the 6190 elution chamber (not defined in Figure 52) is arranged within the corresponding recess 3761. Therefore, when the heater assembly 3700 is positioned at the around each of the cartridges, a side wall of the second portion 3760 of the mounting plate 3754 defining the recesses 3761 is positioned around and / or substantially surrounds a portion of the elution chamber 6190. In this way, the second heating module 3750 it can heat and / or thermally cycle a portion of a sample contained within the elution chamber 6190 of each cartridge.
[000322] In use, the second heating module 3750 can heat a portion of each of the cartridges disposed within the instrument 3001 to promote, perfect and / or facilitate a reaction process that takes place inside the cartridge. For example, in some embodiments, the second 3750 heating module can heat up
Petition 870200011939, of 01/24/2020, p. 143/198
140/184 a portion of a substrate from a PCR module (e.g., substrate 7220 from PCR module 7200 shown and described earlier with reference to Figures 29 to 31). Heating by the second heating module 3750, in one mode, is performed to facilitate a reverse transcription reaction, or for a hot start PCR.
[000323] More particularly, in some embodiments, the second 3750 heating module can facilitate a hot start method associated with a PCR process. The hot start method involves the use of hot start enzymes (polymerase) to reduce the nonspecific primacy of nucleic acids in an amplification reaction. More particularly, when the enzymes are kept at room temperature (for example, below approximately 50 ° C), non-specific hybridization can occur, which can lead to non-specific primacy in the presence of the polymerase. Therefore, hot-starting enzymes are enzymes that are inactive at room temperature, and do not become active until they are heated to a predetermined temperature. This predetermined temperature can be a temperature above approximately 40 ° C, 50 ° C, 70 ° C or 95 ° C. To facilitate the hot start method, the second 3750 heating module can heat an elution chamber (for example, 7190 elution chamber) to keep the eluted nucleic acid sample at an elevated temperature (for example, at a temperature above approximately 40 ° C, 50 ° C, 70 ° C or 95 ° C) before adding the master mix to the amplification reaction inside the PCR flask (for example, the 7260 PCR flask). In some embodiments, for example, the second heating module 3750 can maintain the temperature of the sample inside the elution chamber 7190 up to a temperature between approximately 50 ° C and approximately 95 ° C. By heating the eluted nucleic acid in this way, one can eliminate
Petition 870200011939, of 01/24/2020, p. 144/198
141/184 and / or reduce non-specific hybridization and / or false primacy in the presence of polymerase.
[000324] Reaction reagents (for example, substance R2 contained within the 7270b reagent module shown above in Figures 30 and 31) can then be added to the PCR vial (for example, the 7260 PCR vial) to the lyophilized master mix contained therein. The nucleic acid sample heated from the elution chamber (for example, the 7190 elution chamber) can then be transferred to the PCR flask as described above. In addition, the second heating module 7250 can also heat a flow path between the elution chamber and the PCR flask (for example, passage 7222) in such a way that the contents (for example, the eluted nucleic acid sample that being transferred from the elution chamber to the PCR flask) can be kept at an elevated temperature (for example, at a temperature approximately above 40 ° C, 50 ° C, 70 ° C or 95 ° C). In some embodiments, for example, the second 3750 heating module can maintain the sample temperature within the flow passage at a temperature between approximately 50 ° C and approximately 95 ° C. After the heated elution sample is transported in the PCR flask, the solution is mixed by temperature cycling (produced by the first 3730 heating module), and then the PCR reaction is started.
[000325] The third heating module 3780 includes at least one heater (not shown) and a heater block 3784. As shown in Figure 63, heater block 3784 defines a series of recesses and / or cavities 3786a, 3786b, 3786c , 3786d, 3786e, 3786f, each of which corresponds to each of the cartridges within the 3350 tank). In use, when the 3700 heater assembly is moved into position around each of the
Petition 870200011939, of 01/24/2020, p. 145/198
142/184 cartridges within instrument 3002, a portion of the cartridge is disposed within the corresponding recess (for example, recess 3786a) of heater block 3784. More particularly, as shown in Figure 52, a portion of the insulation module (for example, example, the insulation module 6100) corresponding to the lysis chamber 6114 (not identified in Figure 52) is arranged inside the corresponding recess. Therefore, when the heater assembly 3700 is positioned around each of the cartridges, the side wall of the heater block 3784 that defines the recess 3786 is positioned around and / or substantially surrounds a portion of the lysis chamber 6114. In this way , the third heating module 3780 can heat and / or thermally cycle a portion of a sample contained within the lysis chamber 6114 of each cartridge. In one embodiment, heating by the third heating module 3780 occurs during a reverse transcription and / or PCR reaction.
[000326] Figures 64 to 70 show several views of the 3800 optical assembly of the 3002 instrument. The 3800 optical assembly is configured to monitor a reaction that occurs with a cartridge disposed within the 3002 instrument. More specifically, the 3800 optical assembly is configured to detect one or more different analytes and / or targets within a test sample before, during and / or after a PCR reaction occurs within the PCR vial (for example, the 6260 PCR vial) of the cartridge. As described in this document, the optical mount 3800 can analyze the samples sequentially and / or cyclic and / or in real time. The 3800 optical assembly includes a 3860 excitation module, a 3850 detection module, a 3870 sliding assembly and a 3830 optical fiber assembly.
[000327] For example, in a modality, the optical assembly is
Petition 870200011939, of 01/24/2020, p. 146/198
143/184 used to monitor a nucleic acid amplification reaction in real time. In an additional embodiment, the amplification reaction is a PCR. In another embodiment, the optical assembly is used to measure the results of the binding assays, for example, the bond between the enzyme and the substrate or ligand and receptor.
[000328] The 3830 optical fiber assembly includes a series of excitation optical fibers (identified as excitation fibers 3831a, 3831b, 3831c, 3831d, 3831e, 3831f, 3831g in Figure 64). Each of the excitation fibers 3831a, 3831b, 3831c, 3831d, 3831e and 3831f is configured to carry a beam of light and / or an optical signal from the excitation module 3860 to the corresponding receiving block 3710. Consequently, a first end portion of each excitation fiber 3831a, 3831b, 3831c, 3831d, 3831e and 3831f is arranged within the lumen 3711 of the receiving block 3710, as described above. The 3831g excitation fiber is a calibration fiber and is configured to carry a beam of light and / or an optical signal from the 3860 excitation module to an optical calibration module (not shown). The 3831 excitation optical fibers can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber.
[000329] The 3830 optical fiber assembly includes a series of optical detection fibers (identified as detection fibers 3832a, 3832b, 3832c, 3832d, 3832e, 3832f, 3832g in Figure 64). Each of the detection fibers 3832a, 3832b, 3832c, 3832d, 3832e and 3832f is configured to carry a beam of light and / or optical signal from the receiving block 3710 to the detection module 3850. Consequently, the first portion of end of each detection fiber 3832a, 3832b, 3832c, 3832d, 3832e and 3832f is disposed within the lumen 3712 of the receiving block 3710, as described
Petition 870200011939, of 01/24/2020, p. 147/198
144/184 previously. The 3832g detection fiber is a calibration fiber that is configured to receive a beam of light and / or an optical signal from the optical calibration module (not shown). The 3832 detection optical fibers can be any optical fiber suitable for carrying a beam of light, such as, for example, a multi-mode fiber or a single-mode fiber.
[000330] The 3830 fiber optic mount also includes a 3820 fiber mount block. As shown in Figure 70, the 3820 fiber mount block defines a series of lumens 3825a-3825g and a series of lumens 3824a-3824g. Each of the 3824 lumens is configured to receive a second end portion of the corresponding excitation fiber (for example, the excitation fiber 3831a, as identified in Figure 65). Similarly, each of the 3825 lumens is configured to receive the second end portion of the corresponding detection optical fiber (for example, the 3832a detection fiber, as identified in Figure 65). The fiber mounting block 3820 is coupled to the movable needle 3890 of the sliding assembly 3870 to optically couple excitation fibers 3831 to excitation module 3860 and optically couple detection fibers 3832 to detection module 3850, as described in more detail below .
[000331] As shown in Figure 65, the 3830 optical fiber assembly includes a series of spacers, lenses and sealing members to facilitate the optical connections described here, and / or modify, condition and / or transform a beam of light carried by the 3830 optical fiber assembly. More particularly, the 3830 optical fiber assembly includes a series of excitation spacers 3833 and detection spacers 3834 configured to be arranged within the fiber mounting block 3820 and / or the sliding plate 3890. A 3830 fiber optic mount also includes a
Petition 870200011939, of 01/24/2020, p. 148/198
145/184 series of 3835 excitation lenses and 3836 detection lenses configured to be arranged within the 3820 fiber mounting block and / or the 3890 sliding plate. The 3830 optical fiber assembly also includes a series of sealing members. excitation 3837 and detection seal members 3838 configured to be disposed within the fiber mounting block 3820 and / or sliding plate 3890. Excitation seal members 3837 and detection seal members 3838 are configured to seal and / or prevent contamination from entering the optical paths defined by the 3800 optical assembly.
[000332] As shown in Figures 64 to 66, optical assembly 3800 includes an excitation module 3860 configured to produce a series of excitation light beams (and / or optical signals, not shown). The 3860 excitation module includes a 3861 excitation circuit board on which a series of 3862 excitation light sources are mounted. The 3862 light sources can be any device and / or mechanism suitable for producing a series of excitation light beams. excitation, such as, for example, a laser, a light-emitting diode (LED), a flash lamp, or the like. In some embodiments, the light beam produced by each of the 3862 light sources may have substantially the same characteristics (for example, wavelength, amplitude and / or energy) as the light beams produced by other 3862 light sources. other modalities, however, a first 3862 light source can produce a light beam having a first set of characteristics (for example, a wavelength associated with a red light beam) and a second 3862 light source can produce a beam of light having a second different set of characteristics (for example, a wavelength associated with a beam of green light). This arrangement allows each of the different beams of light (that is, the
Petition 870200011939, of 01/24/2020, p. 149/198
146/184 beams having different characteristics) be transported to each of the 3710 receiving blocks in a sequential manner, as described in greater detail in this document. As shown in Figure 65, excitation module 3860 includes a series of spacers 3863, filters 3864 and lenses 3865 to facilitate the optical connections described here, and / or modify, condition and / or transform a beam of light produced by the excitation module 3860 and carried by the 3831 excitation fibers.
[000333] As shown in Figures 64 to 66, optical assembly 3800 includes a 3850 detection module configured to receive and / or detect a series of emission light beams (and / or optical signals, not shown). The 3850 detection module includes a 3851 detection circuit board on which a series of 3852 emission light detectors are mounted. The 3852 emission light detectors can be any device and / or mechanism suitable for detecting a series of light beams. emission light, such as, for example, an optical detector, a photoresistor, a photovoltaic cell, a photodiode, a phototube, a CCD camera or the like. In some embodiments, each 3852 detector can be configured to selectively receive an emission light beam regardless of the characteristics (e.g., wavelength, amplitude and / or energy) of the emission light beam. In other embodiments, however, detector 3852 can be configured (or tuned) to correspond to a beam of emitting light having a particular set of characteristics (for example, a wavelength associated with a beam of red light). In some embodiments, for example, each of the 3852 detectors can be configured to receive an emission light produced by exciting a portion of the sample when excited by a corresponding light source 3862 of the 3860 excitation module. This arrangement allows each of the different beams of light
Petition 870200011939, of 01/24/2020, p. 150/198
147/184 of emission (i.e., the beams having different characteristics) is received from each of the 3710 receiving blocks in a sequential manner, as described in greater detail in the present document. As shown in Figure 65, the 3850 detection module includes a series of 3853 spacers, 3854 filters and 3855 lenses to facilitate the optical connections described here, and / or modify, condition and / or transform an emission light beam received by the module 3850 detection.
[000334] The 3870 slide assembly includes a 3840 mounting member, a 3880 shoe and a 3890 movable needle. The 3880 shoe is coupled to the 3840 mounting member, and slidably mounted to the 3890 movable needle. As described in more detail below , in use, a conveyor thread 3872, which is rotated by a stepper motor 3873 can rotate within a portion of the shoe 3880 to cause the shoe 3880 (and therefore the mounting member 3840) to move relative to to the 3890 mobile needle, as shown by the HHH arrow in Figures 64 and 66. In this way, the 3840 mounting member can be moved relative to the 3890 mobile needle to sequentially move each of the 3862 excitation light sources and light detectors. emission 3852 in optical communication with the second end of each excitation fiber 3831 and emission fiber 3832, respectively. Additional details on the 3870 slide assembly and the operation of the 3800 optical module are provided below.
[000335] As shown in Figure 67, the mounting member 3840 defines a series of excitation lumens 3844a-3844f and a series of emission lumens 3845a-3845f. As shown in Figure 65, each 3862 excitation light source is disposed within the corresponding excitation lumen 3844, and each 3852 emission light detector is disposed within the corresponding emission lumen 3845. The
Petition 870200011939, of 01/24/2020, p. 151/198
148/184 3840 mounting member is coupled to the 3880 shoe in such a way that the movement of the 3880 shoe causes the 3840 member to move (and therefore the 3862 excitation light sources and 3852 emission light detectors) .
[000336] As shown in Figure 68, shoe 3880 includes a first portion 3881 and a second portion 3882. The first portion 3881 includes a guide protrusion 3886 and defines a series of excitation lumens 3884a-3884f and a series of emission lumens 3855a3855f. When the shoe 3880 is coupled to the mounting member 3840, each of the excitation lumens 3884 of the shoe 3880 is aligned with the corresponding excitation lumen 3844 of the mounting member 3840. Similarly, each of the 3885 emission lumens of the shoe 3880 it is aligned with the corresponding emission lumen 3845 of the mounting member 3840. The guide protuberance is configured so that it is slidably arranged within the corresponding groove 3896 on the 3890 movable needle.
[000337] The second portion 3882 of the 3880 shoe defines a pair of guide lumens 3887 and a lead screw lumen 3888. In use, the conveyor thread 3872 is rotated inside the lead screw lumen 3888 to move the 3880 shoe relative to to the mobile needle 3890. The movement of the shoe 3880 is guided by the guide rails 3871, which are slidably arranged within the corresponding guide lumen 3887.
[000338] As shown in Figure 69, the mobile needle 3890 defines seven excitation openings 3894a, 3894b, 3894c, 3894d, 3894e, 3894f and 3894g, and seven detection openings 3895a, 3895b, 3895c, 3895d, 3895e, 3895f and 3895g . The fiber mounting block 3820 is coupled to the movable needle 3890 in such a way that the excitation fibers 3831 are in optical communication with each corresponding excitation opening, and the detection fibers 3832 are in communication.
Petition 870200011939, of 01/24/2020, p. 152/198
149/184 optics with each corresponding excitation opening. In this way, when the shoe 3880 and the mounting member 3840 are collectively moved relative to the movable needle 3890, each of the excitation openings and detection openings of the shoe 3880 and the mounting member 3840 are aligned with each of the openings. excitation 3894 and detection openings 3895, respectively, of the mobile needle 3890.
[000339] In use, during or after the amplification process, the sliding assembly 3870 can move, in a controlled manner, the 3880 shoe in such a way that each pair of light source 3862 and optical detector 3852 sequentially pass each pair of fiber excitation 3831 and detection fibers 3832. In this way, the optical assembly 3800 can analyze the samples inside each of the six PCR flasks (e.g. PCR flask 6260) in a cyclic and / or multiplexed manner.
[000340] Figures 71 to 73 are schematic block diagrams of the electronic control and computer system for the 3002 instrument. [000341] Although the 3800 optical assembly is shown including the 3850 detection module adjacent to the 3860 excitation module, in other modalities , an optical instrument assembly may include a detection module located in a position relative to an excitation module. For example, Figures 74 to 76 are schematic illustrations of an optical assembly 4800 configured to perform cyclic optical detection of a series of samples, as described above with reference to optical assembly 3800. The optical assembly 4800 is a portion of an instrument ( such as, for example, any of the instruments shown and described in this document) that are configured to contain six 260 reaction vials. The 4800 optical assembly includes a 4860 excitation module, a 4850 detection module and a 4830 fiber assembly The 4860 excitation module includes four excitation light sources
Petition 870200011939, of 01/24/2020, p. 153/198
150/184
4862a, 4862b, 4862c and 4862d. Each of the excitation light sources is configured to produce an excitation light beam having a different wavelength. For example, the light source 4862a is configured to produce a light beam having color # 1 (for example, red), the light source 4862b is configured to produce a light beam having color # 2 (for example, green), the 4862c light source is configured to produce a light beam having color # 3 (for example, blue) and the 4862d light source is configured to produce a light beam having color # 4 (for example, Yellow).
[000342] The 4850 detection module includes four detectors 4852a, 4852b, 4852c and 4865d. Each of the detectors is configured to receive a beam of emission light having a different wavelength. For example, detector 4852a is configured to receive a beam of light resulting from the excitation of an analyte with an excitation color # 1, detector 4852b is configured to receive a beam of light resulting from the excitation of an analyte with an excitation color. # 2, detector 4852hp is configured to receive a beam of light resulting from the excitation of an analyte with an excitation color # 3 and detector 4852d is configured to receive a beam of light resulting from the excitation of an analyte with an excitation color # 4.
[000343] The 4830 fiber assembly includes a series of excitation fibers 4831 and a series of detection fibers 4832. In particular, an excitation fiber is used to optically couple each reaction vial 260 to the excitation module 4860 and a fiber detection sensor 4832 is used to optically couple each reaction vial 260 to the detection module 4850. The excitation module 4860 and detection module 4850 are configured to move relative to the fiber assembly 4830. In this way, each source light and its corresponding detector (for example, the 4862a light source and the 4852a detector) can be sequentially aligned with the
Petition 870200011939, of 01/24/2020, p. 154/198
151/184 excitation and detection for a particular 260 reaction vial.
[000344] In use, when the optical assembly 4800 is in a first configuration, as shown in Figure 74, the light source 4862a and detector 4852a are in optical communication with the first reaction bottle 260. Therefore, the sample contained within of the first reaction flask can be analyzed with an excitation light having color # 1. The excitation module 4860 and detection module 4850 are then moved, as shown by arrows III in Figure 75 to place the optical assembly in a second configuration. When the optical mount 4800 is in the second configuration, as shown in Figure 75, the light source 4862a and detector 4852a are in optical communication with the second reaction bottle 260, and light source 4862b and detector 4852b are in communication optics with the first reaction flask 260. Therefore, the sample contained within the first reaction flask can be analyzed with an excitation light having color # 2 and the sample contained within the second reaction flask can be analyzed with a excitement having color # 1. The excitation module 4860 and detection module 4850 are then moved, as shown by the arrows JJJ in Figure 76 to place the optical assembly in a third configuration. When optical assembly 4800 is in the third configuration, as shown in Figure 76, light source 4862a and detector 4852a are in optical communication with third reaction flask 260, light source 4862b and detector 4852b are in optical communication with the second reaction flask 260, and the light source 4862c and detector 4852c are in optical communication with the first reaction flask 260. Therefore, the sample contained within the first reaction flask can be analyzed with an excitation light having color # 3, the sample contained within the second reaction flask can be analyzed with an excitation light having color # 2, and the sample contained within the third
Petition 870200011939, of 01/24/2020, p. 155/198
152/184 reaction flask can be analyzed with an excitation light having color # 1.
[000345] Figure 75 is a flow chart of a method 100 for detecting nucleic acids in a biological sample according to one embodiment. In particular, the illustrated method is a one-stage target detection method, which can be performed using any of the cartridges shown and described in this document, and any of the instruments shown and described in this document. More particularly, the operations of method 100 described below can be performed on a cartridge without opening the cartridge and / or otherwise exposing the samples, reagents and / or PCR mixture to external conditions. As stated similarly, the method 100 operations described below can be performed in a cartridge without the need for human intervention to transfer samples and / or reagents. For the purposes of the description, method 100 is described as being carried out with insulation module 7100 and PCR module 7200 of cartridge 7001 shown and described previously with reference to Figures 25 to 33.
[000346] The method includes eluting the nucleic acid from the magnetic capture microspheres into an elution chamber, 102. This process can take place, for example, within the 7190 elution chamber of the 7100 isolation module. More particularly, with Referring to Figures 29 to 31, an elution buffer can be stored within the reagent module 7270a, and can be transferred in the 7190 elution chamber, as described above, to complete the elution operation. The elution buffer can be any suitable elution buffer described herein and / or that is compatible with nucleic acid amplification (for example, by PCR and reverse transcription).
[000347] Then, the eluted nucleic acid is transferred from the
Petition 870200011939, of 01/24/2020, p. 156/198
153/184 elution chamber for the PCR chamber, 104. The PCR chamber may, for example, be the 7260 PCR bottle shown in Figures 29 to 31. Although the 7190 elution chamber and the 7260 PCR bottle are shown previously extending in different modules and / or compartments, in other modalities, the elution chamber and the PCR chamber can be located within a monolithically constructed compartment or structure. As previously described, in some embodiments, the PCR chamber may include lyophilized amplification reagents, such that, upon transfer of the nucleic acid, the reagents are reconstituted. The eluted nucleic acid is then transferred into the 7260 PCR flask using the 7235 transfer mechanism, as described above, or any other suitable mechanism.
[000348] The PCR mixture is then thermally cycled and / or heated inside the PCR chamber, 106. The PCR mixture can be cycled between any suitable temperature range using the 3002 instrument, as shown above. In some embodiments, the PCR mix can be raised to a constant temperature to activate enzymes for amplification.
[000349] The amplification reaction is monitored in real time, 108. In some embodiments, the amplification reaction can be monitored by minor groove ligands (MGB) with fluorescent tags and / or any other affinity-based hybridization interactions that to bind to the product (ie, amplicon). Monitoring can be performed using the optical mount 3800 of the instrument 3002 shown and described previously.
[000350] Upon completion of the amplification, the detection probes (for example, MGB) can bind to the target amplicons, 110. This provides a titration detection.
[000351] In some modalities, the method includes performing a
Petition 870200011939, of 01/24/2020, p. 157/198
154/184 fusion analysis and / or temper analysis, 112. This operation can be performed to identify or confirm molecular targets of specific or non-compatible sequences.
[000352] Figure 76 is a flow chart of a method 200 for detecting nucleic acids in a biological sample according to one embodiment. In particular, the illustrated method is a two-stage target detection method, which can be performed using any of the cartridges shown and described in this document, and any of the instruments shown and described in this document. More particularly, the method 200 operations described below can be performed on a cartridge without opening the cartridge and / or otherwise exposing the PCR samples, reagents and / or mixture to external conditions. As stated similarly, the method 200 operations described below can be performed in a cartridge without the need for human intervention to transfer samples and / or reagents. For the purposes of the description, method 200 is described as being carried out with insulation module 6100 and PCR module 6200 shown and described in the present document with reference to Figures 8 to 24.
[000353] The method includes eluting the nucleic acid from the magnetic capture microspheres within an elution chamber, 202. This process can take place, for example, within the 6190 elution chamber of the 6100 isolation module. More particularly, referring to Referring to Figures 8 to 10, an elution buffer can be stored inside reagent chamber 6213c, and can be transferred into the elution chamber, as described above, to complete the elution operation. The elution buffer can be any suitable elution buffer described herein and / or that is compatible with nucleic acid amplification (for example, by PCR and reverse transcription).
Petition 870200011939, of 01/24/2020, p. 158/198
155/184
[000354] The eluted nucleic acid is then transferred from the elution chamber to a PCR chamber, 204. The PCR chamber can, for example, be the 6260 PCR flask shown in Figure 8. As previously described, in some embodiments, the PCR chamber may include lyophilized amplification reagents, such that upon transfer of the nucleic acid, the reagents are reconstituted. The eluted nucleic acid is then transferred using the 6235 transfer mechanism, as described above, or any other suitable mechanism.
[000355] The PCR mixture is then thermally cycled and / or heated inside the PCR chamber, 206. The PCR mixture can be cycled between any suitable temperature range using the 3002 instrument, as shown above. In some embodiments, the PCR mix can be raised to a constant temperature to activate enzymes for amplification.
[000356] The amplification reaction is monitored in real time, 208. In some embodiments, the amplification reaction can be monitored by minor groove ligands (MGB) with fluorescent tags and / or any other affinity-based hybridization interactions that to bind to the product (ie, amplicon). Monitoring can be performed using the optical mount 3800 of the instrument 3002 shown and described previously.
[000357] Upon completion of the amplification, the detection probes (for example, MGB) can bind to the target amplicons, 210. This provides a titration detection. In some embodiments, the method includes performing a fusion analysis and / or a quench analysis, 212. This operation can be performed to identify or confirm molecular targets of specific or non-compatible sequences. Depending on the use in question, a MGB can be used alone as a probe, or it can be conjugated to another molecule and used
Petition 870200011939, of 01/24/2020, p. 159/198
156/184 as a probe. For example, a MGB in one embodiment is coupled to the 5 'termination of a specific DNA oligonucleotide probe, along with a fluorescent dye. The probe, in this embodiment, comprises a non-fluorescent suppressor at the 3 'termination. The fluorescence of the 5 'fluorescent dye is suppressed when the probe is in solution. However, when the probe attaches to its complement, fluorescence is no longer dissipated. Consequently, the degree of fluorescence generated by the probe is directly proportional to the amount of target generated. These probes can be multiplexed in one reaction by conjugating a different fluorescent dye (that is, each fluorescent dye will emit a different wavelength of light when excited, or can be excited at a unique wavelength) to each probe.
[000358] A second set of probes is then distributed to the PCR chamber, 214. In some embodiments, the second set of probes may include a second set of MGB probes or other general probes formulated to bind to specific target sequences or non-compatible ones that melt (dissociation energy to break the affinity interaction) at a temperature approximately above 70 degrees Celsius. In some embodiments, the second set of MGB probes is formulated to bind to specific or non-compatible target sequences that melt at a temperature approximately above 75 degrees Celsius. In other embodiments, the second set of MGB probes is formulated to bind to specific or non-compatible target sequences that melt at a temperature approximately above 80 degrees Celsius. In yet other embodiments, the second set of MGB probes is formulated to bind to specific or non-compatible target sequences that melt at a temperature approximately above 85 degrees Celsius.
Petition 870200011939, of 01/24/2020, p. 160/198
157/184
[000359] In some embodiments, the second set of probes can be stored inside the reagent chamber 6213b, and can be transferred in the PCR vial 6260, either directly or through the elution chamber 6190, as previously described. In this way, the second set of probes can be added to the PCR mix without opening the PCR bottle or cartridge, or otherwise exposing the PCR mix to contaminants.
[000360] Then, the method includes performing a second fusion analysis and / or temper analysis, 216. This operation can be performed to identify or confirm the molecular targets of specific or non-compatible sequences.
[000361] Figure 77 is a flow chart of a method 300 for detecting nucleic acids in a biological sample according to one embodiment. In particular, the illustrated method is a two-step reverse transcription PCR (RT-PCR) method, with single-stage target detection, which can be performed using any of the cartridges shown and described in this document, and any one of the instruments shown and described in this document. More particularly, the method 300 operations described below can be performed on a cartridge without opening the cartridge and / or otherwise exposing the PCR samples, reagents and / or mixture to external conditions. As stated similarly, the method 300 operations described below can be performed on a cartridge without the need for human intervention to transfer samples and / or reagents. For the purposes of the description, method 200 is described as being carried out with isolation module 6100 and PCR module 6200 shown and described previously with reference to Figures 8 to 24.
[000362] The method includes eluting the nucleic acid from the magnetic capture microspheres within an elution chamber,
Petition 870200011939, of 01/24/2020, p. 161/198
158/184
302. This process can take place, for example, inside the elution chamber 6190 of the isolation module 600. More particularly, referring to Figures 8 to 10, an elution buffer can be stored inside the reagent chamber 6213c, and can be transferred in the elution chamber, as previously described, to complete the elution operation. The elution buffer can be any suitable elution buffer described herein and / or that is compatible with nucleic acid amplification (for example, by PCR and reverse transcription).
[000363] The eluted nucleic acid is then transferred from the elution chamber to a PCR chamber, 304. The PCR chamber can, for example, be the 6260 PCR flask shown in Figure 8. As previously described, in some embodiments, the PCR chamber may include lyophilized amplification reagents, such that upon transfer of the nucleic acid, the reagents are reconstituted. The eluted nucleic acid is then transferred using a syringe pump, as described above, or any other suitable mechanism.
[000364] The mixture is then heated inside the PCR chamber to a substantially constant temperature, 306. In this way, enzymes for reverse transcription can be activated.
[000365] Upon completion of reverse transcription, the PCR reagents are distributed to the PCR chamber, 308. The PCR reagents can be stored inside the reagent chamber 6213b and / or 6213a, and can be transferred in the PCR bottle 6260 , either directly or through the 6190 elution chamber, as previously described. In this way, the PCR reagents can be added to the PCR mixture after the end of reverse transcription without opening the PCR cartridge or bottle, or otherwise exposing the PCR mixture to contaminants.
Petition 870200011939, of 01/24/2020, p. 162/198
159/184
[000366] The amplification reaction is monitored in real time, 310. In some embodiments, the amplification reaction can be monitored by minor groove ligands (MGB) with fluorescent tags and / or any other affinity-based hybridization interactions that to bind to the product (ie, amplicon). However, any DNA binding agent can be used for real-time monitoring of a PCR reaction. Monitoring can be performed using the optical mount 3800 of the instrument 3002 shown and described previously.
[000367] Depending on the use in question, the DNA binding agent refers to any detectable molecule, for example, detectable by fluorescence, capable of binding double-stranded or single-stranded DNA. In one embodiment, the DNA binding agent is a fluorescent dye or other chromophore, enzyme, or agent capable of producing a signal, directly or indirectly, when bound to double-stranded or single-stranded DNA. The agent can bind indirectly, that is, the DNA binding agent can be attached to another agent that binds DNA directly. It is only necessary that the agent be able to produce a detectable signal when bound to a double-stranded nucleic acid or to a single-stranded DNA that is distinguishable from the signal produced when the same agent is in solution.
[000368] In one embodiment, the DNA binding agent is an intercalating agent. Interleaving agents, such as ethidium bromide and green SYBR, fluoresce more intensely when interleaved in double-stranded DNA than when bound to single-stranded DNA, RNA, or in solution. Other intercalating agents exhibit a change in the fluorescence spectrum when bound to double-stranded DNA. For example, actinomycin D fluoresces red when bound to single-stranded nucleic acids, and green when bound to a double-stranded template. Want the sign
Petition 870200011939, of 01/24/2020, p. 163/198
160/184 detectable increase, decrease or shift, as in the case of actinomycin D, any intercalating agent that provides a detectable signal that is distinguishable when the agent is bound to double-stranded or unbound DNA is suitable for practice of the invention described.
[000369] In another embodiment, the DNA binding agent is an exonuclease probe that employs fluorescent resonance energy transfer. For example, the DNA binding agent, in one embodiment, is an oligonucleotide probe with a reporter and a suppressor dye at the 5 'and 3' ends, respectively, and specifically binds to a target nucleic acid molecule. In solution, and when intact, the fluorescence of the reporter dye is dissipated. However, the exonuclease activity of a given Taq polymerase serves to cut the probe during PCR, and the reporter is no longer dissipated. Therefore, the fluorescence emission is directly proportional to the amount of target generated.
[000370] In another embodiment, the DNA binding agent employs an MGB conjugated to the 5 'termination of an oligonucleotide probe. In addition to the MGB at the 5 'end, a reporter dye is also coupled to the 5' end of the probe, and a suppressor dye is positioned at the 3 'end. For example, in one embodiment, the DNA probes described by Lukhtanov are employed (Lukhtavon (2007). Nucleic Acids Research 35, p. E30). MGB, in one embodiment, is directly conjugated to the oligonucleotide probe. In another modality, MGB is conjugated to the reporter dye. The fluorescence of the 5 'fluorescent dye is suppressed when the probe is in solution. However, when the probe attaches to its complement, fluorescence is no longer dissipated. Consequently, the degree of fluorescence generated by the probe is directly proportional to the amount of target generated. These probes
Petition 870200011939, of 01/24/2020, p. 164/198
161/184 can be multiplexed in one reaction by conjugating a different fluorescent dye (that is, each fluorescent dye will emit a different wavelength of light when excited, or can be excited at a unique wavelength) to each probe.
[000371] In yet another modality, a smaller groove ligand is used to monitor the PCR reaction in real time. For example, Hoechst 33258 (Searle & Embrey, 1990, Nuc. Acids Res. 18 (13): 37533762) exhibits an altered fluorescence with increasing amount of target. Other MGBs for use in the present invention include distamycin and netropsin.
[000372] According to the modalities described here, a DNA binding agent produces a detectable signal directly or indirectly. The signal is directly detectable, such as by fluorescence or absorbance, or indirectly via a substituted label portion or agglutination ligand attached to the DNA binding agent.
[000373] According to the modalities described here, a DNA binding agent produces a detectable signal directly or indirectly. The signal is directly detectable, such as by fluorescence or absorbance, or indirectly via a substituted label portion or agglutination ligand attached to the DNA binding agent. For example, in one embodiment, a DNA probe conjugated to a fluorescent reporter dye is used. The DNA probe has a suppressor dye at the opposite end of the reporter dye, and will only fluoresce when linked to its complementary sequence. In an additional embodiment, the DNA probe has both an MGB and a fluorescent dye at the 5 'termination.
[000374] Other non-limiting DNA binding agents for use in the invention include, but are not limited to, Molecular Beacons, Scorpions and FRET probes.
Petition 870200011939, of 01/24/2020, p. 165/198
162/184
[000375] Upon completion of the amplification, the detection probes (for example, MGB) can bind to target amplicons, 312. This provides a titration detection. The method includes performing a fusion analysis and / or temper analysis, 314. This operation can be performed to identify or confirm the molecular targets of specific or non-compatible sequences.
[000376] Figure 78 is a flow chart of a 400 method for detecting nucleic acids in a biological sample according to one embodiment. In particular, the illustrated method is an alternative stage detection method to the method 100 shown and described earlier. Method 400 can be performed using any of the cartridges shown and described in this document, and any of the instruments shown and described in this document. More particularly, the method 400 operations described below can be performed on a cartridge without opening the cartridge and / or otherwise exposing the PCR samples, reagents and / or mixture to external conditions. As stated similarly, the 400 method operations described below can be performed on a cartridge without the need for human intervention to transfer samples and / or reagents. For the purposes of the description, method 400 is described as being carried out with isolation module 10100 and PCR module 10200 shown and described in the present document with reference to Figures 85 to 87.
[000377] Method 400 is different from method 100 in that the elution buffer is stored inside the elution chamber of the compartment, rather than being stored in the reagent chamber 6213c, as described for method 100. Therefore, the method includes eluting the nucleic acid from the magnetic capture microspheres inside an elution chamber, 402. This process takes place inside the elution chamber of the 10100 isolation module.
Petition 870200011939, of 01/24/2020, p. 166/198
163/184 elution can be any suitable elution buffer that is compatible with nucleic acid amplification (for example, by PCR and reverse transcription).
[000378] The eluted nucleic acid is then transferred from the elution chamber to a PCR chamber, 404. The PCR chamber can, for example, be the 10260 PCR flask shown in Figures 85 to 87. Although the elution 10190 and the PCR vial 10260 are shown to be in different modules and / or compartments, in other embodiments, the elution chamber and the PCR chamber may be located within a monolithically constructed compartment or structure. As previously described, in some embodiments, the PCR chamber may include lyophilized amplification reagents, such that upon transfer of the nucleic acid, the reagents are reconstituted. The eluted nucleic acid is then transferred using a syringe pump, as described above, or any other suitable mechanism.
[000379] The PCR mixture is then thermally cycled and / or heated inside the 406 PCR chamber. The PCR mixture can be cycled between any suitable temperature range using the 3002 instrument, as shown above. In some embodiments, the PCR mix can be raised to a constant temperature to activate enzymes for amplification.
[000380] The amplification reaction is monitored in real time, 408. In some embodiments, the amplification reaction can be monitored by minor groove ligands (MGB) with fluorescent tags and / or any other affinity-based hybridization interactions that to bind to the product (ie, amplicon). Monitoring can be performed using the optical mount 3800 of the instrument 3002 shown and described previously.
Petition 870200011939, of 01/24/2020, p. 167/198
164/184
[000381] Upon completion of the amplification, the detection probes (for example, MGB) can bind to the target amplicons, 410. This provides a titration detection. In some embodiments, the method includes performing a fusion analysis and / or a quench analysis, 412. This operation can be performed to identify or confirm molecular targets of specific or non-compatible sequences.
[000382] The data produced using the systems and methods described here can be analyzed using any number of different methods. For example, data can be analyzed to identify amplified nucleic acid sequence through fusion or quench analysis using affinity probes. The Fusion / Hardening-Molecular Profile with exclusive affinity probes or molecular tags (consists of modified bases and MGB-fluorine with affinity-directed affinity binding nucleic acid - Kd) indicates / generates spectra of a specific genetic state. For example, Figure 81 is a graph of a spectrum indicating a molecular signature generated from a set of probes that bind to an amplified nucleic acid from a biological sample. The molecular signature represents a diseased state (or presence of unique nucleic acid sequences) referring back to the biological sample. The signature or molecular profile depends on the specific interaction of the molecular tags with the target nucleic acid that can only be generated with the molecular tags inside the cartridge. In other words, the spectrum is a fingerprint trace (that is, a unique sequence of spectral peaks or responses that indicate a diseased state (oncology, infectious disease) or genetic status).
[000383] Multiplex in a spectral multiplexing in more than one sick state (Multiple Markers) with temperature and time (within
Petition 870200011939, of 01/24/2020, p. 168/198
165/184 of a specific wavelength), with exclusive probes or multiple probes (molecular entities-exclusive molecular reagents, indicators, tags).
[000384] Multiple Channel Approach: More than one fingerprint trace (sets of fingerprints) can be used in the identification process. The Spectral Arrangement of Fingerprints in Multiple Fingerprints can be used to determine the result. The variables used to generate the data from multiple channels or arrangement are the wavelength difference fluorescence, the temperature ranges for quenching or dissociation (melting), and the data acquisition rate (time dependent domain).
[000385] The heating and cooling control of the affinity probes and amplified target can be used to produce the desired fingerprint to identify the disease. The temperature range can be within the range of 70 to 100 degrees Celsius for data generation (tempering and melting)
[000386] Although the previous 6001 insulation module is shown including a 6100 insulation module with a 6181 mixing pump to facilitate the lysis process, in other embodiments, any suitable mechanism for transferring energy into a solution to promote and / or enhance cell lysis can be used. For example, in some modalities, you can use acoustic energy.
[000387] For example, Figure 82 shows a second compartment 8160 of an isolation module according to a modality configured to transmit ultrasonic energy in the sample contained within an isolation chamber (not shown) of the isolation module (for example, isolation module 6100, isolation module 7100 or the like) to promote cell lysis and / or isolation of nucleic acids contained therein. The second
Petition 870200011939, of 01/24/2020, p. 169/198
166/184 compartment 8160 can be coupled and / or disposed within a corresponding first compartment (not shown in Figure 82), in a manner similar to that described previously with reference to Figure 11. More particularly, the second compartment 8160 includes a seal (not shown) similar to the 6172 seal shown and described earlier that substantially acoustic isolates the second compartment 8160 from the first compartment.
[000388] The second compartment 8160 defines a series of holding chambers 8163a, 8163b, 8163c and 8163d that contain the reagents and / or other substances used in the isolation process. In particular, the holding chambers may contain a protease (for example, Proteinase K), a lysis solution to solubilize the bulky material, a binding solution to magnetically charge the nucleic acid, and a solution of magnetic microspheres that bind to the Magnetically charged nucleic acid to aid in transporting the nucleic acid within the isolation module and / or the first compartment.
[000389] The second compartment 8160 also defines an opening 8185 within which a portion of an ultrasonic transducer 8195 can be arranged. An acoustic coupling member 8182 is coupled to a portion of the side wall of the second compartment 8160 within the opening 8185. Consequently, in use, at least a portion of an acoustic transducer 8195 can be arranged within the opening 8185 and be in contact with the acoustic coupling member 8182. In this way, the acoustic and / or ultrasonic energy produced by the transducer 8195 can be transported through the acoustic coupling member 8182 and the side wall of the second compartment 8160, and in the solution inside the isolation chamber. The 8195 acoustic transducer can be any suitable acoustic transducer, and can be configured to resonate between 20 kHz and 300 kHz.
Petition 870200011939, of 01/24/2020, p. 170/198
167/184
[000390] The 8195 ultrasonic transducer can be moved in the 8185 opening by an instrument actuator, such as the 3002 instrument described in this document. This actuator may include, for example, a stepper motor configured to move the 8195 ultrasonic transducer by a predetermined distance in contact with the 8182 acoustic coupling member. In some embodiments, for example, an instrument may include an actuator assembly that is similar to the first 3400 actuator assembly shown and described earlier with reference to Figures 37 to 40. In such an embodiment, the first actuator assembly may include a series of ultrasonic transducers that are moved through the opening through a draw bar similar to the drawbar 3445.
[000391] In some embodiments, the actuator can be configured to vary the force exerted by the 8195 ultrasonic transducer on the 8182 acoustic coupling member. This can be accomplished, for example, by moving the 8195 ultrasonic transducer in relation to coupling member 8182 while the ultrasonic transducer is being driven. This arrangement can allow the transmission of ultrasonic energy through the acoustic coupling member 8182 and / or the heat generated by the transmission of ultrasonic energy through the acoustic coupling member 8182 to be dynamically adjusted.
[000392] In some embodiments, the 8182 acoustic coupling member is constructed from a thermally insulating material. In this way, the heat transfer from the acoustic coupling member 8182 to the adjacent side wall of the second compartment 8160 can be minimized. This arrangement can minimize and / or prevent deformation and / or melting of the side wall of the second compartment 8160 when the 8195 acoustic transducer is activated when in contact with the side wall.
Petition 870200011939, of 01/24/2020, p. 171/198
168/184
Additionally, in some embodiments, the acoustic coupling member 8182 can be configured and / or constructed to have an acoustic impedance to promote the transfer of ultrasonic energy through the acoustic coupling member 8182 and in the isolation chamber.
[000393] Figure 83 shows a second compartment 9160 of an isolation module according to a modality configured to transmit ultrasonic energy in the sample contained within an isolation chamber (not shown) of the isolation module to promote cell lysis and / or isolating the nucleicides contained therein. The second compartment 9160 can be coupled and / or disposed within a corresponding first compartment (not shown in Figure 83), in a similar manner as described previously. More particularly, the second compartment 9160 includes a seal (not shown) similar to the seal 6172 shown and described above that substantially acoustic isolates the second compartment 9160 from the first compartment.
[000394] The second compartment 9160 defines a series of holding chambers 9163a, 9163b, 9163c and 9163d that contain the reagents and / or other substances used in the isolation process. The second compartment 9160 also defines an opening 9185 into which a portion of an ultrasonic transducer 9195 can be arranged. Unlike the opening 8185 described above, the opening 9185 can be in fluid communication with the isolation chamber through an opening in the side wall of the second compartment 9160.
[000395] An acoustic coupling member 9183 is disposed within opening 9185 and through a portion of the side wall of the second compartment 9160. More particularly, the acoustic coupling member 9183 is coupled to the side wall in such a way that
Petition 870200011939, of 01/24/2020, p. 172/198
169/184 the first portion 9186 of the acoustic coupling member 9183 is within the opening 9185 and a second portion 9187 of the acoustic coupling member 9183 is within the isolation chamber. A seal 9184 is disposed between the side wall of the second compartment 9160 and the acoustic coupling member 9183 to substantially fluidly isolate the isolation chamber and / or substantially acoustic isolate the acoustic coupling member 9183 from the second compartment.
[000396] In use, at least a portion of an acoustic transducer 8195 can be arranged within opening 9185 and be in contact with the first portion 9186 of acoustic coupling member 9183. In this way, acoustic and / or ultrasonic energy produced by the 9195 transducer can be transported through the acoustic coupling member 9183 in the solution inside the isolation chamber. [000397] The 8195 ultrasonic transducer can be moved within the 9185 aperture by an instrument actuator, such as the 3002 instrument described in this document. This actuator may include, for example, a stepper motor configured to move the 9195 ultrasonic transducer by a predetermined distance in contact with the 9183 acoustic coupling member. In some embodiments, for example, an instrument may include an actuator assembly that is similar to the first 3400 actuator assembly shown and described earlier with reference to Figures 37 to 40. In such an embodiment, the first actuator assembly may include a series of ultrasonic transducers that are moved through the opening through a drawbar similar to the 3445 drawbar.
[000398] In some embodiments, the actuator can be configured to vary the force exerted by the ultrasonic transducer 5195 on the acoustic coupling member 5183. This can be accomplished, for example, by moving the ultrasonic transducer 8195 in relation to to
Petition 870200011939, of 01/24/2020, p. 173/198
170/184 9183 coupling member while the ultrasonic transducer is being driven. This arrangement can allow the transmission of ultrasonic energy through the acoustic coupling member 9183 and / or the heat generated by the transmission of ultrasonic energy through the acoustic coupling member 9183 to be dynamically adjusted.
[000399] As previously described, in some embodiments, the acoustic coupling member 5183 can be configured to have an acoustic impedance to promote the transfer of ultrasonic energy through the acoustic coupling member 9183 and into the isolation chamber.
[000400] Although Figures 82 and 83 show the second compartment of an isolation module configured to transmit ultrasonic energy in the sample contained within the isolation module, in other embodiments, any portion of a cartridge can be configured to transmit ultrasonic energy in the sample. For example, Figure 84 shows the insulation module 7100 (see, for example, Figures 26 to 28) and an ultrasonic transducer 7195. In particular, as described above, compartment 7110 includes an acoustic coupling portion 7182. In use, at least a portion of the acoustic transducer 7195 can be arranged in contact with the acoustic coupling portion 7182. In this way, the acoustic and / or ultrasonic energy produced by the transducer can be transported through the acoustic coupling portion 7182 and the side wall of the first compartment 7110, and the solution inside the lysis chamber 7114.
[000401] The ultrasonic transducer 7195 can be moved in contact with the acoustic coupling portion 7182 by an actuator of an instrument, such as the instrument 3002 described in this document. This actuator can include, for example, a stepper motor
Petition 870200011939, of 01/24/2020, p. 174/198
171/184 configured to move the 7195 ultrasonic transducer by a predetermined distance in contact with the acoustic coupling portion 7182. In some embodiments, for example, an instrument may include an actuator assembly that is similar to the first 3400 actuator assembly shown and described previously with reference to Figures 37 to 40. In such an embodiment, the first actuator assembly may include a series of ultrasonic transducers that are moved in contact with the acoustic coupling portion 7182 through a drawbar similar to the drawbar 3445.
[000402] In some embodiments, the actuator can be configured to vary the force exerted by the ultrasonic transducer 7195 on the acoustic coupling portion 7182. This can be accomplished, for example, by moving the ultrasonic transducer 7195 in relation to to the acoustic coupling portion 7182 while the ultrasonic transducer is being driven. This arrangement can allow the transmission of ultrasonic energy through the acoustic coupling portion 7182 and / or the heat generated by the transmission of ultrasonic energy through the acoustic coupling portion 7182 to be dynamically adjusted. As shown in Figure 83, the ultrasonic transducer 7195 may include a spring 7196 or other guiding member configured to hold and / or orient the ultrasonic transducer with respect to the actuator assembly of the instrument.
[000403] Although PCR module 6200 is shown and described previously including three reagent chambers 6213a, 6213b and 1213c where PCR reagents, elution buffers and the like can be stored, in other embodiments, a PCR module may include any number of reagent chambers. In some embodiments, a PCR module may be devoid of reagent chambers. For example, Figures 85 to 87 show a 10001 cartridge
Petition 870200011939, of 01/24/2020, p. 175/198
172/184 according to one modality. The 10001 cartridge includes a 10100 nucleic acid isolation module and a 10200 amplification (or PCR) module coupled to form the 10001 integrated cartridge. The 10001 integrated cartridge is similar in many ways to the 6001 cartridge and / or the 7001 cartridge. shown and described earlier and therefore will not be described in detail. As shown in Figure 86, which shows the cartridge without the 10005 coating, the 10200 PCR module includes a 10210 compartment, a 10260 PCR bottle and a 10250 transfer tube. The 10200 amplification module is coupled to the 10100 isolation module. such that at least a portion of the transfer tube is disposed within the elution chamber of the insulation module 10100.
[000404] The 10210 compartment includes a 10270 transfer port. The 10270 transfer port defines one or more lumens and / or passages through which isolated nucleic acid and / or other substances or reagents can be transported in the 10260 PCR bottle. The 10210 compartment and / or the 10270 transfer port can define one or more ventilation passages for fluidly coupling the elution chamber and / or the 10260 PCR flask to the atmosphere. In some embodiments, any of these ventilation passages may include a frit, valve and / or other suitable mechanism to minimize and / or prevent loss of sample and / or reagents from the elution chamber and / or the PCR 10260 bottle [000405] The first end portion 10271 of the transfer port 10270 is disposed outside the PCR bottle 10260, and a second end portion 10272 of the transfer port 10270 is disposed inside the PCR bottle. More particularly, the second end portion 10272 is arranged within the PCR vial 10260 such that the volume V of the PCR vial 10260 within which the sample can be disposed is not greater than a magnitude
Petition 870200011939, of 01/24/2020, p. 176/198
173/184 predetermined. In this way, due to the fact that there is an empty space above the sample inside the PCR 10260 flask, condensation that can form on the wall of the 10260 PCR flask during thermal cycling can be minimized and / or eliminated.
[000406] The PCR module 10200 includes a transfer piston 10240 configured to produce a pressure and / or vacuum inside the elution chamber and / or the PCR bottle 10260 to transfer at least a portion of the sample and / or reagents inside the elution chamber to the PCR 10260 flask, as previously described.
[000407] The elution buffer used with the 10001 cartridge is stored in the elution chamber (not shown in Figures 85 to 87) of the 10100 isolation module. The PCR reagents are stored in the 10260 PCR vial in a lyophilized form, as described earlier. In use, the isolated nucleic acid is eluted from the capture microspheres in the elution chamber. The eluted nucleic acid is then transferred into the 10260 PCR flask, as described above, and mixed with the PCR reagents inside the 10260 PCR flask.
[000408] Although the PCR module 6200 is shown and described including three reagent chambers 6213a, 6213b and 6213c which are arranged adjacent to the first end portion 6211 of compartment 6210 (see, for example, Figure 8), in other embodiments , a PCR module can include any number of reagent chambers or modules arranged in any position and / or orientation. In addition, in some embodiments, reagent plungers (for example, plunger 6214a) and / or any of the transfer mechanisms described in this document can be targeted. For example, Figure 88 is a cross-sectional view of a PCR module 11200 coupled to an insulation module 6100 '. The 11200 PCR module includes a compartment
Petition 870200011939, of 01/24/2020, p. 177/198
174/184
11210 which defines three reagent chambers 11213, within which substances and / or reagents of the types described in this document can be stored. A plunger 11214 and a spring 11215 (only one of which is shown and labeled in Figure 88) are arranged within each of the reagent chambers 11213. In this way, the plunger (or transfer mechanism) is oriented in the non-actuated position. In other embodiments, however, the plunger can be oriented in a driven position and can be held in position by a locking tab or the like. In this way, the actuation of the plunger can be aided by the spring force.
[000409] The PCR module also includes a 11130 mixing mechanism (or transfer mechanism) that is fluidly communicating with the 6190 'elution chamber via a 11131 nozzle. A 11250 pipette tube places the 6190 elution chamber in fluid communication with the 11260 PCR bottle.
[000410] In some embodiments, a PCR module may include a PCR vial or a reaction chamber that is disposed adjacent to an isolation module elution chamber. For example, Figure 89 shows a cartridge 12001 having isolation module 6100 'coupled to a PCR module 12200. PCR module 12200 includes a PCR chamber 12260 that is adjacent to elution chamber 6190'. As similarly stated, when PCR module 12200 is coupled to isolation module 6100 ', PCR bottle 12260 is disposed between reagent PCR chamber 12231 and isolation module 6100'.
[000411] Although the cartridges shown and described in this document include an isolation module and an elution chamber (for example, the 7190 elution chamber) coupled to a PCR module such that in use, a portion of a sample isolated is transferred into a PCR vial (eg, PCR 7260 vial),
Petition 870200011939, of 01/24/2020, p. 178/198
175/184 in other embodiments, a PCR module does not need to include a PCR bottle. For example, in some embodiments, a cartridge may include an elution chamber that is also configured to be the reaction volume at which a PCR can occur. For example, Figure 90 shows a cartridge 13001 according to an embodiment that includes an isolation module 6100 'and a PCR module 13200. The PCR module 13200 includes a substrate 13220 and a series of reagent modules 13270. In use , the 13270 reagent modules are configured to transfer one or more reagents and / or substances of the types shown and described in this document in the 6190 'elution chamber of the 6100' isolation module through the 13229 flow tubes. In this way, the PCR can occur in the 6190 'elution chamber. In such embodiments, an instrument similar to instrument 3002 can be configured to thermally cycle elution chamber 6190 'to facilitate PCR. In addition, the instrument may include an optical assembly configured to monitor the reaction optically within the 6190 'elution chamber. In some embodiments, compartment 6110 'may include an optical excitation member (not shown) and / or an optical sensing member (not shown) disposed adjacent to elution chamber 6190'.
[000412] Although the cartridges shown and described in this document generally include a PCR module that is coupled in series to an isolation module, in other embodiments, a cartridge can include a PCR module coupled to an isolation module in any orientation , position and / or location. As similarly stated, although the cartridges are shown and described in this document including a PCR module that is coupled to an end portion of an isolation module, in other embodiments, a PCR module can be integrated and / or
Petition 870200011939, of 01/24/2020, p. 179/198
176/184 coupled to an isolation module in any way. For example, Figure 91 shows a cartridge 14001 that includes an insulation module 14100 and a PCR module 14200. The insulation module 14100 includes a series of washing mechanisms 14130, similar to those previously described. The PCR module includes a series of 14270 reagent modules. The 14270 reagent modules are arranged adjacent and / or between the 14130 flushing mechanisms. [000413] In use, the 14270 reagent modules are configured to transfer one or more reagents and / or substances of the types shown and described in this document in the elution chamber 14190 of the isolation module 14100 through the flow tubes 14229. In this way, PCR can occur in the elution chamber 14190.
[000414] Figures 92 and 93 show another embodiment in which the reagent modules 15270 of the PCR module 15200 are disposed adjacent and / or between the washing mechanisms 15130 of the isolation module 15100. The cartridge 15001 is different from the cartridge 14001 by fact that the substances contained within the 15270 reagent modules are transferred in the PCR bottle 15260 through a series of internal flow paths 15228. The PCR module includes a transfer mechanism 15235 to transfer a portion of the isolated sample from ad elution chamber 15190 in the PCR flask 15260.
[000415] Although the PCR modules shown and described in this document include a single PCR bottle, in other embodiments, a PCR module can include any number of PCR bottles. An example is shown in Figure 94, which shows a 16200 PCR module having four PCR 16260 bottles.
[000416] Although several modalities have been described previously, it must be understood that these were presented
Petition 870200011939, of 01/24/2020, p. 180/198
177/184 only as an example, and without limitation. When the methods and / or schemes described above indicate certain events and / or flow patterns that occur in a certain order, the ordering of certain events and / or flow patterns can be modified. Additionally, certain events can be carried out simultaneously in parallel processes when possible, as well as carried out sequentially. Although the modalities have been particularly shown and described, it is understood that several changes in form and details can be made.
[000417] Although many of the chambers described herein, such as, for example, chamber 6163a, wash buffer module 7130a and reagent module 7270a, are described containing a substance, sample and / or reagent, which is kept in fluidic isolation by a piercing member (for example, piercing member 6170, piercing member 7135a, and piercing member 7275), in some embodiments, any of the chambers can only be partially filled with the desired substance, sample and / or reagent . More particularly, any of the chambers described herein can include a first volume of the desired substance (which is generally in a liquid state) and a second volume of a gas, such as air, oxygen, nitrogen, or the like. This arrangement reduces the force to move a transfer mechanism or piercing member (e.g., piercing portion 6168 of actuator 6166) into the chamber before breaking the piercing member. More particularly, including a portion of the chamber volume as a gas, when the transfer mechanism moves within the chamber, the gas is compressed to reduce the volume of the chamber, thereby allowing the piercing member to contact with the piercable member. In some modalities, any of the
Petition 870200011939, of 01/24/2020, p. 181/198
178/184 chambers described herein can include approximately ten percent of the volume as a gas.
[000418] Although the insulation module 6100 is shown and described previously including a transfer assembly 6140a configured to transfer substances between the lysis chamber 6114 and the washing chamber 6121 while maintaining the lysis chamber 6114 substantially fluidly isolated from the chamber wash 6121, in other embodiments, any of the modules described herein may include a transfer mechanism that transfers substances between the chambers while allowing fluid communication between these chambers. For example, in some embodiments, a module may include a transfer mechanism configured to selectively control the flow of a substance between the first chamber and the second chamber. Such a transfer mechanism may include, for example, a valve.
[000419] Although the cartridges are shown and described in this document including multiple modules (for example, an isolation module and a reaction module) that are coupled together before being disposed within an instrument that handles the cartridge, in other modalities , a cartridge can include multiple modules, at least one of which is configured to mate with another of the modules within and / or through an instrument. Similarly, in some embodiments, an instrument can be configured to couple a module (for example, a reagent module) to another module (for example, a reaction module, an isolation module or the like) as part of the processing of the cartridge.
[000420] Although transfer mechanisms, such as the 6140 transfer assembly, are shown and described in this document using a magnetic force to facilitate the movement of a target portion of the sample within a cartridge, in
Petition 870200011939, of 01/24/2020, p. 182/198
179/184 other modalities, any of the transfer mechanisms shown and described in this document can employ any suitable type of force to facilitate the movement of a target portion of the sample within a cartridge. For example, in some embodiments, a transfer mechanism may include a pump. In other embodiments, a transfer mechanism can produce a peristaltic movement of the target portion of the sample.
[000421] Although the cartridges and / or portions thereof have been described primarily for use with nucleic acid isolation and amplification reactions, and for use with particular instruments described herein, the cartridge is not limited to these. Although the instruments and / or their portions have been described primarily for use with nucleic acid isolation and amplification reactions, and for use with the particular cartridges described herein, the instrument is not limited to these.
[000422] For example, the cartridge, instrument and / or portions thereof provided herein can be used on a next generation sequencing platform (NGS). NGS technologies have been reported to generate three to four orders of magnitude more sequence than the Sanger method, and are also less expensive to perform (Harismendy et al. (2009). Genome Biology 10, pp. R32.1- R32 .13). The application of NGS includes, but is not limited to, genomic shotgun sequencing, terminal sequencing of bacterial artificial chromosome (BAC), single nucleotide polymorphism staining and re-sequencing, other mutation disclosure, chromatin immunoprecipitation (ChIP), micro RNA development, large-scale sequence marker sequencing, primer walking method, or serial gene expression analysis (SAGE).
[000423] In one modality, a module is used to fit a
Petition 870200011939, of 01/24/2020, p. 183/198
180/184 cartridge of the present invention in an NGS platform instrument, for nucleic acid sequence analysis. Alternatively, a sample transfer module (for example, an automated liquid handling instrument) can transfer the nucleic acid amplification product to a flow cell of an NGS instrument.
[000424] In one embodiment, a module is provided such that a cartridge of the present invention is receptable for use with one of the following NGS platforms: Roche 454 GS-FLX platform, Illumina Sequencing Platforms (for example, HiSeq 2000 , HiSeq 1000, MiSeq, Genome Analyzer IIx), Illumina Solexa IG Genome Analyzer, Applied Biosystems 3730xl platform, ABI SOLiD ™ (for example, 5500xl or 5500 SOLiD ™ System). The method can be attached to one of the devices mentioned above, or it can be a sample transfer module, which moves the product of the nucleic acid amplification reaction to the NGS instrument.
[000425] In one embodiment, the cartridge of the present invention is used for genomic shotgun sequencing, bacterial artificial chromosome (BAC) terminal sequencing, single nucleotide polymorphism staining and resequencing, another mutation disclosure, chromatin immunoprecipitation (ChIP) , micro RNA development, large-scale sequence marker sequencing, primer walking method, or serial gene expression analysis (SAGE).
[000426] In one embodiment, the isolation and / or amplification of nucleic acid (for example, PCR) is performed in a cartridge and instrument of the invention, as described in this document. In an additional modality, upon completion of the amplification reaction, a sample transfer module transfers the amplification product to the flow cell of the respective measurement instrument.
Petition 870200011939, of 01/24/2020, p. 184/198
181/184
NGS, for library preparation, and subsequent sequencing. [000427] In another embodiment, the isolation and / or amplification of nucleic acid (for example, PCR) is performed in a cartridge and / or instrument of the invention, as described in this document. In an additional modality, upon completion of the amplification reaction, the cartridge is transferred to a receptive module for use with one of the NGS instruments previously provided. The nucleic acid amplification product is then transferred to the fluid cell of the respective NGS instrument, for library preparation, and subsequent sequencing.
[000428] In some embodiments, an apparatus includes a first module, a second module and a third module. The first module defines a first chamber and a second chamber, in which at least the first chamber is configured to contain the sample. The second module defines a first volume configured to contain a first substance. A portion of the second module is configured so that it is disposed within the first chamber of the first module when the second module is coupled to the first module in such a way that the first volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber and a second volume configured to contain a second substance. A portion of the third module is disposed within the second chamber of the first module when the third module is coupled to the first module in such a way that the reaction chamber and the second volume are in fluid communication with the second chamber of the first module.
[000429] In some embodiments, any of the modules described in this document may include an acoustic coupling member configured to carry acoustic energy in a chamber defined by the module.
Petition 870200011939, of 01/24/2020, p. 185/198
182/184
[000430] In some embodiments, any of the modules described here may include a transfer mechanism configured to transfer a sample between a first chamber within the module and a second chamber within the module. Such transfer mechanisms can use any suitable mechanism to transfer substances, including fluid from a solution, a magnetic force or the like.
[000431] In some embodiments, any of the modules described here may include a valve configured to transfer a sample between a first chamber within the module and a second chamber within the module. In some embodiments, such a valve can be configured to maintain fluidic isolation between the first chamber and the second chamber.
[000432] In some embodiments, an apparatus includes a first module, a second module and a third module. The first module defines a first chamber and a second chamber. The first module that includes a first transfer mechanism is configured to transfer a sample between the first chamber and the second chamber while maintaining fluidic isolation between the first chamber and the second chamber. The second module defines a volume configured to contain a substance. A portion of the second module is configured so that it is disposed within the first chamber of the first module when the second module is coupled to the first module in such a way that the volume is configured to be selectively placed in fluid communication with the first chamber. The third module defines a reaction chamber, in which the third module is configured so that it is coupled to the first module in such a way that the reaction chamber is in fluid communication with the second chamber. The third module includes a second transfer mechanism configured to transfer a portion of the
Petition 870200011939, of 01/24/2020, p. 186/198
183/184 sample between the second chamber and the reaction chamber.
[000433] In some embodiments, a device includes a first module and a second module. The first module includes a reaction flask, a substrate and a first transfer mechanism. The reaction flask defines a reaction chamber. The first transfer mechanism includes a plunger movably arranged within a compartment such that the compartment and plunger define a first volume, wherein the first volume contains a first substance. The substrate defines at least a portion of a first flow path and a second flow path. The first flow path is configured so that it is in fluid communication with the reaction chamber. The first volume and an isolation chamber of an isolation module, the second flow path is configured so that it is in fluid communication with the isolation chamber. A portion of the plunger is arranged within the first flow path such that the first volume is fluidly isolated from the reaction chamber when the plunger is in a first position within the compartment. The plunger portion is arranged separate from the first flow path in such a way that the first volume is in fluid communication with the reaction chamber when the plunger is in a second position within the compartment. The plunger is configured to produce a vacuum inside the reaction chamber to transfer a sample from the isolation chamber to the reaction chamber when the plunger is moved from the first position to the second position. The second module includes a second transfer mechanism and defines a second volume configured to contain a second substance. The second module is configured so that it is coupled to the first module in such a way that the second volume can be selectively placed in fluid communication with the isolation chamber through the second path of
Petition 870200011939, of 01/24/2020, p. 187/198
184/184 flow. The second transfer mechanism is configured to transfer the second substance from the second volume to the isolation chamber when the second transfer mechanism is activated.
[000434] In some embodiments, an instrument includes a block, a first optical member, a second optical member and an optical assembly. The block defines a reaction volume configured to receive at least a portion of a reaction vessel. The first optical member is arranged at least partially within the block in such a way that the first optical member defines a first light path and is in optical communication with the reaction volume. The second optical member is arranged at least partially within the block in such a way that the second optical member defines a second light path and is in optical communication with the reaction volume. A first plane that includes the first light path and a second plane that includes the second light path that defines an angle greater than about 75 degrees. The optical assembly is coupled to the first optical member and the second optical member in such a way that a beam of excitation light can be transported in the reaction volume and a beam of emission light can be received from the reaction volume.
[000435] Although several modalities have been described as having particular resources and / or combinations of components, other modalities are possible having a combination of any resources and / or components from any of the modalities as previously discussed.

权利要求:
Claims (13)
[1]
1. Apparatus, comprising:
an insulation module (1100, 5100, 6100, 7100) that includes a first compartment (1110, 5110, 6110, 7110) and a second compartment (1160, 6160, 7160, 8160), in which the first compartment (1110, 5110 , 6110, 7110) defines a first chamber (1114, 5111,6114, 7114) and a second chamber (1190, 6190, 7190), the first chamber (1114, 5111, 6114, 7114) is configured to contain a sample, in whereas the second compartment (1160, 6160, 7160, 8160) includes a side wall and a piercable member (1170, 6170, 7170), wherein the side wall and the piercable member (1170, 6170, 7170) collectively define a first volume (6163a-6163d) configured to contain a first substance (R1), a portion of the second compartment (1160, 6160, 7160, 8160) is configured to be disposed within the first compartment (1110, 5110, 6110, 7110) in such a way so that the first volume (6163a-6163d) is in fluid communication with the first chamber (1114, 5111, 6114, 7114) when a portion of the member perforable ro (1170, 6170, 7170) is perforated; and a reaction module (1200, 6200, 7200) that defines a reaction chamber (1262, 6262) and a second volume (1213, 6213a 6123c) configured to contain a second substance (R2), in which the reaction module ( 1200, 6200, 7200) is configured to be coupled to the isolation module (1100, 5100, 6100, 7100) in such a way that the reaction chamber (1262, 6262) and the second volume (1213, 6213a - 6123c) are in fluid communication with the second chamber (1190, 6190, 7190) of the first compartment (1110, 5110, 6110, 7110, characterized by the fact that the reaction module (1200, 6200, 7200) includes a plunger (6214a - 6214c) the second volume (1213, 6213a - 6213c), the
Petition 870200011939, of 01/24/2020, p. 189/198
[2]
2/4 piston (6214a - 6214c) configured to exert a force on the second substance (R2) to transfer the second substance (R2) from the second volume (1213, 6213a - 6213c) to one of the reaction chambers (1262, 6262) or the second chamber (1190, 6190, 7190) when the plunger (6214a - 6214c) is moved within the second volume (1213, 6213a 6213c).
2. Apparatus, according to claim 1, characterized by the fact that the reaction module (1200, 6200, 7200) is configured to be removably coupled to the isolation module (1100, 5100, 6100, 7100 ).
[3]
3. Apparatus according to claim 1, characterized in that a portion of the reaction module (1200, 6200, 7200) is arranged inside the second chamber (1190, 6190, 7190) of the first compartment (1110, 5110, 6110, 7110) when the reaction module (1200, 6200, 7200) is coupled to the isolation module (1100, 5100, 6100, 7100).
[4]
4. Apparatus, according to claim 1, characterized by the fact that:
the reaction module (1200, 6200, 7200) includes a substrate that partially defines a first flow path and a second flow path, in which the first flow path is configured so that it is in fluid communication with the reaction chamber ( 1262, 6262) and the second chamber (1190, 6190, 7190) of the first compartment (1110, 5110, 6110, 7110), in which the second flow path is configured to be in fluid communication with the second volume (1213) and the second chamber (1190) of the first compartment (1110).
[5]
5. Apparatus according to claim 1, characterized by the fact that the isolation module (1100, 5100, 6100, 7100) includes a transfer mechanism (6140) configured to transfer
Petition 870200011939, of 01/24/2020, p. 190/198
3/4 the sample between the first chamber (1114, 5111, 6114, 7114) and the second chamber (1190, 6190, 7190).
[6]
6. Apparatus according to claim 1, characterized by the fact that the isolation module (1100, 5100, 6100, 7100) includes a valve partially arranged inside the first compartment (1110, 5110, 6110, 7110), in which the valve is configured to transfer the sample between the first chamber (1114, 5111, 6114, 7114) and the second chamber (1190, 6190, 7190) while maintaining fluidic isolation between the first chamber (1114, 5111, 6114, 7114) and the second chamber (1190, 6190, 7190).
[7]
7. Apparatus according to claim 1, characterized by the fact that the insulation module (1100, 5100, 6100, 7100) includes a plunger partially arranged within the first volume (6163a-6163d) of the second compartment (1160, 6160 , 7160, 8160), wherein the plunger is configured to pierce the pierced member portion (1170, 6170, 7170) when moved within the first volume (6163a-6163d).
[8]
8. Apparatus according to claim 1, characterized by the fact that the insulation module (1100, 5100, 6100, 7100) includes an acoustic coupling member (7182, 8182) configured to conduct ultrasonic energy in the first chamber (1114, 7114).
[9]
Apparatus according to claim 1, characterized in that the first volume (6163a-6163d) contains a first substance (R1) comprising a lysis buffer, a nucleic acid isolating reagent or a combination thereof.
[10]
10. Apparatus according to claim 1, characterized by the fact that the second volume (1213, 6213) comprises a second substance (R2) which comprises reagents to perform a PCR.
[11]
11. Apparatus according to claim 10,
Petition 870200011939, of 01/24/2020, p. 191/198
4/4 characterized by the fact that the second substance (R2) also comprises a reverse transcriptase enzyme.
[12]
12. Apparatus according to claim 1, characterized by the fact that the reaction chamber (1262, 6262) comprises a master mix for lyophilized PCR.
[13]
13. Apparatus according to claim 12, characterized by the fact that the reaction chamber (1262, 6262) further comprises a reverse transcriptase enzyme.

类似技术:

---

公开号 | 公开日 | 专利标题

---

US10646875B2|2020-05-12|Apparatus and methods for integrated sample preparation, reaction and detection

---

US9931636B2|2018-04-03|Apparatus and method for integrated sample preparation, reaction and detection

---

AU2017201579B2|2018-05-17|Apparatus and methods for integrated sample preparation, reaction and detection

---

AU2014224115B2|2015-07-09|Apparatus and methods for integrated sample preparation, reaction and detection

同族专利:

---

公开号 | 公开日

---

EP2539426A4|2014-06-25|

---

EP2539426A1|2013-01-02|

---

CA2796586C|2017-10-24|

---

EP2539426B1|2016-11-09|

---

KR101643196B1|2016-07-27|

---

AU2011220873A1|2012-09-13|

---

EP3150689A1|2017-04-05|

---

US20150258548A1|2015-09-17|

---

CN105004596B|2018-12-21|

---

EP3150690A1|2017-04-05|

---

US20170080427A1|2017-03-23|

---

KR20180088489A|2018-08-03|

---

US9539577B2|2017-01-10|

---

AU2011220873B2|2014-07-10|

---

JP2016163576A|2016-09-08|

---

JP6576988B2|2019-09-18|

---

HK1215300A1|2016-08-19|

---

CA3084515A1|2011-09-01|

---

JP6285478B2|2018-02-28|

---

CA2977845C|2020-08-04|

---

CN103221529B|2015-05-27|

---

KR101882940B1|2018-07-30|

---

KR20130048200A|2013-05-09|

---

KR20160088958A|2016-07-26|

---

BR112012021202A2|2015-10-06|

---

KR102004319B1|2019-07-26|

---

JP6154137B2|2017-06-28|

---

CN103221529A|2013-07-24|

---

KR20170078857A|2017-07-07|

---

US20180318838A1|2018-11-08|

---

CA2796586A1|2011-09-01|

---

US10040071B2|2018-08-07|

---

CN105004596A|2015-10-28|

---

WO2011106384A1|2011-09-01|

---

US20110236960A1|2011-09-29|

---

HK1187364A1|2014-04-04|

---

US20130266948A1|2013-10-10|

---

JP2017209110A|2017-11-30|

---

US9074250B2|2015-07-07|

---

CA2977845A1|2011-09-01|

---

US10646875B2|2020-05-12|

---

US8372340B2|2013-02-12|

---

JP2013520204A|2013-06-06|

引用文献:

---

公开号 | 申请日 | 公开日 | 申请人 | 专利标题

---

---

US2940448A|1957-04-29|1960-06-14|Jr Norman B Furlong|Disposable blood-gas analyzer|

---

SE327840B|1968-01-25|1970-08-31|Atuokemi Ab|

---

US3802782A|1970-08-19|1974-04-09|Rohe Scientific Corp|Chemical analyzer performing sequential analysis of samples|

---

US4004150A|1975-05-01|1977-01-18|Samuel Natelson|Analytical multiple component readout system|

---

FR2368709B1|1976-10-19|1979-02-23|Pasteur Institut|

---

US4201578A|1977-11-03|1980-05-06|Eastman Kodak Company|Blocked competing developers for color transfer|

---

US4448534A|1978-03-30|1984-05-15|American Hospital Corporation|Antibiotic susceptibility testing|

---

FI63296C|1980-07-11|1983-05-10|Eflab Oy|fotometer|

---

JPH027420B2|1981-09-18|1990-02-19|Toa Medical Electronics|

---

US4626684A|1983-07-13|1986-12-02|Landa Isaac J|Rapid and automatic fluorescence immunoassay analyzer for multiple micro-samples|

---

US5500188A|1984-03-01|1996-03-19|Molecular Devices Corporation|Device for photoresponsive detection and discrimination|

---

DE3441179C2|1984-11-10|1987-02-26|Dynatech Deutschland Gmbh, 7306 Denkendorf, De|

---

US5656493A|1985-03-28|1997-08-12|The Perkin-Elmer Corporation|System for automated performance of the polymerase chain reaction|

---

US5333675C1|1986-02-25|2001-05-01|Perkin Elmer Corp|Apparatus and method for performing automated amplification of nucleic acid sequences and assays using heating and cooling steps|

---

GB8607975D0|1986-04-01|1986-05-08|Fisons Plc|Devices|

---

US6645758B1|1989-02-03|2003-11-11|Johnson & Johnson Clinical Diagnostics, Inc.|Containment cuvette for PCR and method of use|

---

US5229297A|1989-02-03|1993-07-20|Eastman Kodak Company|Containment cuvette for PCR and method of use|

---

DK374889D0|1989-07-28|1989-07-28|Koege Kemisk Vaerk|PROCEDURE FOR PROCESS MONITORING|

---

JPH06100538B2|1989-08-02|1994-12-12|株式会社日立製作所|Atomic absorption spectrophotometer|

---

US5283624A|1989-10-18|1994-02-01|Hitachi, Ltd.|Multi-element simultaneous analysis atomic absorption spectroscopy photometer and multi-element simultaneous analytic method|

---

US5073029A|1990-02-16|1991-12-17|Eqm Research, Inc.|Multisource device for photometric analysis and associated chromogens|

---

AT188509T|1990-02-16|2000-01-15|Hoffmann La Roche|IMPROVEMENTS IN THE SPECIFICITY AND PURPOSE OF THE POLYMERASE CHAIN REACTION|

---

US5139745A|1990-03-30|1992-08-18|Block Medical, Inc.|Luminometer|

---

JPH0678978B2|1990-05-25|1994-10-05|スズキ株式会社|Aggregation pattern detector|

---

US6787338B2|1990-06-04|2004-09-07|The University Of Utah|Method for rapid thermal cycling of biological samples|

---

US5935522A|1990-06-04|1999-08-10|University Of Utah Research Foundation|On-line DNA analysis system with rapid thermal cycling|

---

US5188455A|1990-11-13|1993-02-23|The Pennsylvania Research Corporation|Apparatus for remote mixing of fluids|

---

KR100236506B1|1990-11-29|2000-01-15|퍼킨-엘머시터스인스트루먼츠|Apparatus for polymerase chain reaction|

---

US5494646A|1993-04-14|1996-02-27|Seymour; Eugene H.|Sampling device and sample adequacy system|

---

US5242837A|1990-12-24|1993-09-07|Slovacek Rudolf E|Method for the rapid detection of analytes involving specific binding reactions and the use of light attenuating magnetic particles|

---

US5994056A|1991-05-02|1999-11-30|Roche Molecular Systems, Inc.|Homogeneous methods for nucleic acid amplification and detection|

---

CA2069537A1|1991-06-07|1992-12-08|Thomas A. Cook|Multiple output referencing system for evanescent wave sensor|

---

DE4123817C2|1991-07-18|1994-06-09|Berthold Lab Prof Dr|Radiation measuring device, in particular for measuring luminescence|

---

JPH05157684A|1991-12-02|1993-06-25|Seikagaku Kogyo Co Ltd|Absorptionmeter|

---

US5348853A|1991-12-16|1994-09-20|Biotronics Corporation|Method for reducing non-specific priming in DNA amplification|

---

US5242660A|1992-02-28|1993-09-07|Paul Hsei|Sample preparation device|

---

WO1993020240A1|1992-04-06|1993-10-14|Abbott Laboratories|Method and device for detection of nucleic acid or analyte using total internal reflectance|

---

US5639423A|1992-08-31|1997-06-17|The Regents Of The University Of Calfornia|Microfabricated reactor|

---

US5897783A|1992-09-24|1999-04-27|Amersham International Plc|Magnetic separation method|

---

US5538849A|1992-12-29|1996-07-23|Toyo Boseki Kabushiki Kaisha|Apparatus for automated assay of DNA probe and method for assaying nucleic acid in sample|

---

EP0682671A4|1993-02-01|1998-01-14|Seq Ltd|Methods and apparatus for dna sequencing.|

---

US5861124A|1993-07-15|1999-01-19|Hamamatsu Photonics Kk|Method and apparatus for detecting denaturation of nucleic acid|

---

GB9314991D0|1993-07-20|1993-09-01|Sandoz Ltd|Mechanical device|

---

EP0636413B1|1993-07-28|2001-11-14|PE Corporation |Nucleic acid amplification reaction apparatus and method|

---

US5436718A|1993-07-30|1995-07-25|Biolumin Corporation|Mutli-functional photometer with movable linkage for routing optical fibers|

---

US5397709A|1993-08-27|1995-03-14|Becton Dickinson And Company|System for detecting bacterial growth in a plurality of culture vials|

---

JP3326708B2|1993-08-31|2002-09-24|日水製薬株式会社|Optical measuring device and method thereof|

---

CA2130013C|1993-09-10|1999-03-30|Rolf Moser|Apparatus for automatic performance of temperature cycles|

---

US5525300A|1993-10-20|1996-06-11|Stratagene|Thermal cycler including a temperature gradient block|

---

US5645801A|1993-10-21|1997-07-08|Abbott Laboratories|Device and method for amplifying and detecting target nucleic acids|

---

US5415839A|1993-10-21|1995-05-16|Abbott Laboratories|Apparatus and method for amplifying and detecting target nucleic acids|

---

US5503805A|1993-11-02|1996-04-02|Affymax Technologies N.V.|Apparatus and method for parallel coupling reactions|

---

US5580523A|1994-04-01|1996-12-03|Bard; Allen J.|Integrated chemical synthesizers|

---

US5541072A|1994-04-18|1996-07-30|Immunivest Corporation|Method for magnetic separation featuring magnetic particles in a multi-phase system|

---

AT198511T|1994-04-29|2001-01-15|Perkin Elmer Corp|METHOD AND DEVICE FOR REAL-TIME DETECTION OF PRODUCTS OF NUCLEIC ACID AMPLIFICATION|

---

SE9401594D0|1994-05-06|1994-05-06|Pharmacia Lkb Biotech|Method of nucleic acid transfer|

---

US5511558A|1994-06-06|1996-04-30|Becton, Dickinson And Company|Blood collection assembly having additive dispensing means and method for sample collection using same|

---

US5976896A|1994-06-06|1999-11-02|Idexx Laboratories, Inc.|Immunoassays in capillary tubes|

---

DE4420732A1|1994-06-15|1995-12-21|Boehringer Mannheim Gmbh|Device for the treatment of nucleic acids from a sample|

---

US5837144A|1994-06-16|1998-11-17|Boehringer Mannheim Gmbh|Method of magnetically separating liquid components|

---

US5508197A|1994-07-25|1996-04-16|The Regents, University Of California|High-speed thermal cycling system and method of use|

---

US5627041A|1994-09-02|1997-05-06|Biometric Imaging, Inc.|Disposable cartridge for an assay of a biological sample|

---

US5705628A|1994-09-20|1998-01-06|Whitehead Institute For Biomedical Research|DNA purification and isolation using magnetic particles|

---

US5576197A|1995-04-07|1996-11-19|Molecular Bio-Products|Polymerase chain reaction container and methods of using the same|

---

US5578818A|1995-05-10|1996-11-26|Molecular Dynamics|LED point scanning system|

---

US5589136A|1995-06-20|1996-12-31|Regents Of The University Of California|Silicon-based sleeve devices for chemical reactions|

---

US5856174A|1995-06-29|1999-01-05|Affymetrix, Inc.|Integrated nucleic acid diagnostic device|

---

US20020022261A1|1995-06-29|2002-02-21|Anderson Rolfe C.|Miniaturized genetic analysis systems and methods|

---

US5686300A|1995-09-11|1997-11-11|Becton Dickinson And Company|Fluorescence detector|

---

US5639668A|1995-09-14|1997-06-17|Boehringer Mannheim Corporation|Optical apparatus for performing an immunoassay|

---

US6004512A|1995-12-08|1999-12-21|Mj Research|Sample cartridge slide block|

---

US5657118A|1996-01-23|1997-08-12|Lee; John T. S.|Device and method for detection/measurement of light|

---

US5661301A|1996-02-07|1997-08-26|The Penn State Research Foundation|Spectroscopy and mapping of atoms, molecules and surface features via difference frequency generation with a scanning tunneling microscope or related instruments|

---

US5863801A|1996-06-14|1999-01-26|Sarnoff Corporation|Automated nucleic acid isolation|

---

US6074827A|1996-07-30|2000-06-13|Aclara Biosciences, Inc.|Microfluidic method for nucleic acid purification and processing|

---

US5882903A|1996-11-01|1999-03-16|Sarnoff Corporation|Assay system and method for conducting assays|

---

US5825478A|1996-11-15|1998-10-20|Wilcox; Steven|Multifunctional photometer apparatus|

---

AU5895898A|1996-12-20|1998-07-17|Gamera Bioscience Corporation|An affinity binding-based system for detecting particulates in a fluid|

---

US6027945A|1997-01-21|2000-02-22|Promega Corporation|Methods of isolating biological target materials using silica magnetic particles|

---

DE19707226A1|1997-02-24|1998-08-27|Bodenseewerk Perkin Elmer Co|Light scanner|

---

US6369893B1|1998-05-19|2002-04-09|Cepheid|Multi-channel optical detection system|

---

DK2333520T3|1997-02-28|2014-09-29|Cepheid|Heat-exchanging, requested chemical reaction device|

---

US6074868A|1997-03-03|2000-06-13|Regents Of The University Of Minnesota|Alumina plate method and device for controlling temperature|

---

US6143496A|1997-04-17|2000-11-07|Cytonix Corporation|Method of sampling, amplifying and quantifying segment of nucleic acid, polymerase chain reaction assembly having nanoliter-sized sample chambers, and method of filling assembly|

---

US6429007B1|1997-05-02|2002-08-06|BIOMéRIEUX, INC.|Nucleic acid amplification reaction station for disposable test devices|

---

US5989499A|1997-05-02|1999-11-23|Biomerieux, Inc.|Dual chamber disposable reaction vessel for amplification reactions|

---

US5904899A|1997-05-15|1999-05-18|Tosoh Corporation|Assaying apparatus and a vessel holder device in use with the assaying apparatus|

---

JP4278717B2|1997-05-23|2009-06-17|ベクトン・ディキンソン・アンド・カンパニー|Automated microbiological testing apparatus and method|

---

SE9702005D0|1997-05-28|1997-05-28|Alphahelix Ab|New reaction vessel and method for its use|

---

US6982431B2|1998-08-31|2006-01-03|Molecular Devices Corporation|Sample analysis systems|

---

US6071748A|1997-07-16|2000-06-06|Ljl Biosystems, Inc.|Light detection device|

---

US6297018B1|1998-04-17|2001-10-02|Ljl Biosystems, Inc.|Methods and apparatus for detecting nucleic acid polymorphisms|

---

US6043506A|1997-08-13|2000-03-28|Bio-Rad Laboratories, Inc.|Multi parameter scanner|

---

US6061128A|1997-09-04|2000-05-09|Avocet Medical, Inc.|Verification device for optical clinical assay systems|

---

US6597450B1|1997-09-15|2003-07-22|Becton, Dickinson And Company|Automated Optical Reader for Nucleic Acid Assays|

---

US6222619B1|1997-09-18|2001-04-24|University Of Utah Research Foundation|Diagnostic device and method|

---

US6492162B1|1998-10-27|2002-12-10|Hitachi, Ltd.|Apparatus for the recovery of nucleic acids|

---

WO1999033559A1|1997-12-24|1999-07-08|Cepheid|Integrated fluid manipulation cartridge|

---

US6050719A|1998-01-30|2000-04-18|Affymetrix, Inc.|Rotational mixing method using a cartridge having a narrow interior|

---

JP3551293B2|1998-02-02|2004-08-04|東洋紡績株式会社|Nucleic acid extraction device|

---

FI102906B|1998-02-23|1999-03-15|Bio Nobile Oy|Procedure and means for transporting a substance|

---

JP3813730B2|1998-03-19|2006-08-23|浜松ホトニクス株式会社|Fluorescence measuring device|

---

US6057163A|1998-04-28|2000-05-02|Turner Designs|Luminescence and fluorescence quantitation system|

---

EP2316570A3|1998-05-01|2011-07-27|Gen-Probe Incorporated|Automated diagnostic analyzer and method|

---

US7387891B2|1999-05-17|2008-06-17|Applera Corporation|Optical instrument including excitation source|

---

US6818437B1|1998-05-16|2004-11-16|Applera Corporation|Instrument for monitoring polymerase chain reaction of DNA|

---

US7498164B2|1998-05-16|2009-03-03|Applied Biosystems, Llc|Instrument for monitoring nucleic acid sequence amplification reaction|

---

US7423750B2|2001-11-29|2008-09-09|Applera Corporation|Configurations, systems, and methods for optical scanning with at least one first relative angular motion and at least one second angular motion or at least one linear motion|

---

EP1046032A4|1998-05-18|2002-05-29|Univ Washington|Liquid analysis cartridge|

---

US6232608B1|1998-08-18|2001-05-15|Molecular Devices Corporation|Optimization systems in a scanning fluorometer|

---

US6569631B1|1998-11-12|2003-05-27|3-Dimensional Pharmaceuticals, Inc.|Microplate thermal shift assay for ligand development using 5--2-oxazole derivative fluorescent dyes|

---

US6838680B2|1999-05-12|2005-01-04|Aclara Biosciences, Inc.|Multiplexed fluorescent detection in microfluidic devices|

---

US7078224B1|1999-05-14|2006-07-18|Promega Corporation|Cell concentration and lysate clearance using paramagnetic particles|

---

US8815521B2|2000-05-30|2014-08-26|Cepheid|Apparatus and method for cell disruption|

---

US6818185B1|1999-05-28|2004-11-16|Cepheid|Cartridge for conducting a chemical reaction|

---

ES2272289T5|1999-05-28|2011-10-21|Cepheid|CARTRIDGE TO PERFORM A CHEMICAL REACTION.|

---

US20040200909A1|1999-05-28|2004-10-14|Cepheid|Apparatus and method for cell disruption|

---

DE60014676T2|1999-05-28|2005-11-17|Cepheid, Sunnyvale|DEVICE AND METHOD FOR THE ANALYSIS OF LIQUID SAMPLES|

---

US6706519B1|1999-06-22|2004-03-16|Tecan Trading Ag|Devices and methods for the performance of miniaturized in vitro amplification assays|

---

US6353475B1|1999-07-12|2002-03-05|Caliper Technologies Corp.|Light source power modulation for use with chemical and biochemical analysis|

---

AU777180B2|1999-07-19|2004-10-07|Organon Teknika B.V.|Device and method for mixing magnetic particles with a fluid|

---

US6657169B2|1999-07-30|2003-12-02|Stratagene|Apparatus for thermally cycling samples of biological material with substantial temperature uniformity|

---

JP5006494B2|1999-08-13|2012-08-22|バイエル・テクノロジー・サービシーズ・ゲゼルシャフト・ミット・ベシュレンクテル・ハフツング|Device and method for measuring multiple analytes|

---

JP4045475B2|1999-09-06|2008-02-13|東洋紡績株式会社|Nucleic acid / protein purification equipment|

---

JP3551860B2|1999-10-05|2004-08-11|株式会社日立製作所|DNA testing method and DNA testing device|

---

US6431476B1|1999-12-21|2002-08-13|Cepheid|Apparatus and method for rapid ultrasonic disruption of cells or viruses|

---

US6699713B2|2000-01-04|2004-03-02|The Regents Of The University Of California|Polymerase chain reaction system|

---

US6358387B1|2000-03-27|2002-03-19|Caliper Technologies Corporation|Ultra high throughput microfluidic analytical systems and methods|

---

US6783934B1|2000-05-01|2004-08-31|Cepheid, Inc.|Methods for quantitative analysis of nucleic acid amplification reaction|

---

GB0010910D0|2000-05-05|2000-06-28|Jones Osborn|Analytical method and apparatus|

---

US6690467B1|2000-05-05|2004-02-10|Pe Corporation|Optical system and method for optically analyzing light from a sample|

---

US6672458B2|2000-05-19|2004-01-06|Becton, Dickinson And Company|System and method for manipulating magnetically responsive particles fluid samples to collect DNA or RNA from a sample|

---

US6520197B2|2000-06-02|2003-02-18|The Regents Of The University Of California|Continuous laminar fluid mixing in micro-electromechanical systems|

---

US8097471B2|2000-11-10|2012-01-17|3M Innovative Properties Company|Sample processing devices|

---

US6511277B1|2000-07-10|2003-01-28|Affymetrix, Inc.|Cartridge loader and methods|

---

WO2002014926A2|2000-08-15|2002-02-21|Nanostream, Inc.|Optical devices with fluidic systems|

---

US6545758B1|2000-08-17|2003-04-08|Perry Sandstrom|Microarray detector and synthesizer|

---

US7142296B2|2000-10-30|2006-11-28|Sru Biosystems, Inc.|Method and apparatus for detecting biomolecular interactions|

---

US7622083B2|2002-01-28|2009-11-24|Biocal Technology, Inc.|Multi-capillary electrophoresis cartridge interface mechanism|

---

DK1354192T3|2001-01-26|2011-12-12|Qiagen Sciences Llc|Multichannel cassette for bioseparation|

---

US6576459B2|2001-03-23|2003-06-10|The Regents Of The University Of California|Sample preparation and detection device for infectious agents|

---

GB0110476D0|2001-04-30|2001-06-20|Secr Defence|Reagent delivery system|

---

US6955589B2|2001-05-22|2005-10-18|Qed Technologies, Inc.|Delivery system for magnetorheological fluid|

---

WO2003002959A1|2001-06-15|2003-01-09|Mj Research, Inc.|Controller for a fluorometer|

---

US20030073110A1|2001-07-03|2003-04-17|Masaharu Aritomi|Method for isolating nucleic acid and a cartridge for chemical reaction and for nucleic acid isolation|

---

US7521179B2|2001-08-21|2009-04-21|Lukas Bestmann|Thermo-optical analysis system for biological reactions|

---

JP4558995B2|2001-09-12|2010-10-06|ベックマンコールター，インコーポレイテッド|Transfer unit and automatic analyzer equipped with the transfer unit|

---

JP5193408B2|2001-09-13|2013-05-08|ベックマンコールター，インコーポレイテッド|Automatic analyzer|

---

WO2003029397A1|2001-10-02|2003-04-10|Stratagene|Side-wall heater for thermocycler device|

---

US6739531B2|2001-10-04|2004-05-25|Cepheid|Apparatus and method for rapid disruption of cells or viruses|

---

US7344894B2|2001-10-16|2008-03-18|Agilent Technologies, Inc.|Thermal regulation of fluidic samples within a diagnostic cartridge|

---

US20030100125A1|2001-10-19|2003-05-29|Monogen, Inc.|Flow control metering system and method for controlling filtration of liquid-based specimens|

---

KR100445560B1|2001-10-31|2004-08-21|바이오넥스|Method of manufacturing kit for isolating nucleic acids or biological materials, kit manufactured by the method, and apparatus using the kit|

---

JP2003139783A|2001-11-01|2003-05-14|Fuji Photo Film Co Ltd|Biochemical analyzing system and biochemical analyzing unit handling device used in the same|

---

IL146404D0|2001-11-08|2002-07-25|E Afikin Computerized Dairy Ma|Spectroscopic fluid analyzer|

---

WO2003049530A2|2001-12-07|2003-06-19|Dyax Corporation|Method and apparatus for washing magnetically responsive particles|

---

US7373253B2|2002-02-12|2008-05-13|Idaho Technology|Multi-test analysis of real-time nucleic acid amplification|

---

US8048386B2|2002-02-25|2011-11-01|Cepheid|Fluid processing and control|

---

US20030203491A1|2002-04-26|2003-10-30|Andrevski Zygmunt M.|Gravitational flow purification system|

---

EP1499415A4|2002-04-26|2009-10-28|Abbott Lab|Structure and method for handling magnetic particles in biological assays|

---

WO2003104770A2|2002-06-01|2003-12-18|Chempaq A/S|A disposable cartridge for characterizing particles suspended in a liquid|

---

JP2005532072A|2002-07-10|2005-10-27|マサチューセッツ・インスティテュート・オブ・テクノロジー|Apparatus and method for isolating nucleic acids from a sample|

---

WO2004029195A2|2002-09-24|2004-04-08|U.S. Government As Represented By The Secretary Of The Army|Portable thermocycler|

---

US6730883B2|2002-10-02|2004-05-04|Stratagene|Flexible heating cover assembly for thermal cycling of samples of biological material|

---

GB0227765D0|2002-11-28|2003-01-08|Secr Defence|Apparatus for processing a fluid sample|

---

US7682565B2|2002-12-20|2010-03-23|Biotrove, Inc.|Assay apparatus and method using microfluidic arrays|

---

US7584019B2|2003-12-15|2009-09-01|Dako Denmark A/S|Systems and methods for the automated pre-treatment and processing of biological samples|

---

US20060094108A1|2002-12-20|2006-05-04|Karl Yoder|Thermal cycler for microfluidic array assays|

---

KR100483684B1|2003-01-29|2005-04-18|바이오넥스|Kit for separating and purifying nucleic acids or various biological materials, and system for automatically performing separation or purification of biological materials using the same|

---

AU2004220626B2|2003-02-05|2010-07-29|Iquum Inc.|Sample processing tubule|

---

WO2004087950A2|2003-04-04|2004-10-14|Roche Diagnostics Gmbh|Improved system for multi color real time pcr|

---

KR100528561B1|2003-04-21|2005-11-16|엘지전자 주식회사|Electromagnetically actuated valve for fluidic mass flow control, manufacturing method thereof and heat exchanger using the same|

---

DE10319045A1|2003-04-25|2004-12-09|november Aktiengesellschaft Gesellschaft für Molekulare Medizin|Device and method for processing liquids containing biopolymers|

---

TW579430B|2003-05-02|2004-03-11|Dr Chip Biotechnology Inc|Automatic micro-fluid hybridization chip platform|

---

US7148043B2|2003-05-08|2006-12-12|Bio-Rad Laboratories, Inc.|Systems and methods for fluorescence detection with a movable detection module|

---

WO2005010147A2|2003-06-13|2005-02-03|The General Hospital Corporation|Device and method for contacting pocoliter volumes of fluids|

---

EP2402089A1|2003-07-31|2012-01-04|Handylab, Inc.|Processing particle-containing samples|

---

EP1510577A1|2003-08-29|2005-03-02|Qiagen GmbH|Method for magnetic bead isolation of nucleic acids|

---

GB0323043D0|2003-09-24|2003-11-05|Lux Biotechnology Ltd|Biochip|

---

US7387485B2|2003-09-29|2008-06-17|Quantum Corporation|Cartridge transport assembly|

---

US7394547B2|2003-11-06|2008-07-01|Fortebio, Inc.|Fiber-optic assay apparatus based on phase-shift interferometry|

---

US20070281288A1|2004-01-27|2007-12-06|Shimshon Belkin|Method and System for Detecting Analytes|

---

US20060257991A1|2004-02-27|2006-11-16|Mcdevitt John T|Integration of fluids and reagents into self-contained cartridges containing particle-based sensor elements and membrane-based sensor elements|

---

US7223949B2|2004-04-21|2007-05-29|Beckman Coulter, Inc.|Analysis apparatus having improved temperature control unit|

---

US20050244837A1|2004-04-28|2005-11-03|Cepheid|Method and device for sample preparation control|

---

WO2005108620A2|2004-05-03|2005-11-17|Handylab, Inc.|Processing polynucleotide-containing samples|

---

WO2005111243A2|2004-05-07|2005-11-24|Cepheid|Multiplexed detection of biological agents|

---

EP1771786B1|2004-06-07|2017-12-20|Iquum, Inc.|Sample multiprocessing|

---

US7585663B2|2004-08-26|2009-09-08|Applied Biosystems, Llc|Thermal device, system, and method, for fluid processing device|

---

EP1794581A2|2004-09-15|2007-06-13|Microchip Biotechnologies, Inc.|Microfluidic devices|

---

JP4999103B2|2004-10-27|2012-08-15|セフィード|Multistage nucleic acid amplification reaction in closed system|

---

US20060165558A1|2004-12-21|2006-07-27|Thomas Witty|Cartridge for diagnostic assays|

---

JP5165383B2|2004-12-23|2013-03-21|アイ−スタツト・コーポレイシヨン|Molecular diagnostic system and method|

---

US7699979B2|2005-01-07|2010-04-20|Board Of Trustees Of The University Of Arkansas|Separation system and efficient capture of contaminants using magnetic nanoparticles|

---

WO2006085443A1|2005-02-10|2006-08-17|Matsushita Electric Industrial Co., Ltd.|Fluid chip, control method for movement of fluid employing it, and chemical reactor|

---

EP1856282A4|2005-02-15|2009-05-13|Univ Virginia|Nucleic acid isolation methods and materials and devices thereof|

---

EP2348321A3|2005-03-10|2014-06-11|Gen-Probe Incorporated|System and methods to perform assays for detecting or quantifying analytes within samples|

---

US7507575B2|2005-04-01|2009-03-24|3M Innovative Properties Company|Multiplex fluorescence detection device having removable optical modules|

---

WO2006122311A2|2005-05-11|2006-11-16|The Trustees Of The University Of Pennsylvania|Microfluidic chip|

---

US20070036026A1|2005-05-16|2007-02-15|Laibinis Paul E|Magnetic Particle Systems and Methods|

---

AR054363A1|2005-05-23|2007-06-20|Astrazeneca Ab|COMPOUNDS THAT DISPLAY MODULATORY ACTIVITY IN THE 5-HYDROXY-TRIPTAMINE 6 RECEIVER|

---

CA2614180A1|2005-07-06|2007-01-11|The Regents Of The University Of California|Apparatuses, systems, and methods for isolating and separating biological materials|

---

JP2007043998A|2005-08-11|2007-02-22|Tosoh Corp|Improved micro fluid chip|

---

US20070054293A1|2005-08-30|2007-03-08|California Institute Of Technology|Microfluidic chaotic mixing systems and methods|

---

JP4682008B2|2005-10-04|2011-05-11|キヤノン株式会社|Biochemical treatment equipment, containers and inspection equipment used therefor|

---

US8372340B2|2005-10-19|2013-02-12|Luminex Corporation|Apparatus and methods for integrated sample preparation, reaction and detection|

---

US7727473B2|2005-10-19|2010-06-01|Progentech Limited|Cassette for sample preparation|

---

US8337755B2|2006-03-13|2012-12-25|Veridex, Llc|Operator independent programmable sample preparation and analysis system|

---

WO2007125468A2|2006-05-01|2007-11-08|Koninklijke Philips Electronics N.V.|Fluid sample transport device with reduced dead volume for processing, controlling and/or detecting a fluid sample|

---

US8232091B2|2006-05-17|2012-07-31|California Institute Of Technology|Thermal cycling system|

---

WO2007139747A1|2006-05-22|2007-12-06|Biovest International Inc.|Interface of a cultureware module in a cell culture system and installation method thereof|

---

US20080038163A1|2006-06-23|2008-02-14|Applera Corporation|Systems and Methods for Cooling in Biological Analysis Instruments|

---

US7888107B2|2006-07-24|2011-02-15|Nanosphere, Inc.|System using self-contained processing module for detecting nucleic acids|

---

GB0618966D0|2006-09-26|2006-11-08|Iti Scotland Ltd|Cartridge system|

---

CN101568636A|2006-11-02|2009-10-28|威腾技术公司|Cartridge for conducting diagnostic assays|

---

US8895267B2|2006-11-03|2014-11-25|Siemens Healthcare Diagnostics Inc.|Random access system and method for polymerase chain reaction testing|

---

US7754148B2|2006-12-27|2010-07-13|Progentech Limited|Instrument for cassette for sample preparation|

---

US7863035B2|2007-02-15|2011-01-04|Osmetech Technology Inc.|Fluidics devices|

---

WO2008109878A2|2007-03-07|2008-09-12|California Institute Of Technology|Testing device|

---

CN101541962A|2007-03-23|2009-09-23|株式会社东芝|Nucleic acid detection cassette and nucleic acid detection apparatus|

---

CN101078028B|2007-03-30|2011-08-31|东华大学|Twice bio-bar-code nucleic acid detecting technique|

---

CN103157400B|2007-06-21|2014-12-24|简·探针公司|Instrument and method for exposing container to multiple areas|

---

EP2214824A4|2007-11-28|2015-08-19|Smart Tube Inc|Devices, systems and methods for the collection, stimulation, stabilization, and analysis of a biological sample|

---

US9409166B2|2007-12-10|2016-08-09|The Trustees Of The University Of Pennsylvania|Integrated PCR reactor for cell lysis, nucleic acid isolation and purification, and nucleic acid amplication related applications|

---

US20090186344A1|2008-01-23|2009-07-23|Caliper Life Sciences, Inc.|Devices and methods for detecting and quantitating nucleic acids using size separation of amplicons|

---

AU2009217355A1|2008-02-21|2009-08-27|Avantra Biosciences Corporation|Assays based on liquid flow over arrays|

---

EP2430177B1|2009-05-14|2018-09-26|Icubate, Inc.|Apparatus for performing amplicon rescue multiplex pcr|

---

KR20120030130A|2009-06-02|2012-03-27|인터젠엑스 인크.|Fluidic devices with diaphragm valves|

---

CN101586162B|2009-06-10|2011-02-09|戴立忠|Method of extracting target nucleic acid and performing PCR amplification|

---

CN107338189B|2011-05-04|2021-02-02|卢米耐克斯公司|Apparatus and method for integrated sample preparation, reaction and detection|US8372340B2|2005-10-19|2013-02-12|Luminex Corporation|Apparatus and methods for integrated sample preparation, reaction and detection|

---

US7727473B2|2005-10-19|2010-06-01|Progentech Limited|Cassette for sample preparation|

---

US7754148B2|2006-12-27|2010-07-13|Progentech Limited|Instrument for cassette for sample preparation|

---

US20110312635A1|2010-06-17|2011-12-22|Geneasys Pty Ltd|Microfluidic device with flow-channel structure for capillary-driven fluidic propulsion without trapped air bubbles|

---

CN103555558B|2010-07-23|2016-09-28|贝克曼考尔特公司|Container for real-time PCR|

---

CN103534575B|2011-02-04|2016-08-10|环球生物研究株式会社|Automatically reaction/light measurement device and method thereof|

---

CN107338189B|2011-05-04|2021-02-02|卢米耐克斯公司|Apparatus and method for integrated sample preparation, reaction and detection|

---

US8586387B2|2011-08-30|2013-11-19|Supernova Diagnostics, Inc.|Methods of triggering activation of encapsulated signal-generating substances and apparatus utilising activated signal-generating substances|

---

US9664702B2|2011-09-25|2017-05-30|Theranos, Inc.|Fluid handling apparatus and configurations|

---

US9632102B2|2011-09-25|2017-04-25|Theranos, Inc.|Systems and methods for multi-purpose analysis|

---

US20140170735A1|2011-09-25|2014-06-19|Elizabeth A. Holmes|Systems and methods for multi-analysis|

---

US10012664B2|2011-09-25|2018-07-03|Theranos Ip Company, Llc|Systems and methods for fluid and component handling|

---

US8475739B2|2011-09-25|2013-07-02|Theranos, Inc.|Systems and methods for fluid handling|

---

JP6217637B2|2012-07-25|2017-10-25|ソニー株式会社|Nucleic acid amplification reaction sample preparation method and nucleic acid amplification reaction sample preparation apparatus|

---

AU2013202808B2|2012-07-31|2014-11-13|Gen-Probe Incorporated|System and method for performing multiplex thermal melt analysis|

---

TWI528823B|2012-08-28|2016-04-01|緯創資通股份有限公司|Device and method for displaying and adjusting image information|

---

US20140322706A1|2012-10-24|2014-10-30|Jon Faiz Kayyem|Integrated multipelx target analysis|

---

CN103776660B|2012-10-24|2016-03-09|艾博生物医药有限公司|A kind of device|

---

CN105748108B|2012-10-24|2019-04-05|艾博生物医药有限公司|A kind of device|

---

USD881409S1|2013-10-24|2020-04-14|Genmark Diagnostics, Inc.|Biochip cartridge|

---

CA2889415C|2012-10-24|2020-06-02|Genmark Diagnostics, Inc.|Integrated multiplex target analysis|

---

US9347056B2|2012-10-26|2016-05-24|Seiko Epson Corporation|Nucleic acid extraction device, and nucleic acid extraction method, nucleic acid extraction kit, and nucleic acid extraction apparatus, each using the same|

---

US9810704B2|2013-02-18|2017-11-07|Theranos, Inc.|Systems and methods for multi-analysis|

---

US9481903B2|2013-03-13|2016-11-01|Roche Molecular Systems, Inc.|Systems and methods for detection of cells using engineered transduction particles|

---

MX348826B|2013-03-15|2017-06-30|Theranos Inc|Devices, systems and methods for sample preparation.|

---

US9359632B2|2013-03-15|2016-06-07|Theranos, Inc.|Devices, systems and methods for sample preparation|

---

US9222623B2|2013-03-15|2015-12-29|Genmark Diagnostics, Inc.|Devices and methods for manipulating deformable fluid vessels|

---

US9540675B2|2013-10-29|2017-01-10|GeneWeave Biosciences, Inc.|Reagent cartridge and methods for detection of cells|

---

GB201401584D0|2014-01-29|2014-03-19|Bg Res Ltd|Intelligent detection of biological entities|

---

US10195610B2|2014-03-10|2019-02-05|Click Diagnostics, Inc.|Cartridge-based thermocycler|

---

CN204727866U|2014-03-31|2015-10-28|博奥生物集团有限公司|A kind ofly collect the micro fluidic device that nucleic acid amplification and microarray are detected on one|

---

US9827567B2|2014-04-22|2017-11-28|Nanosphere, Inc.|Diagnostic cartridges having flexible seals|

---

ES2749925T3|2014-04-24|2020-03-24|Lucira Health Inc|Colorimetric detection of nucleic acid amplification|

---

JP6602796B2|2014-06-16|2019-11-06|ライフテクノロジーズコーポレーション|Reagent mixer and fluid control device|

---

US9598722B2|2014-11-11|2017-03-21|Genmark Diagnostics, Inc.|Cartridge for performing assays in a closed sample preparation and reaction system|

---

US9498778B2|2014-11-11|2016-11-22|Genmark Diagnostics, Inc.|Instrument for processing cartridge for performing assays in a closed sample preparation and reaction system|

---

US10005080B2|2014-11-11|2018-06-26|Genmark Diagnostics, Inc.|Instrument and cartridge for performing assays in a closed sample preparation and reaction system employing electrowetting fluid manipulation|

---

CN107429281A|2014-12-31|2017-12-01|易捷仪器诊断股份有限公司|Apparatus and method for molecule diagnostic test|

---

EP3302802B1|2015-06-05|2019-09-04|Douglas Scientific, LLC|Sample processing devices, and methods of use thereof|

---

US11260386B2|2015-06-05|2022-03-01|The Emerther Company|Component of a device, a device, and a method for purifying and testing biomolecules from biological samples|

---

WO2017009002A1|2015-07-13|2017-01-19|Philip Morris Products S.A.|Producing an aerosol-forming composition|

---

US10351893B2|2015-10-05|2019-07-16|GeneWeave Biosciences, Inc.|Reagent cartridge for detection of cells|

---

US9513220B1|2015-12-29|2016-12-06|International Business Machines Corporation|On-chip molecule fluorescence detection|

---

CN113567525A|2016-01-13|2021-10-29|普诺森公司|Systems and methods for capillary electrophoresis, isoelectric point and molecular weight analysis|

---

CA3015376A1|2016-03-14|2017-09-21|Diassess Inc.|Devices and methods for biological assay sample preparation and delivery|

---

EP3429752A4|2016-03-14|2019-10-30|Lucira Health, Inc.|Systems and methods for performing biological assays|

---

JP6920334B2|2016-03-25|2021-08-18|ザ ジェネラル ホスピタル コーポレイション|Polarization-based fluorescent nucleic acid detection|

---

JP6982338B2|2016-04-20|2021-12-17|Ｂｌｕｅ Ｉｎｄｕｓｔｒｉｅｓ株式会社|Pretreatment kit for gene analysis, chip for nucleic acid analysis, analysis system, chip for biomaterial analysis|

---

WO2017185067A1|2016-04-22|2017-10-26|Click Diagnostics, Inc.|Printed circuit board heater for an amplification module|

---

WO2017197040A1|2016-05-11|2017-11-16|Click Diagnostics, Inc.|Devices and methods for nucleic acid extraction|

---

JP2017225366A|2016-06-21|2017-12-28|大研医器株式会社|Genetic testing apparatus|

---

USD800331S1|2016-06-29|2017-10-17|Click Diagnostics, Inc.|Molecular diagnostic device|

---

EP3478857A1|2016-06-29|2019-05-08|Click Diagnostics, Inc.|Devices and methods for the detection of molecules using a flow cell|

---

USD800914S1|2016-06-30|2017-10-24|Click Diagnostics, Inc.|Status indicator for molecular diagnostic device|

---

USD800913S1|2016-06-30|2017-10-24|Click Diagnostics, Inc.|Detection window for molecular diagnostic device|

---

US11156298B2|2016-08-11|2021-10-26|Pall Corporation|Front-loading valve assembly for manifold for processing fluid sample|

---

CN107964507A|2016-10-18|2018-04-27|财团法人工业技术研究院|Thermal convection current polymerase chain reaction device and its optical detecting method|

---

NL2017705B1|2016-11-02|2018-05-18|Lumicks Tech B V|Method and system for studying biological cells|

---

PL235210B1|2016-12-21|2020-06-15|Genomtec Spolka Akcyjna|Method for detection of genetic material in a biological specimen the device for the execution of this method|

---

US10471425B2|2017-02-16|2019-11-12|International Business Machines Corporation|Automated machine for sorting of biological fluids|

---

US11080848B2|2017-04-06|2021-08-03|Lucira Health, Inc.|Image-based disease diagnostics using a mobile device|

---

US11077444B2|2017-05-23|2021-08-03|Roche Molecular Systems, Inc.|Packaging for a molecular diagnostic cartridge|

---

DE102017005835B4|2017-06-20|2020-04-02|Diehl Metering Gmbh|Device for the mobile determination of a property of a liquid, solid or gaseous sample|

---

WO2019038437A1|2017-08-24|2019-02-28|Innatoss Laboratories B.V.|Liquid handling system and method|

---

CN111356768A|2017-09-15|2020-06-30|生米公司|Method and system for automated sample processing|

---

CN107523487A|2017-09-18|2017-12-29|星源智（珠海）生物科技有限公司|A kind of integrated tubular reactor device|

---

US11162130B2|2017-11-09|2021-11-02|Visby Medical, Inc.|Portable molecular diagnostic device and methods for the detection of target viruses|

---

GB2572408B|2018-03-29|2022-03-16|Herriot Watt Univ|Fluidic device|

---

CN110699432A|2018-07-10|2020-01-17|Tdk株式会社|Device and method for detecting nucleic acid by constant temperature amplification technology|

---

KR101996617B1|2018-10-11|2019-07-04|주식회사 엘지화학|Integrated cartridge|

---

DE102018130082A1|2018-11-28|2020-05-28|Bundesrepublik Deutschland, vertreten durch das Bundesministerium für Gesundheit, dieses vertreten durch das Robert-Koch-Institut, vertreten durch seinen Präsidenten|Sample reaction vessel and its use|

---

USD910200S1|2018-12-21|2021-02-09|Lucira Health, Inc.|Test tube|

---

KR102281116B1|2019-06-28|2021-07-27|주식회사 엘지화학|integrated cartridge|

---

WO2021000750A1|2019-07-01|2021-01-07|申翌生物科技有限公司|Novel method for performing pcr reaction using comprehensive pcr reaction system|

---

CN112301097A|2019-07-26|2021-02-02|申翌生物科技有限公司|Sample lysis and PCR reaction composition|

---

WO2021101334A1|2019-11-22|2021-05-27|Seegene, Inc.|Sample holder transferring apparatus used for thermal cycler|

---

KR20210085832A|2019-12-31|2021-07-08|프리시젼바이오 주식회사|Apparatus for Heating Sample Solution for In Vitro Diagnostics|

---

CN111235639B|2020-03-06|2021-04-20|通用生物系统（安徽）有限公司|Phage antibody elution device|

---

CN112322473B|2020-11-11|2021-07-20|苏州雅睿生物技术有限公司|One-step POCTreal-time nucleic acid detection device|

法律状态:
2016-09-13| B25A| Requested transfer of rights approved|Owner name: LUMINEX CORPORATION (US) |

---

2017-12-12| B06F| Objections, documents and/or translations needed after an examination request according [chapter 6.6 patent gazette]|

---

2018-10-02| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

---

2019-10-29| B06A| Patent application procedure suspended [chapter 6.1 patent gazette]|

---

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

---

2020-06-09| B16A| Patent or certificate of addition of invention granted [chapter 16.1 patent gazette]|Free format text: PRAZO DE VALIDADE: 20 (VINTE) ANOS CONTADOS A PARTIR DE 23/02/2011, OBSERVADAS AS CONDICOES LEGAIS. |

优先权:

---

申请号 | 申请日 | 专利标题

---

US30728110P| true| 2010-02-23|2010-02-23|

---

PCT/US2011/025871|WO2011106384A1|2010-02-23|2011-02-23|Apparatus and methods for integrated sample preparation, reaction and detection|

---