Methods and systems for sample analysis

Abstract

The present disclosure provides methods, systems, and / or kits for performing a polymerase chain reaction (PCR). A method may comprise providing a second reaction mixture comprising a second analyte to a second chamber of the substrate. The first chamber and the second chamber may be individually addressable. The first reaction mixture may be subjected to first conditions sufficient to perform the first PCR on the first analyte. The second reaction mixture may be subjected to second conditions sufficient to perform the second PCR on the second analyte. A coefficient of variation of a yield of the first PCR and the second PCR may be less than about 35%. A difference of a temperature between the first chamber and the second chamber may be at least about 30 °C.

Description

Atty Dkt No.: 61197-702601METHODS AND SYSTEMS FOR SAMPLE ANALYSISCROSS-REFERENCE

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 664,557 filed June 26, 2024, which application is incorporated herein by reference in its entirety.BACKGROUND

[0002] Diagnostic testing methods and devices have become an important part of modern medical care. Polymerase chain reaction (PCR) based diagnostics have grown in importance as global attention has been drawn to the health problems created by hard to identify pathogens.SUMMARY

[0003] In some cases, next-generation sequencing workflows can utilize tightly controlled nucleic acid amplification to provide optimal yields and data quality. Over-amplification can lead to reactant depletion, skewed representation of input material, or increased PCR duplicates, or any combination thereof. These results can negatively affect sequencing accuracy or efficiency. Under-amplification can result in insufficient library material, which can waste sequencing capacity. Experimental designs (e g., design of experiments (DOE)) may use the ability to generate different amplification profiles across multiple samples in a single PCR run. Some thermocyclers can lack well to well control that can enable such flexibility, which can force users to run multiple well plates or use complex workflows to achieved variability, which can waste time and reagents or introduce additional variability.

[0004] The methods and systems of the present disclosure can address these challenges by enabling independent control of each chamber (e g., well) of a substrate (e g., a 96 well plate). This can enable precise, chamber specific amplification tailored to each sample’s input and predetermined endpoint. Users can prevent reagent depletion by halting amplification at an optimal point or explore multiple amplification conditions simultaneously for DOE without additional instrumentation or runs. The level of individualization and control can dramatically improve workflow efficiency, reproducibility, and data quality in sample preparation (e g., nextgeneration sequencing sample preparation).

[0005] In an aspect, the present disclosure provides a method, comprising: (a) providing (i) a first reaction mixture comprising a first analyte to a first chamber of a substrate and (ii) a second reaction mixture comprising a second analyte to a second chamber of the substrate, wherein the first chamber and the second chamber are each individually addressable; and (b) subjecting (i) the first reaction mixture to first conditions sufficient to perform a first polymerase chainAtty Dkt No.: 61197-702601 reaction (PCR) on the first analyte and (ii) the second reaction mixture to second conditions sufficient to perform a second PCR on the second analyte, wherein (1) a coefficient of variation of a yield of the first PCR and the second PCR is less than about 35% or (2) a difference of a temperature between the first chamber and the second chamber is at least about 30 °C.

[0006] In some embodiments, the coefficient of variation of a yield of the first PCR and the second PCR is less than about 35%. In some embodiments, the coefficient of variation of a yield of the first PCR and the second PCR is less than about 35% in an absence of depletion of a reagent of the first reaction mixture or the second reaction mixture. In some embodiments, the difference of a temperature between the first chamber and the second chamber is at least about 30 °C. In some embodiments, the method further comprises, (i) during the first PCR, detecting one or more first signals or first signal changes from the first reaction mixture or (ii) during the second PCR, detecting one or more second signals or second signal changes from the second reaction mixture. In some embodiments, a first thermal element is in operable communication with the first chamber. In some embodiments, the first thermal element is a thermoelectric element. In some embodiments, the first thermal element has a length of at most or a width of at most about 9 millimeters. In some embodiments, a second thermal element is in operable communication with the second chamber. In some embodiments, the second thermal element is a thermoelectric element. In some embodiments, the second thermal element has a length of at most or a width of at most about 9 millimeters. In some embodiments, the first thermal element and the second thermal element are different thermal elements. In some embodiments, the method further comprises, subsequent to (b), extracting, at least a portion of the first reaction mixture from the first chamber or at least a portion of the second reaction mixture from the second reaction chamber. In some embodiments, the at least the portion of the first reaction mixture or the at least the portion of the second reaction mixture are used for a next-generation sequencing application. In some embodiments, the first reaction mixture or the second reaction mixture are not pre-normalized. In some embodiments, the method further comprises adjusting the first conditions to decrease a rate of or halt the first PCR. In some embodiments, the second PCR continues after the rate of the first PCR is decreased or halted. In some embodiments, the adjusting is in response to one or more signals from the first reaction mixture. In some embodiments, during the subjecting of (b), (i) a thermal conductivity between a first thermal element associated with the first chamber and a heat sink in thermal contact with the first thermal element is at least about 5 W / Km; or (ii) a thermal conductivity between the first chamber and a first sensor associated with the first chamber is at least about 5 W / Km. In some embodiments, the thermal conductivity between the first thermal element associated with the first chamber andAtty Dkt No.: 61197-702601 the heat sink in thermal contact with the first thermal element is at least about 5 W / Km. In some embodiments, the thermal conductivity between the first chamber and the first sensor associated with the first chamber is at least about 5 W / Km. In some embodiments, the method further comprises measuring, using the first sensor, a property of the first chamber. In some embodiments, the property of the first chamber comprises a temperature of the first chamber. In some embodiments, the first thermal element, the heat sink, the first chamber, or the first sensor are in contact with a thermally conductive element. In some embodiments, during the subjecting of (b), (i) a thermal conductivity between a second thermal element associated with the second chamber and a heat sink in thermal contact with the second thermal element is at least about 5 W / Km; or (ii) a thermal conductivity between the second chamber and a second sensor associated with the second chamber is at least about 5 W / Km. In some embodiments, the thermal conductivity between the second thermal element associated with the second chamber and the heat sink in thermal contact with the second thermal element is at least about 5 W / Km. In some embodiments, the thermal conductivity between the second chamber and the second sensor associated with the second chamber is at least about 5 W / Km. In some embodiments, the method further comprises measuring, using the second sensor, a property of the second chamber. In some embodiments, the property of the second chamber comprises a temperature of the second chamber. In some embodiments, the second thermal element, the heat sink, the second chamber, or the second sensor are in contact with a thermally conductive element. In some embodiments, the thermally conductive element has a thermal conductivity of at least about 5 W / Km. In some embodiments, the method further comprises transferring heat through the thermally conductive element. In some embodiments, the thermally conductive element comprises at least about 2 layers. In some embodiments, a difference in a temperature between the first chamber and a temperature of the first sensor is at most about 0.1 °C. In some embodiments, a difference in a temperature between the second chamber and a temperature of the second sensor is at most about 0. 1 °C. In some embodiments, during the subjecting of (b), (i) a thermal conductivity between a second thermal element associated with the second chamber and a heat sink in thermal contact with the second thermal element is at least about 5 W / Km; or (ii) a thermal conductivity between the second chamber and a second sensor associated with the second chamber is at least about 5 W / Km. In some embodiments, during (b), (i) a first volume of the first reaction mixture or (ii) a second volume of the second reaction mixture changes by less than about 10%. In some embodiments, the first volume and the second volume stay within about 10% of one another during the subjecting of (b). In some embodiments, the method further comprises, prior to (b), bringing the substrate in contact with a cover plate. In some embodiments, the cover plateAtty Dkt No.: 61197-702601 comprises a first feature corresponding to the first chamber and a second feature corresponding to the second chamber. In some embodiments, the feature comprises a first hole disposed above the first chamber and a second hole disposed above the second chamber. In some embodiments, the cover plate comprises a thermal element. In some embodiments, the cover plate comprises one or more air deflectors. In some embodiments, the cover plate is connected to a top plate. In some embodiments, one or more springs are disposed between the cover plate and the top plate. In some embodiments, a contact force between the substrate and a cover plate disposed above the substrate during the subjecting of (b) is at least about 30 pounds. In some embodiments, (i) a first thermal element is used to affect a first temperature condition of the first conditions and (ii) a second thermal element is used to affect a second temperature condition of the second conditions each have a heating output and a cooling output that differ by at most about 0.5 W. In some embodiments, a system comprising the substrate has an area of at most about 2,500 square centimeters (cm2). In some embodiments, a thermal conductivity between the first chamber and the second chamber is at most about 1 W / Km. In some embodiments, one or more thermal management elements are disposed between the first chamber and the second chamber. In some embodiments, the one or more thermal management elements comprise a thermal insulator. In some embodiments, the one or more thermal management elements comprise a gap. In some embodiments, the individual addressability comprises use of control circuitry. In some embodiments, the control circuitry comprises a converter. In some embodiments, the converter comprises a buck-boost converter or a buck converter. In some embodiments, the buck-boost converter is a current sinking buck-boost converter.

[0007] In another aspect, the present disclosure provides a method for determining a property of an analyte, comprising: (a) providing a reaction mixture comprising an analyte to a chamber of a substrate, wherein the chamber is individually addressable; (b) subjecting the reaction mixture to conditions sufficient to perform a polymerase chain reaction (PCR) on the analyte; (c) during the PCR, detecting one or more signals or signal changes from the reaction mixture, and (d) determining a property of the analyte with an accuracy of at least 90% within at most 60 min based at least in part on the one or more signals or signal changes.

[0008] In some embodiments, the method further comprises, during the PCR, detecting one or more signals or signal changes from the reaction mixture. In some embodiments, a thermal element of the system has a thermal element with a maximum length or maximum width of at most about 9 millimeters. In some embodiments, the reaction mixture is not pre-normalized. In some embodiments, the method further comprises, in (b), using a first element disposed adjacent to the chamber to affect the conditions in the chamber to thereby initiate the PCR reaction. InAtty Dkt No.: 61197-702601 some embodiments, the method further comprises using a second element operably coupled to a first element to adjust an operating condition of the first element at least partially in response to the signal or signal changes, to thereby affect the condition of the chamber. In some embodiments, the chamber comprises a first thermal element and an additional chamber of the substrate comprises a second thermal element. In some embodiments, the chamber is operably coupled to a first sensor and an additional chamber is operably coupled to a second sensor. In some embodiments, a thermal element and an additional thermal element share a heatsink. In some embodiments, during the subjecting of (b), (i) a thermal conductivity between a thermal element associated with the chamber and a heat sink in thermal contact with the thermal element is at least about 5 watts / kelvin / meter (W / Km); or (ii) a thermal conductivity between the chamber and a sensor associated with the chamber is at least about 5 watts / kelvin / meter (W / Km). In some embodiments, a volume of the reaction mixture within the chamber changes by at most about 10% during the PCR. In some embodiments, a contact force between the substrate and a cover plate disposed above the substrate during the subjecting of (b) is at least about 30 pounds. In some embodiments, a thermal element used to affect a temperature condition of the conditions has a heating output and cooling output that differ by at most about 0.5 W. In some embodiments, an alignment between the chamber and a feature of a cover plate corresponding to the chamber has an error of at most about 250 micrometers. In some embodiments, an alignment between the chamber and a thermal element corresponding to the chamber has an error of at most about 250 micrometers. In some embodiments, a system comprising the substrate has an area of at most about 2,500 square centimeters (cm2). In some embodiments, an additional chamber of the substrate comprises an additional reaction mixture. In some embodiments, an additional PCR reaction continues within the additional chamber after the PCR reaction in the first chamber is stopped. In some embodiments, the chamber and the additional chamber do not share a sensor. In some embodiments, the method further comprises, prior to (a), extracting at least a portion of the reaction mixture from a sample. In some embodiments, the method further comprises adjusting the operating condition of the first element to decrease a rate of the PCR reaction upon the one or more signal or signal changes reaching a predetermined threshold. In some embodiments, the chamber and an additional chamber of the substrate share a thermal element. In some embodiments, the chamber and an additional chamber of the substrate share a printed circuit board (PCB) support. In some embodiments, the thermal conductivity between the thermal element associated with the chamber and the heat sink in thermal contact with the thermal element is at least about 5 W / Km. In some embodiments, the thermal conductivity between the chamber and the sensor associated with the chamber is at least about 5 W / Km. In someAtty Dkt No.: 61197-702601 embodiments, the thermal element comprises a thermoelectric thermal element. In some embodiments, the method further comprises transferring heat between the thermal element and the heat sink. In some embodiments, the method further comprises measuring, using the sensor, a property of the chamber. In some embodiments, the property of the chamber comprises a temperature of the chamber. In some embodiments, the thermal element, the heat sink, the chamber, or the sensor are in contact with a thermally conductive element. In some embodiments, the thermally conductive element comprises graphite, copper, or indium, or any combination thereof. In some embodiments, the thermally conductive element has a thermal conductivity of at least about 5 W / Km. In some embodiments, the method further comprises transferring heat through the thermally conductive element. In some embodiments, the thermally conductive element comprises at least about 2 layers. In some embodiments, the thermally conductive element has an area smaller than the chamber, the sensor, or the heatsink. In some embodiments, a difference in a temperature of the chamber and a temperature of the sensor is at most about 0. 1 °C. In some embodiments, (i) the thermal element and the heat sink or (ii) the chamber and the sensor are separated by a printed circuit board (PCB). In some embodiments, the method further comprises, prior to (b), bringing the substrate in contact with a cover plate. In some embodiments, the cover plate comprises a feature corresponding to the chamber. In some embodiments, the feature comprises a hole disposed above the chamber. In some embodiments, the cover plate comprises a thermal element. In some embodiments, the cover plate comprises one or more air deflectors. In some embodiments, the cover plate is connected to a top plate. In some embodiments, one or more springs are disposed between the cover plate and the top plate. In some embodiments, one or more alignment members are configured to align the chamber and the temperature control element. In some embodiments, the one or more alignment members comprise one or more of rail guides, alignment rods, pins, grooves, or wheels, or any combination thereof. In some embodiments, a thermal conductivity between the chamber and the additional chamber is at most about 1 W / Km. In some embodiments, one or more thermal management elements are disposed between the chamber and the additional chamber. In some embodiments, the one or more thermal management elements comprise a thermal insulator. In some embodiments, the one or more thermal management elements comprise a gap. In some embodiments, a volume of the reaction mixture and the additional reaction mixture differs by less than about 10% over the course of the PCR and an additional PCR of the additional reaction mixture. In some embodiments, a cover plate is disposed above and in contact with the substrate prior to (b). In some embodiments, the cover plate comprises a thermal control element. In some embodiments, the thermal control element corresponds to the chamber and an additional thermalAtty Dkt No.: 61197-702601 control element corresponds to the additional chamber. In some embodiments, the thermal control element comprises a heater. In some embodiments, the heater comprises a resistive heater or a thermoelectric heater. In some embodiments, the cover plate comprises one or more air deflectors. In some embodiments, the cover plate is connected to a top plate. In some embodiments, one or more springs are disposed between the cover plate and the top plate.

[0009] In another aspect, the present disclosure provides a method for performing a polymerase chain reaction (PCR), comprising: (a) providing a reaction mixture comprising an analyte to a chamber on a substrate, wherein the chamber is individually addressable; (b) subjecting the reaction mixture to conditions sufficient to perform the PCR on the analyte; (c) during the PCR, having a volume change of the reaction mixture of at most about 10%.

[0010] In some embodiments, the volume change of the reaction mixture is at most about 1 microliter. In some embodiments, the method further comprises, in (b), using a first element disposed adjacent to the chamber to affect the conditions in the chamber to thereby initiate the PCR reaction. In some embodiments, the method further comprises using a second element operably coupled to a first element to adjust an operating condition of the first element at least partially in response to a signal or signal changes from the PCR, to thereby affect the condition of the chamber. In some embodiments, the method further comprises adjusting the operating condition of a first element to decrease a rate of the PCR reaction upon one or more signal or signal changes from the PCR reaching a predetermined threshold. In some embodiments, the chamber and an additional chamber of the substrate share a printed circuit board (PCB) support. In some embodiments, a thermal element and an additional thermal element share a heatsink. In some embodiments, a cover plate in contact with the substrate comprises a feature corresponding to the chamber. In some embodiments, the feature comprises a hole disposed above the chamber. In some embodiments, a cover plate in contact with the substrate comprises a thermal element. In some embodiments, a cover plate in contact with the substrate comprises one or more air deflectors. In some embodiments, a cover plate in contact with the substrate is connected to a top plate. In some embodiments, one or more springs are disposed between the cover plate and the top plate. In some embodiments, a contact force between a cover plate in contact with the substrate and the substrate during the subjecting of (b) is at least about 30 pounds. In another aspect, the present disclosure provides a system for performing a polymerase chain reaction (PCR), comprising: a substrate comprising a chamber; a cover plate disposed above the substrate, wherein the cover plate is configured to, when engaged with the substrate, cover the chamber, wherein the cover plate is configured to maintain a volume of a reaction mixture within the reaction chamber within about 10% of a starting volume of the reaction mixture. In someAtty Dkt No.: 61197-702601 embodiments, the volume change of the reaction mixture is at most about 1 microliter. In some embodiments, the cover plate is disposed against a top plate configured to hold the cover plate above the substrate. In some embodiments, the method further comprises one or more springs disposed between the cover plate and the top plate. In some embodiments, the one or more springs provide a contact force between the cover plate and the substrate. In some embodiments, the one or more springs comprise one or more calibrated springs. In some embodiments, the cover plate comprises a thermal element. In some embodiments, the thermal element comprises a heater. In some embodiments, the thermal element is configured to control a temperature of the cover plate. In some embodiments, the cover plate comprises one or more air deflectors. In some embodiments, the substrate comprises a plurality of chambers. In some embodiments, the plurality of chambers comprises at least about 96 chambers. In some embodiments, the cover plate comprises a feature. In some embodiments, the cover plate comprises a plurality of features. In some embodiments, when the substrate and the cover plate are engaged, the plurality of features align with a plurality of chambers comprising the chamber. In some embodiments, the plurality of features comprise a plurality of access holes through the cover plate. In some embodiments, the method further comprises a thermoelectric element in thermal communication with the chamber. In some embodiments, the system further comprises a plurality of thermoelectric elements in thermal communication with a plurality of chambers comprising the chamber. In some embodiments, a sample evaporation rate of the PCR is less than about 1 microliters / hour. In some embodiments, a temperature of the chamber may be at most about 0.2 °C from a set temperature of the chamber. In some embodiments, the method further comprises one or more air deflectors configured to deflect air around or away from the cover plate and the substrate. In some embodiments, the volume change comprises evaporation. In some embodiments, the volume change comprises condensation. In some embodiments, the method further comprises, prior to (a), extracting at least a portion of the reaction mixture from a sample. In some embodiments, the reaction mixture is not pre-normalized. In some embodiments, the chamber comprises a first thermal element and an additional chamber of the substrate comprises a second thermal element. In some embodiments, the chamber and an additional chamber of the substrate share a thermal element. In some embodiments, the chamber comprises a first sensor and an additional chamber comprises a second sensor.

[0011] In another aspect, the present disclosure provides a method for performing a polymerase chain reaction (PCR) analysis of a sample containing or suspected of containing an analyte, comprising: (a) providing the sample to a location on a substrate, wherein the location is operably coupled to a thermally conductive element disposed in at least a portion of a support,Atty Dkt No.: 61197-702601 wherein the location has a volume sufficient to retain the sample; and (b) using a thermal element disposed adjacent to the location to initiate a PCR reaction using the sample, wherein the PCR reaction generates one or more signals or signal changes which is indicative of a property associated with the analyte, and wherein the thermal element is in thermal contact with the thermally conductive element. In some embodiments, the thermal element is a thermoelectric element. In some embodiments, the method further comprises (c) using a second element operably coupled to the thermal element to adjust an operating condition of the thermal element in response at least partially to the signal or signal change, to thereby affect a condition in the location. In some embodiments, the support comprises a second thermally conductive element disposed through at least a portion of the support, and wherein the second element is in thermal contact with the second thermally conductive element. In some embodiments, the thermally conductive element is electrically insulated from the thermal element. In some embodiments, the support is a printed circuit board. In some embodiments, the printed circuit board is a multilayered metal printed circuit board. In some embodiments, the at least a portion of the support is an entire thickness of the support. In some embodiments, the method further comprises a heatsink disposed on an opposite side of the support from the thermal element. In some embodiments, the thermal element is in thermal communication with the heatsink at least partially through the thermally conductive element. In some embodiments, the method further comprises a thermal conductor placed between the thermal element and the thermally conductive element. In some embodiments, thermal conductor is a graphite sheet or a thermal compound. In some embodiments, the thermally conductive element comprises one or more metals. In some embodiments, the one or more metals comprises copper. In some embodiments, the one or more metals comprises aluminum. In some embodiments, the method further comprises performing an isothermal amplification reaction within the location. In some embodiments, said one or more springs comprises at least about 4 springs. In some embodiments, said one or more springs comprises at least about 6 springs. In some embodiments, said one or more springs comprises at least about 8 springs. In some embodiments, the contact force is a constant engagement force. In some embodiments, the contact force is greater than about 30 pounds. In some embodiments, the contact force is less than about 80 pounds. In some embodiments, the contact force across the cover plate has a variation of at most about 10%. In some embodiments, the one or more springs are one or more calibrated springs. In some embodiments, the one or more calibrated springs have a variation of diameters, lengths, or spring constants of at most about 10%. In some embodiments, the heater is a resistive heater. In some embodiments, the heater is a thermoelectric heater. In some embodiments, said substrate is removable from said system. InAtty Dkt No.: 61197-702601 some embodiments, each chamber of said plurality of chambers is in thermal communication with a different thermoelectric element of said plurality of thermoelectric elements. In some embodiments, an engagement force between the substrate and the cover plate is less than a damage threshold for said plurality of thermoelectric elements. In some embodiments, said one or more air deflectors comprise one or more features. In some embodiments, said one or more features are configured to perform said deflecting said air. In some embodiments, a sample evaporation rate of a PCR of said plurality of PCRs is less than about 1 microliters / hour.

[0012] In another aspect, the present disclosure provides a method, comprising: (a) providing a device comprising (i) a power supply comprising a circuit comprising a current sinking converter, and (ii) a thermoelectric element in electrical communication with the power supply; and (b) using the power supply to provide power to the thermoelectric element, wherein when power is supplied to the thermoelectric element, the current sinking converter removes current from the circuit that is in excess of a current sufficient to operate the thermoelectric element.

[0013] In some embodiments, the method further comprises (c) adjusting a temperature of a chamber disposed in thermal contact with the thermoelectric element using at least in part the thermoelectric element. In some embodiments, the adjusting the temperature of the chamber comprises adjusting a power output of the power supply. In some embodiments, the current sinking converter is a current sinking buck-booster converter. In some embodiments, the method further comprises performing a polymerase chain reaction within a chamber in thermal contact with the thermoelectric element. In some embodiments, the chamber is a well. In some embodiments, the method further comprises using another power supply to provide power to another thermoelectric element. In some embodiments, the another power supply comprises another current sinking converter. In some embodiments, the another current sinking converter is a current sinking buck-boost converter. In some embodiments, the method further comprises adjusting a temperature of another chamber using at least in part the second thermoelectric element. In some embodiments, the temperature of the another chamber is different from a temperature of a chamber in thermal contact with the thermoelectric element. In some embodiments, the thermoelectric element and the another thermoelectric element are individually addressable. In some embodiments, the method further comprises performing an isothermal amplification reaction within a chamber in thermal contact with the thermoelectric element.

[0014] In another aspect, the present disclosure provides a method, comprising: (a) providing (i) a plurality of chambers, (ii) a thermoelectric plate comprising a plurality of thermoelectric elements, wherein each thermoelectric element of the plurality of thermoelectric elements corresponds to a different chamber of the plurality of chambers, (iii) a movable assembly inAtty Dkt No.: 61197-702601 contact with the thermoelectric plate; and (b) moving, via the movable assembly, the thermoelectric plate, thereby contacting the plurality of thermoelectric elements to the plurality of chambers, wherein a chamber of the plurality of chambers is aligned to a thermoelectric element of the plurality of thermoelectric elements with an offset of at most about 250 micrometers.

[0015] In some embodiments, the plurality of chambers are a plurality of wells. In some embodiments, the contacting the plurality of thermoelectric elements with the plurality of chambers is an automated contacting. In some embodiments, the thermoelectric plate is contacted to the plurality of chambers with at least about 30 pounds of force. In some embodiments, the thermoelectric plate is contacted to the plurality of chambers with at most about 80 pounds of force. In some embodiments, the device has a footprint of at most about 540 square millimeters (mm2). In some embodiments, the method further comprises a lid positioned above the plurality of chambers. In some embodiments, the lid comprises a heating element. In some embodiments, the movable assembly comprises a lever. In some embodiments, the lever has a movement ratio of the lever to the movable assembly of about 2: 1. In some embodiments, the movable assembly comprises a plurality of pivoting points. In some embodiments, the movable assembly comprises at least three pivoting points. In some embodiments, the method further comprises a limit switch configured to detect a position of the movable assembly. In some embodiments, the limit switch is an optical limit switch. In some embodiments, the limit switch is a mechanical limit switch. In some embodiments, the movable assembly is positioned below the thermoelectric plate. In some embodiments, the method further comprises performing an isothermal amplification reaction within a chamber of the plurality of chambers. In some embodiments, the method further comprises performing an independent isothermal amplification reaction within each chamber of the plurality of chambers.

[0016] In another aspect, the present disclosure provides a device, comprising: a thermoelectric element operably coupled to a support, wherein the support comprises one or more thermally conductive elements, which one or more thermally conductive elements are disposed in at least a portion of the support, wherein the thermoelectric element is in thermal contact with the one or more thermally conductive elements.

[0017] In some embodiments, the one or more thermally conductive elements are electrically insulated from the thermoelectric element. In some embodiments, the support is a printed circuit board. In some embodiments, the printed circuit board is a multi-layered metal printed circuit board. In some embodiments, the one or more thermally conductive elements are metallic. In some embodiments, the at least a portion of the support is an entire thickness of the support. InAtty Dkt No.: 61197-702601 some embodiments, the device further comprises a heatsink disposed on an opposite side of the support from the thermoelectric element. In some embodiments, the thermoelectric element is in thermal communication with the heatsink. In some embodiments, the device further comprises a thermal conductor placed between the thermoelectric element and the one or more thermally conductive elements. In some embodiments, the thermal conductor is a graphite sheet or a thermal compound. In some embodiments, the one or more thermally conductive elements are metal. In some embodiments, the metal is copper. In some embodiments, the metal is aluminum.

[0018] In another aspect, the present disclosure provides a device, comprising: a thermoelectric element; and a power supply configured to provide power to the thermoelectric element, wherein the power supply comprises a current sinking converter in an electrical path of the thermoelectric element.

[0019] In some embodiments, the current sinking converter is a current sinking buck-booster converter. In some embodiments, the current sinking converter comprises a feedback signal loop configured to adjust power provided by the power supply to the thermoelectric element. In some embodiments, the current sinking converter makes a power range supplied from the power supply to the thermoelectric element symmetrical.

[0020] In another aspect, the present disclosure provides a device, comprising: a plurality of chambers; a thermoelectric plate comprising a plurality of thermoelectric elements, wherein each thermoelectric element of the plurality of thermoelectric elements corresponds to a different chamber of the plurality of chambers; and a movable assembly in contact with the thermoelectric plate, wherein the movable assembly is configured to, through movement, contact the plurality of thermoelectric elements to the plurality of chambers, wherein a chamber of the plurality of chambers is aligned to a thermoelectric element of the plurality of thermoelectric elements with an offset of at most about 250 micrometers.

[0021] In some embodiments, the plurality of chambers are a plurality of wells. In some embodiments, the contact is an automated contact. In some embodiments, the thermoelectric plate is contacted to the plurality of chambers with at least about 30 pounds of force. In some embodiments, the thermoelectric plate is contacted to the plurality of chambers with at most about 80 pounds of force. In some embodiments, the device has a footprint of at most about 540 square millimeters (mm2). In some embodiments, the device further comprises a lid positioned above the plurality of chambers. In some embodiments, the lid comprises a heating element. In some embodiments, the movable assembly comprises a lever. In some embodiments, the lever has a movement ratio of the lever to the movable assembly of about 2: 1. In some embodiments, the movable assembly comprises a plurality of pivoting points. In some embodiments, theAtty Dkt No.: 61197-702601 movable assembly comprises at least three pivoting points. In some embodiments, the device further comprises a limit switch configured to detect a position of the movable assembly. In some embodiments, the limit switch is an optical limit switch. In some embodiments, the limit switch is a mechanical limit switch. In some embodiments, the movable assembly is positioned below the thermoelectric plate.

[0022] In another aspect, the method, comprising: (a) providing (i) a first reaction mixture comprising a first analyte to a first chamber of a substrate and (ii) a second reaction mixture comprising a second analyte to a second chamber of the substrate, wherein the first chamber and the second chamber are each individually addressable; and (b) subjecting (i) the first reaction mixture to first conditions sufficient to perform a first PCR on the first analyte and (ii) the second reaction mixture to second conditions sufficient to perform a second PCR on the second analyte, wherein the subjecting of the first reaction mixture to the first conditions is independent from the subjecting the second reaction mixture to the second conditions.

[0023] In some embodiments, a plurality of reaction conditions across the substrate comprising the first set of conditions and the second set of conditions are in a gradient. In some embodiments, a plurality of reaction conditions across the substrate comprising the first set of conditions and the second set of conditions are discrete reaction conditions. In some embodiments, the method further comprises (i) during the first PCR, detecting one or more first signals or first signal changes from the first reaction mixture or (ii) during the second PCR, detecting one or more second signals or second signal changes from the second reaction mixture. In some embodiments, a first thermal element is in operable communication with the first chamber. In some embodiments, the first thermal element is a thermoelectric element. In some embodiments, the first thermal element has a length of at most about or a width of at most about of at most about 9 millimeters. In some embodiments, a second thermal element is in operable communication with the second chamber. In some embodiments, the first thermal element and the second thermal element are different thermal elements. In some embodiments, the method further comprises, subsequent to (b), extracting at least a portion of the first reaction mixture or the second reaction mixture. In some embodiments, the first reaction mixture or the second reaction mixture are not pre-normalized. In some embodiments, the method further comprises adjusting the first conditions to decrease a rate of or halt the first PCR. wherein the second PCR continues after the rate of the first PCR is decreased or halted, wherein the adjusting is in response to one or more signals from the first reaction mixture. In some embodiments,, during the subjecting of (b), (i) a thermal conductivity between a first thermal element associated with the first chamber and a heat sink in thermal contact with the first thermal element is at least aboutAtty Dkt No.: 61197-7026015 W / Km; or (ii) a thermal conductivity between the first chamber and a first sensor associated with the first chamber is at least about 5 W / Km. In some embodiments, the thermal conductivity between the first thermal element associated with the first chamber and the heat sink in thermal contact with the first thermal element is at least about 5 W / Km. In some embodiments, the thermal conductivity between the first chamber and the first sensor associated with the first chamber is at least about 5 W / Km. In some embodiments, the method further comprises measuring, using the first sensor, a property of the first chamber. In some embodiments, the property of the first chamber comprises a temperature of the first chamber. In some embodiments, the first thermal element, the heat sink, the first chamber, or the first sensor are in contact with a thermally conductive element. In some embodiments, the thermally conductive element has a thermal conductivity of at least about 5 W / Km. In some embodiments, the method further comprises transferring heat through the thermally conductive element. In some embodiments, the thermally conductive element comprises at least about 2 layers. In some embodiments, a difference in a temperature between the first chamber and a temperature of the first sensor is at most about 0.1 °C. In some embodiments,, during the subjecting of (b), (i) a thermal conductivity between a second thermal element associated with the second chamber and a heat sink in thermal contact with the second thermal element is at least about 5 W / Km; or (ii) a thermal conductivity between the second chamber and a second sensor associated with the second chamber is at least about 5 W / Km. In some embodiments, during (b), (i) a first volume of the first reaction mixture or (ii) a second volume of the second reaction mixture changes by less than about 10%. In some embodiments, the first volume and the second volume stay within about 10% of one another during the subjecting of (b). In some embodiments, the method further comprises, prior to (b), bringing the substrate in contact with a cover plate. In some embodiments, the cover plate comprises a first feature corresponding to the first chamber and a second feature corresponding to the second chamber. In some embodiments, the feature comprises a first hole disposed above the first chamber and a second hole disposed above the second chamber. In some embodiments, the cover plate comprises a thermal element. In some embodiments, the cover plate comprises one or more air deflectors. In some embodiments, the cover plate is connected to a top plate. In some embodiments, one or more springs are disposed between the cover plate and the top plate. In some embodiments, a contact force between the substrate and a cover plate disposed above the substrate during the subjecting of (b) is at least about 30 pounds. In some embodiments, (i) a first thermal element used to affect a first temperature condition of the first conditions and (ii) a second thermal element used to affect a second temperature condition of the second conditions each have a heating output and a coolingAtty Dkt No.: 61197-702601 output that differ by at most about 0.5 W. In some embodiments, a system comprising the substrate has an area of at most about 2,500 square centimeters (cm2). In some embodiments, a thermal conductivity between the first chamber and the second chamber is at most about 1 W / Km. In some embodiments, one or more thermal management elements are disposed between the first chamber and the second chamber. In some embodiments, the one or more thermal management elements comprise a thermal insulator. In some embodiments, the one or more thermal management elements comprise a gap. In some embodiments, the individual addressability comprises use of control circuitry. In some embodiments, the control circuitry comprises a converter. In some embodiments, the converter comprises a buck-boost converter. In some embodiments, the buck-boost converter is a current sinking buck-boost converter.

[0024] In another aspect, the present disclosure provides a system for performing a polymerase chain reaction (PCR), comprising: a first location and a second location; a support disposed between the first location and the second location; and a thermal management element or a thermally conductive element disposed in thermal communication with and between the first location and the second location.

[0025] In some embodiments, the first location and the second location are a first chamber and a second chamber. In some embodiments, the thermal management element comprises a hole disposed in the support. In some embodiments, the first chamber or the second chamber is affixed to the support via one or more fasteners. In some embodiments, the one or more fasteners comprise one or more screws. In some embodiments, a thermal conductivity between the first location and the second location is at most about 1 W / Km. In some embodiments, the support comprises a printed circuit board (PCB). In some embodiments, the PCB comprises one or more holes comprising one or more thermally conductive elements. In some embodiments, the PCB has a thickness of at least about 2 mm. In some embodiments, the PCB is a metal printed PCB. In some embodiments, the PCB is a multi-layer PCB. In some embodiments, the multi-layer PCB comprises at least two conductor layers and at least two insulating layers. In some embodiments, the PCB is at least a portion of a PCB assembly (PCBA). In some embodiments, the thermally conductive element comprises one or more of graphite, copper, or indium, or any combination thereof. In some embodiments, the thermally conductive element comprises a foil. In some embodiments, the first location is a chamber and the second location is a sensor associated with the chamber. In some embodiments, the thermally conductive element comprises a thermally conductive element configured to reduce a thermal resistance between the chamber and the sensor.Atty Dkt No.: 61197-702601

[0026] Another aspect of the present disclosure provides a non-transitory computer readable medium comprising machine executable code that, upon execution by one or more computer processors, implements any of the methods above or elsewhere herein.

[0027] Another aspect of the present disclosure provides a system comprising one or more computer processors and computer memory coupled thereto. The computer memory comprises machine executable code that, upon execution by the one or more computer processors, implements any of the methods above or elsewhere herein.

[0028] Additional aspects and advantages of the present disclosure will become readily apparent to those skilled in this art from the following detailed description, wherein only illustrative embodiments of the present disclosure are shown and described. As will be realized, the present disclosure is capable of other and different embodiments, and its several details are capable of modifications in various obvious respects, all without departing from the disclosure.Accordingly, the drawings and description are to be regarded as illustrative in nature, and not as restrictive.INCORPORATION BY REFERENCE

[0029] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. To the extent publications and patents or patent applications incorporated by reference contradict the disclosure contained in the specification, the specification is intended to supersede and / or take precedence over any such contradictory material.BRIEF DESCRIPTION OF THE DRAWINGS

[0030] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings (also “Figure” and “FIG.” herein), of which:

[0031] FIG. l is a flow chart of a method for performing a polymerase chain reaction (PCR) analysis of a sample containing or suspected of containing an analyte, according to an embodiment of the present disclosure.

[0032] FIG. 2 is a schematic of a device, according to an embodiment.

[0033] FIG. 3 is a schematic of a device, according to an embodiment.Atty Dkt No.: 61197-702601

[0034] FIG. 4A is a schematic of a thermoelectric control architecture without a current sink capacity, according to an embodiment.

[0035] FIG. 4B is a schematic of a thermoelectric control architecture with a current sink capacity, according to an embodiment.

[0036] FIG. 5 is a schematic of an addressable control array, according to an embodiment.

[0037] FIG. 6 shows a computer system that is programmed or otherwise configured to implement methods provided herein.

[0038] FIG. 7 is a flow chart of control logic for a chamber, according to an embodiment.

[0039] FIG. 8 is an example of a normalization based on a fluorescence endpoint, according to an embodiment.

[0040] FIG. 9 is an example of a plurality of views of a controller daughterboard, according to some embodiments.

[0041] FIG. 10 is an example of a control circuitry, according to some embodiments.

[0042] FIG. 11 is a cut away example of a system as described elsewhere herein, according to some embodiments

[0043] FIGs. 12A-12B show example schematics of control architectures, according to some embodiments.

[0044] FIG. 13 is an example of the thermal performance of a plurality of adjacent individually controlled wells, according to some embodiments.

[0045] FIG. 14 shows the performance comparison of individually controlled wells versus non- individually controlled wells, according to some embodiments.

[0046] FIGs. 15A-15C are examples of a pillar design, according to some embodiments.

[0047] FIGs. 16A-16B show different views of an example of a device, according to some embodiments.

[0048] FIG. 17 shows an example of an optics assembly, according to some embodiments.

[0049] FIG. 18 shows an example of a configuration of a plurality of optical assemblies, according to some embodiments.

[0050] FIG. 19 shows an example of a unit comprising a plurality of optics assemblies positioned adjacent to a plurality of chambers, according to some embodiments.

[0051] FIG. 20 shows an example unit comprising 8 optics assemblies, according to some embodiments.

[0052] FIG. 21 shows an example unit comprising 16 optics assemblies in a single unit, according to some embodiments.Atty Dkt No.: 61197-702601

[0053] FIG. 22 shows an example of an annealing temperature optimization experiment, according to some embodiments.

[0054] FIG. 23 shows a plot of temperature profiles obtained within 24 wells executing a protocol with a 65 degree Celsius linear gradient, according to some embodiments.

[0055] FIGs. 24A - 24B show software setting for the experiment performed in FIG. 23, according to some embodiments.

[0056] FIG. 25 is a flow chart of a method of performing a PCR analysis of a sample containing or suspected of containing an analyte, according to some embodiments.

[0057] FIG. 26 is a flow chart of a method, according to some embodiments.

[0058] FIG. 27 is a flow chart of a method, according to some embodiments.

[0059] FIG. 28 shows an example of a support comprising one or more thermally conductive elements, according to some embodiments.

[0060] FIGs. 29A and 29B examples of power supplies configured to provide power to a thermoelectric element, according to some embodiments.

[0061] FIGs. 30A and 30B show an example of a system comprising a thermoelectric plate and a plurality of chambers, according to some embodiments.

[0062] FIG. 31 shows an example of input and output conditions of a serial dilution amplification example, according to some embodiments.

[0063] FIGs. 32A and 32B show examples of the fluorescent signal from a fixed number amplification reaction and a dynamic number amplification reaction, respectively, according to some embodiments.

[0064] FIG. 33 shows an example of cover plate interfacing with a top plate and a support, according to some embodiments.

[0065] FIG. 34 shows an example of a cover plate, according to some embodiments.

[0066] FIG. 35A shows an example of a plurality of chambers and a plurality of sensors operably coupled to a support, according to some embodiments. FIG. 35B shows an example of a detail of a plurality of chambers and a plurality of sensors operably coupled to a support, according to some embodiments.

[0067] FIG. 36 is a flow chart of a method, according to some embodiments.

[0068] FIGs. 37A - 37B show an example of a control interface, according to some embodiments.

[0069] FIGs. 38A - 38C show a front view (FIG. 38A), an isometric view (FIG. 38B), and a rear view (FIG. 38C) of a system, according to some embodiments.Atty Dkt No.: 61197-702601

[0070] FIGs. 39A - 39G show examples of portions of a user interface, according to some embodiments.

[0071] FIGs. 40A - 40C show examples of auto-normalization interfaces, according to some embodiments.

[0072] FIGs. 41A - 41C show examples of a chamber selection user interface, according to some embodiments.

[0073] FIGs. 42A - 42C shows an example of a system with a movable assembly and substrate, according to some embodiments.

[0074] FIGs. 43A - 43B show examples of a system comprising a movable assembly, according to some embodiments.

[0075] FIG. 44 shows an example of an alignment feature, according to some embodiments

[0076] FIG. 45 shows an example of a system of the present disclosure, according to some embodiments.

[0077] FIG. 46 shows an example of a system and associated computer system, according to some embodiments.DETAILED DESCRIPTION

[0078] While various embodiments of the invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions may occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed.

[0079] As used herein, the singular forms “a,” “an,” and “the” include plural references unless the context clearly dictates otherwise. Any reference to “or” herein is intended to encompass “and / or” unless otherwise stated.

[0080] Whenever the term “at least,” “greater than,” or “greater than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “at least,” “greater than” or “greater than or equal to” applies to each of the numerical values in that series of numerical values. For example, greater than or equal to 1, 2, or 3 is equivalent to greater than or equal to 1, greater than or equal to 2, or greater than or equal to 3.

[0081] Whenever the term “no more than,” “less than,” or “less than or equal to” precedes the first numerical value in a series of two or more numerical values, the term “no more than,” “less than,” or “less than or equal to” applies to each of the numerical values in that series of numerical values. For example, less than or equal to 3, 2, or 1 is equivalent to less than or equal to 3, less than or equal to 2, or less than or equal to 1.Atty Dkt No.: 61197-702601

[0082] Certain inventive embodiments herein contemplate numerical ranges. When ranges are present, the ranges include the range endpoints. Additionally, every sub range and value within the range is present as if explicitly written out. The term “about” or “approximately” may mean within an acceptable error range for the particular value, which will depend in part on how the value is measured or determined, e.g., the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art. Alternatively, “about” may mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value may be assumed.

[0083] In some cases, a device as described elsewhere herein may comprise one or more chambers. The one or more chambers may be locations as described elsewhere herein. For example, a device can comprise a plurality of locations that are a plurality of chambers.

[0084] In another aspect, the present disclosure provides a method for performing a first PCR and a second PCR. A first reaction mixture comprising a first analyte can be provided to a first chamber of a substrate. A second reaction mixture comprising a second analyte can be provided to a second chamber of the substrate. The first chamber and the second chamber may be individually addressable. The first reaction mixture may be subjected to first conditions sufficient to perform the first PCR on the first analyte. The second reaction mixture may be subjected to second conditions sufficient to perform the second PCR on the second analyte. A coefficient of variation of a yield of the first PCR and the second PCR may be as described elsewhere herein. A difference in temperature between the first chamber and the second chamber may be as described elsewhere herein. The subjecting of the first reaction mixture to the first conditions may be independent from the subjecting the second reaction mixture to the second conditions.

[0085] In another aspect, the present disclosure provides methods, systems, and / or kits for determining one or more properties of an analyte. A sample or a reaction mixture comprising an analyte may be provided to a location or chamber of a substrate. The location or the chamber may be individually addressable. The sample or the reaction mixture may be subjected to conditions sufficient to perform a polymerase chain reaction (PCR) on the analyte. During the PCR, one or more signals or signal changes may be detected from the reaction mixture. The one or more properties of the analyte may be determined based at least in part on the one or more signals or signal changes. In another aspect, the present disclosure provides methods, systems, and / or kits for performing a PCR reaction on an analyte.Atty Dkt No.: 61197-702601

[0086] In some cases, the kits may comprise components and / or reagents in a suitable container (e.g., tube, bottle). The kits may be supplied by a user. The kits may be commercial kits. The kit may comprise instructions for use of the kit in accordance with one or more of the methods and / or systems of the present disclosure. The kit can comprise one or more substrates as described elsewhere herein, one or more seals as described elsewhere herein, ore or more sensors as described elsewhere herein, one or more sensor arrays as described elsewhere herein, one or more nanopores (e g., sequencing nanopores), or one or more devices as described elsewhere herein, or any combination thereof.

[0087] In another aspect, the present disclosure can provide methods, systems, and / or kits for determining one or more properties of an analyte. A reaction mixture can be provided comprising an analyte to a chamber of a substrate. The chamber can be individually addressable. The reaction mixture can be subjected to conditions sufficient to perform a PCR on the analyte, thereby resulting in exponential amplification of the analyte.

[0088] In another aspect, the present disclosure can provide methods, systems, and / or kits for determining a property of an analyte. A reaction mixture comprising the analyte can be provided to a chamber of a substrate. The chamber can be individually addressable. The reaction mixture can be subjected to conditions sufficient to perform a PCR on the analyte. The PCR may have an efficiency of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or more percent. The PCR may have an efficiency of at most about 99, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less percent. The PCR may have an efficiency from about 10 to about 99, about 50 to about 99, about 50 to about 80, or about 80 to about 99 percent. The PCR efficiency may be a measurement of how many of the nucleic acid molecules within the reaction mixture are amplified during the PCR. For example, in a reaction mixture with 100 nucleic acid molecules where 90 of the nucleic acid molecules undergo amplification, the PCR efficiency can be 90%. In some cases, a PCR efficiency of a plurality of chambers (e g., a first chamber and an additional chamber) may have a difference of at least about 0.01, 0.05, 0. 1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more percent. The difference in the PCR efficiency of the plurality of chambers may be at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less percent. The difference in the PCR efficiency of the plurality of chambers may be from about 0.01 to about 10, about 0.01 to about 1, about 0. 1 to about 2, about 0.5 to about 1, or about 0.5 to about 3 percent. The PCR efficiency or low variation in PCR efficiency between the plurality of chambers may be related to the consistency of the reaction parameters (e.g., temperature, volume), which may be, in turn, affected by the use of a cover plate (e.g., to maintain a volume of the reaction mixture), thermally conductive element (e g., to improve the accuracy ofAtty Dkt No.: 61197-702601 measurement of the reaction conditions of the reaction mixture), thermal management element (e.g., to improve thermal isolation between chambers), or the control circuitry (e.g., to accurately control the reaction conditions of the chamber), or any combination thereof. During the PCR, one or more signals or signal changes can be detected from the reaction mixture.

[0089] In another aspect, the present disclosure can provide methods, systems, and / or kits for performing a PCR. A reaction mixture comprising an analyte can be provided to a chamber on a substrate. The chamber can be individually addressable. The reaction mixture may be subjected to conditions sufficient to perform the PCR on the analyte. During the PCR, the reaction mixture can have a volume change as described elsewhere herein.

[0090] In another aspect, the present disclosure can provide methods, systems, and / or kits for performing a PCR. A system may comprise a substrate comprising a chamber. A system may comprise a cover plate disposed above the substrate. The cover plate may be configured to, when engaged with the substrate, cover the chamber. The cover plate can be configured to maintain a volume of a reaction mixture within the reaction chamber within a percentage of a starting volume of the reaction mixture as described elsewhere herein.

[0091] In another aspect, the present disclosure can provide methods, systems, and / or kits for performing a PCR. The system may comprise a first location and a second location. The system may comprise a support disposed between the first location and the second location. The system may comprise a thermal management element and / or a thermally conductive element disposed in thermal communication with and between the first location and the second location.

[0092] In another aspect, the present disclosure can provide a device. The device may comprise a thermoelectric element operably coupled to a support. The support may comprise one or more thermally conductive elements. The one or more thermally conductive elements may be disposed in at least a portion of the support. The thermoelectric element may be in thermal contact with the one or more thermally conductive elements.

[0093] In another aspect, the present disclosure may provide a method. A device may be provided. The device may comprise a power supply comprising a circuit comprising a current sinking converter and a thermoelectric element in electrical communication with the power supply. The power supply may be used to provide power to the thermoelectric element. The power may be supplied to the thermoelectric element. The current sinking convertor may remove current from the circuit that is in excess of a current sufficient to operate the thermoelectric element. The power supply may comprise a fan.

[0094] In another aspect, the present disclosure may provide a method. A plurality of chambers, a thermoelectric plate comprising a plurality of thermoelectric elements, and a movable assemblyAtty Dkt No.: 61197-702601 in contact with the thermoelectric plate may be provided. Each thermoelectric element of the plurality of thermoelectric elements may correspond to a different chamber of the plurality of chambers. The movable assembly may be used to move the thermoelectric plate, thereby contacting the plurality of thermoelectric elements to the plurality of chambers. A chamber of the plurality of chambers may be aligned to a thermoelectric element of the plurality of thermoelectric elements with an offset as described elsewhere herein.

[0095] In another aspect, the present disclosure can provide a device. The device can comprise a plurality of chambers. The device may comprise a thermoelectric plate comprising a plurality of thermoelectric elements. Each thermoelectric element of the plurality of thermoelectric elements can correspond to a different chamber of the plurality of chambers. The device may comprise a movable assembly in contact with the thermoelectric plate. The movable assembly may be configured to, through movement, contact the plurality of thermoelectric elements to the plurality of chambers. A chamber of the plurality of chambers can be aligned to a thermoelectric element of the plurality of thermoelectric elements with an offset as described elsewhere herein.

[0096] The conditions sufficient to perform the PCR may comprise heating the sample or the reaction mixture to a temperature sufficient to initiate the PCR. The heating may comprise heating to a temperature of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more °C. The heating may comprise heating to a temperature of at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less °C. The heating may comprise heating to a temperature from about 10 to about 100, about 20 to about 100, about 30 to about 100, about 10 to about 90, about 20 to about 90, or about 30 to about 90 °C. The heating may be a portion of a denaturation operation, an annealing operation, or an extension operation, or any combination thereof of the PCR.

[0097] The conditions sufficient to perform the PCR may comprise cooling the reaction mixture to a temperature sufficient to slow or halt the PCR. The cooling may comprise cooling to a temperature of at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more °C. The cooling may comprise cooling to a temperature of at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less °C. The cooling may comprise cooling to a temperature from about 10 to about 100, about 20 to about 100, about 30 to about 100, about 10 to about 90, about 20 to about 90, or about 30 to about 90 °C. The cooling may be a portion of a denaturation operation, an annealing operation, or an extension operation, or any combination thereof of the PCR.

[0098] A temperature condition can be affected by a thermal element disposed in thermal contact with the chamber. Using a thermoelectric element with a symmetrical heating and cooling outputAtty Dkt No.: 61197-702601 as the thermal element can provide increased system efficiency as well as reduced error in the temperature control of the chamber. The symmetrical heating and cooling output may be where a heating output (e.g., watts of heating output) and a cooling output (e.g., watts of cooling output) are the same or substantially the same. The heating output of the thermal element may be used to, for example, heat a sample or reaction mixture to initiate the PCR. The symmetrical heating and cooling output may enable faster heating for the initiation of the PCR. The cooling output of the thermal element may be used to cool the sample or the reaction mixture between PCR operations and / or terminate the PCR. Using a thermal element with a symmetrical heating and cooling output can enable faster cooling and termination of the PCR. The thermal element used to affect a temperature condition of the conditions may have a heating output and a cooling output that differ by at least about 0.001 , 0.005, 0.01 , 0.05, 0.1 , 0.5, 1 , 2, 3, 4, 5, or more watts. The thermal element used to affect a temperature condition of the conditions may have a heating output and a cooling output that differ by at most about 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or less watts. The thermal element used to affect a temperature condition of the conditions may have a heating output and a cooling output that differs by an amount in a range as defined by any two of the preceding values. For example, the thermal element used to affect a temperature condition of the conditions may have a heating output and a cooling output that differs by from about 0.001 to about 5, 0.01 to about 5, 0.1 to about 5, 0.001 to about 1, 0.01 to about 1 , or about 0.1 to about 1 watts. The thermal element may be as described elsewhere herein. For example, the thermal element can be connected to a current sinking buck-boost convertor.

[0099] The conditions sufficient to perform the PCR may comprise providing stirring to the reaction mixture. The stirring may be at a rate of at least about 1, 10, 50, 100, 500, 1,000, or more rotations per minute. The stirring may be at a rate of at most about 1,000, 500, 100, 50, 10, 1, or less rotations per minute. The conditions sufficient to perform the PCR may comprise providing one or more of a temperature, a presence of reagents, or an absence of inhibitors, or any combination thereof. The presence of reagents may comprise a presence of one or more primers (e.g., to bind to the analyte for initiation of the PCR), polymerases (e.g., to synthesize a complement to the analyte), nucleotides (e g., to provide the materials for the PCR extension), buffers (e g., to maintain a pH or ionic strength of the reaction mixture), or solvents (e.g., to adjust the solvation of the analyte or the other reagents), or any combination thereof. The pH of the sample or the reaction mixture may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, or more. The pH of the sample or the reaction mixture may be at most about 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less. The one or more buffers may comprise one or more of Tris-HCl,Atty Dkt No.: 61197-702601 potassium chloride, sodium chloride, magnesium chloride, or phosphate buffers, or any combination thereof. The absence of inhibitors may comprise an absence of may comprise an absence of proteins, phenolic compounds, detergents, alcohols, or formaldehyde, or any combination thereof. A concentration of one or more reagents in the reaction mixture may be at least about 0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, or more micromolar. The concentration of the one or more reagents in the reaction mixture may be at most about 100, 500, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, 0.0005, 0.0001, or less micromolar. The concentration of the one or more reagents may be from about 0.0001 to about 100, about 0.0001 to about 1, 0.001 to about 0.005, or about 0.0001 to about 0.1 micromolar.

[0100] Prior to use in the methods and systems of the present disclosure, at least a portion of a reaction mixture can be extracted from a sample (e g., an analyte can be extracted from the sample to a reaction mixture). A sample as described elsewhere herein can be subjected to one or more pre-processing or extraction operations that prepare the sample for analysis. For example, the sample can be washed, homogenized, or treated, or any combination thereof prior to analysis. A reaction mixture can be generated prior to the methods of the present disclosure. The sample can be combined with one or more reagents (e g., one or more PCR reagents), thereby generating the reaction mixture. The reaction mixture can be placed into a chamber (e g., a well) of a substrate (e.g., a well plate) and placed in a system of the present disclosure. The system can then be configured to subject the chamber and / or the reaction mixture to conditions sufficient to perform the PCR reaction. For example, a temperature of the reaction mixture can be raised to initiate the PCR reaction. The system may be configured to monitor the PCR reaction (e g., by using one or more optical systems of the present disclosure) and detect the one or more signals or signal changes. The one or more signals or signal changes may then, in turn, be processed or analyzed to provide the property of the analyte using, for example, a computer system of the present disclosure.

[0101] FIG. 36 is a flow chart of a method 3600, according to some embodiments. The method may be performed on one or more systems of the present disclosure. The method may be a method of determining one or more properties of an analyte. In an operation 3610, the method 3600 may comprise providing a reaction mixture comprising an analyte to a chamber of a substrate. The chamber of the substrate may be as described elsewhere herein. For example, the chamber of the substrate can be a well of a well plate. The chamber may be individually addressable. For example, the chamber may be addressed individually. The addressing may comprise changing the conditions of the chamber, sampling or interrogating the contents of the chamber, adding or removing a reaction mixture or reagents from the chamber. For example, theAtty Dkt No.: 61197-702601 addressing of a first chamber can be independent from the addressing of a second chamber. In this example, the thermal conditions of the chamber, and / or a measurement of a signal from the chamber, can be performed without changing a thermal condition or measuring a signal from an additional chamber.

[0102] In some cases, prior to operation 3610, the method 3600 may comprise generating the reaction mixture. The generating the reaction mixture may comprise processing a sample as described elsewhere herein. The generating the reaction mixture may comprise adding, to a processed or unprocessed sample, one or more reagents. The one or more reagents may be PCR reagents. Examples of PCR reagents include, but are not limited to, nucleic acid templates, primers, polymerases, nucleotide triphosphates, buffers, cations (e.g., divalent cations), or solvents (e g., water, dimethyl sulfoxide, glycerol), or any combination thereof. After the generating the reaction mixture, the reaction mixture can comprise one or more of the sample, one or more components extracted from the sample, one or more analytes, one or more nucleic acid templates, one or more primers, one or more polymerases, one or more nucleotide triphosphates, one or more buffers, one or more cations, or one or more solvents, or any combination thereof.

[0103] The reaction mixture may have a volume of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1,000, 1,050, 1,100, 1,150, 1,200, 1,250, 1,300, 1,350, 1,400, 1,450, 1,500, 1,550, 1,600, 1,650, 1,700, 1,750, 1,800, 1,850, 1,900, 1,950, 2,000, 2,050, 2, 100, 2,150, 2,200, 2,250, 2,300, 2,350, 2,400, 2,450, 2,500, 2,550, 2,600, 2,650, 2,700, 2,750, 2,800, 2,850, 2,900, 2,950, 3,000, or more microliters. The reaction mixture may have a volume of at most about 3,000, 2,950, 2,900, 2,850, 2,800, 2,750, 2,700, 2,650, 2,600, 2,550, 2,500, 2,450, 2,400, 2,350, 2,300, 2,250, 2,200, 2,150, 2,100, 2,050, 2,000, 1,950, 1,900, 1,850, 1,800, 1,750, 1,700, 1,650, 1,600, 1,550, 1,500, 1,450, 1,400, 1,350, 1,300, 1,250, 1,200, 1,150, 1,100, 1,050, 1,000, 950, 900, 850, 800, 750, 700, 650, 600, 550, 500, 450, 400, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less microliters. The reaction mixture may have a volume in a range as defined by any two of the preceding values. For example, the reaction mixture may have a volume of about 10 to 3,000, 50 to 2,000, 100 to 1,000, 100 to 500, 100 to 400, or 250 to 450 microliters.

[0104] The reaction mixture may have a concentration of one or more analytes of at least about 1 x IO'6, 5 x IO’6, 1 x 10'5, 5 x 10'5, 1 x 10'4, 5 x IO’4, 1 x IO'3, 5 x IO'3, 1 x IO'2, 5 x IO'2, 1 x 101, 5 x 10’1, 1, 5, 10, 50, 100, or more nanograms per microliter. The reaction mixture may have aAtty Dkt No.: 61197-702601 concentration of one or more analytes of at most about 100, 50, 10, 5, 1, 5 x 10’1, 1 x 10'1, 5 x 10‘2, 1 x 10‘2, 5 x 10’3, 1 x 10‘3, 5 x 10‘4, 1 x 10‘4, 5 x I O'5, 1 x 10‘5, 5 x 10‘6, 1 x 10‘6, or less nanograms per microliter. The reaction mixture may have a concentration of one or more analytes in a range as defined by any two of the preceding values. For example, the reaction mixture can have a concentration of about 1 x IO-6to about 100, l x IO-6to about 1, or about 1 x 10’5to about 1 . The reaction mixture and an additional reaction mixture may have an initial concentration difference of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more orders of magnitude. The reaction mixture and an additional reaction mixture may have an initial concentration difference of at most about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less orders of magnitude. A product concentration of the reaction mixture and the additional reaction mixture may differ by at least about 1 , 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more percent. A product concentration of the reaction mixture and the additional reaction mixture may differ by at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, 1, or less percent.

[0105] In another operation 3620, the method 3600 may comprise subjecting the reaction mixture to conditions sufficient to perform a PCR on the analyte. The subjecting may comprise the use of one or more thermal elements as described elsewhere herein. For example, the subjecting can comprise using a thermoelectric element to heat and / or cool the reaction mixture to perform the PCR on the analyte. In another example, the subjecting can comprise using the thermoelectric element to maintain a reaction temperature of a reaction mixture.

[0106] One or more thermally conductive elements can be disposed between various components of the system to increase thermal conductivity during the methods of the present disclosure. For example, the one or more thermally conductive elements can be disposed between (i) a chamber and a sensor, (ii) a thermal element and a heatsink, or (iii) a chamber and a thermal element, or any combination thereof. The increase in thermal conductivity can provide benefits such as increased accuracy of sensing, increased thermal element efficiency, reduced waste heat, or improved operating parameters of thermal elements, or any combination thereof. Heat may be transferred through the thermally conductive element. For example, the contact of the chamber and the sensor to the thermally conducive element can result in convective heat transfer between the chamber and the sensor. The heat transfer may be between the chamber and the sensor. The heat transfer may be between the thermal element and the heatsink. The heat transfer may be via contact with the thermally conductive element. For example, the heat transfer may be conductive. The heat transfer may be radiative. For example, the thermal element can be in line of sight communication with the heatsink, thereby radiatively transferring heat to the heatsink.Atty Dkt No.: 61197-702601

[0107] During the subjecting of 3620, a thermal conductivity between a thermal element associated with the chamber and a heat sink in thermal contact with the thermal element may be at least about 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 600, 700, 800, 900, 1 ,000, 1 , 100, 1 ,200, 1 ,300, 1 ,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, or more watts / kelvin / meter (W / Km). The thermal conductivity between the thermal element and the heat sink may be at most about 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,200, 1,100, 1,000, 900, 800, 700, 600, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 1 10, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less W / Km. The thermal conductivity between the thermal element and the heat sink may have a conductivity from about 50 to about 2,000, about 80 to about 1,000, about 100 to about 1,000, about 150 to about 350, or about 150 to about 250 W / Km.

[0108] In some cases, a thermal element and the heatsink may be separated by a support. In some cases, the support is a printed circuit board (PCB). The PCB may be as described elsewhere herein. The support may be thermally insulating. For example, the support can have a low thermal conductivity. The thermal conductivity of the support may be less than about 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less W / Km. The thermal conductivity of the support may be greater than about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more W / Km. The thermal conductivity of the support may be in a range as defined by any two of the preceding values. For example, the thermal conductivity of the support may be from about 0.1 to about 1, about 0. 1 to about 0.5, about 0.5 to about 1, or about 0.3 to about 0.7 W / Km.

[0109] The support may comprise one or more holes comprising one or more thermally conductive elements. The one or more thermally conductive elements may be disposed within the one or more holes. Each hole of the one or more holes may comprise a different thermally conductive element disposed therein. At least one hole of the one or more holes may comprise a thermally conductive element. The thermal element and / or the heat sink may be operably coupled (e.g., affixed) to the support. The thermal element and / or the heat sink may be operably coupled to the thermally conductive element. The coupling may comprise solder, thermally conductive adhesive, brazing, or mechanical fixation (e g., screws, threads, wires), or any combination thereof. The chamber and / or the sensor may be operably coupled to the support. The chamber and / or the sensor may be operably coupled to the thermally conductive element.Atty Dkt No.: 61197-702601The coupling may comprise solder, thermally conductive adhesive, brazing, or mechanical fixation (e.g., screws, threads, wires), or any combination thereof. In some cases, the one or more holes may comprise one or more sensors. For example, a sensor can be disposed in at least a portion of the support.

[0110] In some cases, heat may be transferred between the thermal element and a heat sink. The thermal element may comprise a thermoelectric element as described elsewhere herein. The thermoelectric element may comprise at least two sides and, when in use, one side can provide heating or cooling to the chamber depending on a direction of a flow of current. For example, one side of the thermoelectric element can provide heating under positive potential and cooling under negative potential. During the use, the second side of the thermoelectric element can undergo the opposite temperature change (e g., cool when heating the chamber, heat when cooling the chamber). In some cases, placing the thermoelectric element on a thermally insulating support (e.g., a PCB) can result in the support undergoing thermal stress from the temperature changes of the second side of the thermoelectric element. To address this, by placing the thermoelectric element in thermal contact with a thermally conductive element, the second side of the thermoelectric element can have excess heating or cooling removed, improving longevity of the thermoelectric element and improving control over the thermal condition of the well.

[0111] The thermal element may have a minimum temperature of at least about -50, -40, -30, - 20, -10, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more °C. The thermal element may have a minimum temperature of at most about 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 0, -10, -20, -30, -40, -50, or less °C. The thermal element may have a minimum temperature in a range as defined by any two of the preceding values. The thermal element may have a maximum temperature of at least about -50, -40, -30, -20, -10, 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, or more °C. The thermal element may have a maximum temperature of at most about 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 0, -10, -20, -30, -40, -50, or less °C. The thermal element may have a maximum temperature in a range as defined by any two of the preceding values. The thermal element may have a temperature ramp rate (e.g., a rate of heating and / or cooling) of at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6,7, 8, 9, 10, or more °C per second. The thermal element may have a temperature ramp rate of at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, or less °C per second. The thermal element may have a temperature ramp rate from about 0.1 to about 10, about 1 to about 10, about 1 to about 5, or about 5 to about 10 °C per second.Atty Dkt No.: 61197-702601

[0112] During the subjecting of 3620, a thermal conductivity between the chamber and a sensor associated with the chamber may be at least about 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15,16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170,180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490, 500, 600, 700, 800, 900, 1,000, 1 ,100, 1,200, 1,300, 1 ,400, 1 ,500, 1 ,600, 1,700, 1,800, 1 ,900, 2,000, or more watts / kelvin (W / Km). The thermal conductivity between the chamber and the sensor may be at most about 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,200, 1, 100, 1,000, 900, 800, 700, 600, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320,310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130,120, 1 10, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9,8, 7, 6, 5, 4, 3, 2, 1, or less W / Km. The thermal conductivity between the chamber and the sensor may have a conductivity from about 50 to about 2,000, about 80 to about 1,000, about 100 to about 1,000, about 150 to about 350, or about 150 to about 250 W / Km. By placing a thermally conductive element in a thermal path between the chamber and the sensor, the thermal conductivity can be increased between the chamber and the sensor.

[0113] Increasing the thermal conductivity between the chamber and the sensor can also result in more accurate monitoring of the chamber by the sensor. The sensor can be used to measure a property of the chamber. The property of the chamber can comprise a temperature of the chamber and / or a reaction mixture within the chamber. Reducing a difference between a temperature of a reaction mixture in the chamber and the temperature of a sensor configured to measure the temperature of the reaction mixture can enable closer, more accurate monitoring of the reaction mixture. This can, in turn, improve control over the reaction and increase reaction efficiency. The property of the chamber can be a property of the conditions sufficient to perform the PCR. For example, the conditions can provide a set temperature for the chamber, and the property can be a real temperature of the chamber. The sensor can be a thermal sensor (e g., a thermocouple, a thermopile, a thermistor, a resistance thermal detector, digital thermal sensor, any combination thereof). The sensor may have an area of at least about 0.0001, 0.001, 0.01, 0.1, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, or more square millimeters. The sensor may have an area of at most about 25, 24, 23, 22, 21, 20, 19, 18,17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.0001, or fewer square millimeters. The sensor may have a thickness of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000,Atty Dkt No.: 61197-7026013.100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100, 4,200, 4,300, 4,400,4.500, 4,600, 4,700, 4,800, 4,900, 5,000, 5,100, 5,200, 5,300, 5,400, 5,500, 5,600, 5,700, 5,800,5.900, 6,000, 6,100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900, 7,000, 7,100, 7,200,7.300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300, 8,400, 8,500, 8,600,8.700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700, 9,800, 9,900, 10,000, or more micrometers. The sensor may have a thickness of at most about 10,000, 9,900, 9,800,9.700, 9,600, 9,500, 9,400, 9,300, 9,200, 9,100, 9,000, 8,900, 8,800, 8,700, 8,600, 8,500, 8,400,8.300, 8,200, 8,100, 8,000, 7,900, 7,800, 7,700, 7,600, 7,500, 7,400, 7,300, 7,200, 7,100, 7,000,6.900, 6,800, 6,700, 6,600, 6,500, 6,400, 6,300, 6,200, 6,100, 6,000, 5,900, 5,800, 5,700, 5,600,5.500, 5,400, 5,300, 5,200, 5,100, 5,000, 4,900, 4,800, 4,700, 4,600, 4,500, 4,400, 4,300, 4,200,4.100, 4,000, 3,900, 3,800, 3,700, 3,600, 3,500, 3,400, 3,300, 3,200, 3,100, 3,000, 2,900, 2,800,2.700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100, 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400,1.300, 1,200, 1,100, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less micrometers. The sensor may have a range as defined by any two of the preceding values.

[0114] A difference in a temperature of (i) the chamber and / or the reaction mixture and (ii) the sensor may be at least about 0.001, 0.002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, or fewer °C. The difference in the temperature of (i) the chamber and / or the reaction mixture and (ii) the sensor may be at most about 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.09, 0.08, 0.07, 0.06, 0.05, 0.04, 0.03, 0.02, 0.01, 0.009, 0.008, 0.007, 0.006, 0.005, 0.004, 0.003, 0.002, 0.001, or less °C. The difference in the temperature of (i) the chamber and / or the reaction mixture and (ii) the sensor may be in a range as defined by any two of the preceding values. For example, the difference in the temperature can be from about 0.001 to about 5, about 0.1 to about 5, about 0.5 to about 5, about 0. 1 to about 1, about 0.5 to about 1, or about 0.01 to about 1 °C. Having a low difference in the temperature between the chamber and / or the reaction mixture and the sensor can provide accurate sensing and improved control over the parameters of the PCR. The low temperature difference can also be influenced by having a low crosstalk between the chambers. For example, having low thermal conductivity between the chambers, especially tightly spaced chambers, can improve the accuracy of the temperature sensing and quality of the PCR

[0115] The PCR may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, 100, or more cycles. TheAtty Dkt No.: 61197-702601PCR may comprise at most about 100, 99, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 cycle. The PCR may comprise a number of cycles in a range as defined by any two of the preceding values. For example, the PCR may comprise from about 1 to about 50, about 5 to about 40, about 10 to about 20, or about 5 to about 15 cycles.

[0116] In some cases, the use of a cover plate (e.g., a heated cover plate) can reduce evaporation and / or condensation from the reaction mixture during the PCR. During the PCR, a volume of the reaction mixture within the chamber may change by at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more percent. The volume of the reaction mixture within the chamber may change by at most about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17,16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, or less percent. The volume of the reaction mixture may change by about 0. 1 to about 50, about 1 to about 50, about 1 to about 20, about 1 to about 10, or about 5 to about 10 percent. The change may be a change from a starting volume of the reaction mixture. For example, the change may be a total change from the initial volume of the reaction mixture at the beginning of the PCR. During the PCR, the volume of the reaction mixture may change by at least about 0.1 , 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more microliters. During the PCR, the volume of the reaction mixture may change by at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12,11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0. 1, or less microliters. During the PCR, the volume may change by about 0.1 to about 100, about 1 to about 100, about 1 to about 50, about 5 to about 100, about 5 to about 50, about 10 to about 50, or about 10 to about 20 microliters. During the PCR, the volume change may comprise evaporation and / or condensation. For example, during a heating cycle of the PCR, the reaction mixture may at least partially evaporate. In another example, during a cooling cycle of the PCR, moisture from the air may condense into the rection mixture. Reducing a rate at which a volume of a reaction mixture changes can improve a consistency of the PCR, reduce variations between different PCRs in the substrate, or improve yields of the PCR, or any combination thereof. A volume change of a reaction mixture can change a concentration of reagents and / or analytes within the reaction mixture, which can change the outcome of the PCR. By reducing the volume change, these errors can be decreased.Atty Dkt No.: 61197-702601

[0117] An evaporation rate of the reaction mixture during the PCR may be at least about 0.001, 0.005, 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 5, 10, 50, 100, or more microliters per hour. The evaporation rate of the reaction mixture during the PCR may be at most about 100, 50, 10, 5, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or less microliters per hour. The evaporation rate may be from about 0.001 to about 10, about 0.01 to about 10, about 0.1 to about 10, about 1 to about 10, about 0.1 to about 5, about 1 to about 5, or about 0.1 to about 1 microliters per hour. The evaporation rate may be reduced through the cover plate coupling with the substrate, reducing a volume of air above the reaction mixture to evaporate into.

[0118] An additional chamber of the substrate may comprise an additional reaction mixture. For example, during the PCR, the reaction mixture can be undergoing the PCR and the additional rection mixture can undergo an additional PCR. Thermally isolating the chamber and the additional chamber can decrease thermal crosstalk, which can improve sensing accuracy, reduce sensor crosstalk, fidelity of applied conditions to input conditions, or improve consistency of the PCRs, or any combination thereof. In some cases, one or more thermal insulators can be disposed between the chamber and the additional chamber. For example, the thermal insulator can reduce a thermal, bleed between the chamber and the additional chamber. Examples of thermal insulators include, but are not limited to, aerogel, fiberglass, a foam, mineral wool, ceramic insulation, or gasses (e.g., air), or any combination thereof.

[0119] A temperature difference between the chamber and the additional chamber may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more degrees Celsius. The temperature difference between the chamber and the additional chamber may be at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less degrees Celsius. The temperature difference may be from about 1 to about 100, about 25 to about 100, about 50 to about 100, about 25 to about 90, about 50 to about 90, or about 75 to about 90 degrees Celsius. The chamber and the additional chamber may be adjacent. For example, the chamber and the additional chamber may not have an intervening chamber. The chamber and the additional chamber may be on different portions of the substrate. For example, each chamber can be on opposite sides of the substrate.

[0120] In some cases, the substrate can comprise an additional chamber comprising an additional reaction mixture. Using one or more cover plates can reduce volume change for the chamber and the additional chamber, thereby providing consistent PCR environments through a portion of or each of the chambers of the substrate. This can, in turn, reduce coefficients of variation of yields of the PCR reactions, or reduce reaction rate changes (e g., due to changes in reaction mixtureAtty Dkt No.: 61197-702601 pH or reagent imbalance). The reduced volume change may reduce sample contamination (e g., sample crosstalk) by reducing evaporation of sample from a first chamber and condensation to a second chamber. A volume of the reaction mixture and the additional reaction mixture may differ by at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, or more percent over the course of the PCR and an additional PCR of the additional reaction mixture. The volume of the reaction mixture and the additional reaction mixture may differ by at least about 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, or less percent over the course of the PCR and an additional PCR of the additional reaction mixture. The volume of the reaction mixture and the additional reaction mixture may change by about 0.1 to 50, 1 to 50, 1 to 20, 1 to 10, or 5 to 10 percent. A single cover plate may be disposed in contact with the chamber and the additional chamber. A first cover plate may be disposed in contact with the chamber and a second cover plate may be disposed in contact with the additional chamber. The cover plate may be as described elsewhere herein.

[0121] A coefficient of variation of a yield of a first PCR in a first chamber and a second PCR in a second chamber may be at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44,45, 46, 47, 48, 49, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more percent. The coefficient of variation of a yield of a first PCR in a first chamber and a second PCR in a second chamber may be at most about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13,12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less percent. The coefficient of variation of the yield of the first PCR reaction and the second PCR reaction may be from about 1 to about 95, about 1 to about 50, about 1 to about 30, about 5 to about 50, about 5 to about 30, about 10 to about 50, about 10 to about 30, or about 25 to about 50 percent. The coefficient of variation may be in an absence of a depletion of a reagent of the first reaction mixture or the second reaction mixture. For example, the first PCR or the second PCR may not be run to completion. In this example, the coefficient of variation can be achieved without overamplifying the first PCR or the second PCR. Additionally, not depleting the reagents of the first PCR or the second PCR may provide additional control over the yield of the first PCR or the second PCR. For example, by not running the first PCR or the second PCR to completion, an amount of PCR product can be selected for downstream applications (e g., next generation sequencing). Low coefficient ofAtty Dkt No.: 61197-702601 variation can be related to, for example, consistent conditions (e.g., reagent loading, chamber temperature, ambient temperature) between the first chamber and the second chamber, low volume change of the first reaction mixture and the second reaction mixture, consistent control of one or more thermal elements associated with the first chamber or the second chamber, or proper sealing of the substrate to a cover plate, or any combination thereof.

[0122] Prior to and / or during operation 3620, the chamber may be brought in contact with one or more cover plates. In some cases, the cover plate can be a lid of the present disclosure. The cover plate may be configured to reduce a volume change of the reaction mixture. For example, the cover plate can interface with the substrate to seal the chamber, heat the local environment around the chamber, or deflect air around the substrate, or any combination thereof. The bringing may comprise raising the substrate into contact with the cover plate. For example, the bringing may comprise lowering the cover plate into contact with the substrate. The bringing may comprise the use of one or more movable assemblies as described elsewhere herein. The contacting may comprise forming a seal. For example, the cover plate and the substrate may form a seal after being contacted. The seal may be a partial seal. For example, the seal may permit a flow of some gasses. The seal may be a hermetic seal. For example, the seal may not permit or substantially not permit a flow of gasses. The cover plate may comprise one or more metals as described elsewhere herein. The seal may comprise use of a film. For example, the film can be disposed between the cover plate and the substrate. The film may form a seal with the cover plate and / or the substrate. The film may comprise a plastic film or a metal film. The force between the cover plate and the substrate can hold the film in place (e g., to seal the chambers of the substrate). The force can be sufficient to seal the film (e.g., to reduce or prevent gas expansion within the chamber during a PCR). The film may form a gas tight (e.g., hermetic) seal on the substrate. For example, the film can provide a seal that seals a rection mixture into the chamber.

[0123] In some cases, the cover plate may be connected to a top plate. The top plate may provide an anchor for the cover plate to attach to. For example, the top plate can provide the connection point for the cover plate to the rest of the system. The top plate may be disposed above the substrate and / or the cover plate. The top plate may be fixed. For example, the top plate may not move during the operation of the system. The cover plate may be mounted to the top plate. The mounting may comprise one or more fasteners (e g., one or more screws). The one or more springs may be disposed between the cover plate and the top plate when the cover plate is mounted to the top plate. The one or more springs can adjust a contact force between the cover plate and the substrate. For example, when the cover plate is engaged with the substrate, theAtty Dkt No.: 61197-702601 presence of the one or more springs can prevent the contact force between the substrate and the cover plate from being too great (e.g., leading to possible damage) and / or too weak (e.g., leading to an improper seal). The one or more springs may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more springs. The one or more springs may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 spring. The one or more springs may comprise from about 1 to about 10 springs, about 2 to about 10 springs, about 4 to about 10 springs, about 6 to about 10 springs, about 8 to about 10 springs, about 2 to about 8 springs, about 4 to about 8 springs, or about 4 to about 6 springs. The one or more springs may comprise a number of springs in a range as defined by any two of the preceding values.

[0124] The one or more springs may comprise one or more calibrated springs. Each spring of the one or more springs may be a calibrated spring. A calibrated spring may provide a correct predetermined amount of force when the cover plate is engaged to the substrate, reducing errors associated with too strong or too weak of springs. The one or more calibrated springs may have a variation of spring force, length, diameter, wire gauge, and / or spring constant of at least about 0.01, 0.05, 0. 1, 0.5, 1, 2, 3, 4, 5, or more percent from one another. The one or more calibrated springs may have a variation of spring force, length, diameter, wire gauge, and / or spring constant of at most about 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less percent from one another. The one or more calibrated springs may have a variation of spring force, length, diameter, wire gauge, and / or spring constant in a range as defined by any two of the preceding values.

[0125] When the substrate and the cover plate are engaged, there may be a contact force between the cover plate and the substrate. The contact force may be selected to correctly seal the cover plate and the substrate while not being too great as to damage the cover plate, the substrate, or a thermal element disposed adjacent to the substrate. The contract force may be calibrated. For example, a force applied on the cover plate and / or substrate may be adjusted (e.g., by changing springs disposed between the top plate and the cover plate, by adjusting a travel distance of the cover plate and / or the substrate). The adjusting may comprise adjusting a force applied and / or a travel distance of the movable assembly. The contact force between the substate and the cover plate during operation 3620 may be at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more pounds. The contact force between the substrate and the cover plate may be at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less pounds. The contact force may be from about 5 to about 100, about 10 to about 90, about 20 to about 70, about 30 to about 80, about 30 to about 50, or about 50 to about 80 pounds. The contact force may have a variation over the cover plate of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, or more percent over the area of the cover plate. The contactAtty Dkt No.: 61197-702601 force may have a variation of at most about 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less percent over the area of the cover plate. The contact force may be adjusted by the one or more springs. For example, the one or more springs may prevent the contact force from being too great or too weak. The one or more springs may be configured to adjust the contacting force during the PCR. For example, during a heating operation the substrate can undergo creep, and the one or more springs can adjust the force in response to the creep. The contacting between the substrate and the cover plate with a given force can reduce thermal resistance between the cover plate and the substrate. The reduced thermal resistance can reduce a temperature difference between the cover plate and the substrate, improving control of the conditions of a chamber of the substrate.

[0126] The contact force may be applied at least in part by one or more movable assemblies. The one or more movable assemblies may be as described elsewhere herein. The bringing the substrate in contact with the cover plate may comprise moving the substrate. For example, the substrate may be moved to couple with the cover plate. The bringing may not comprise moving the cover plate. For example, the cover plate may stay in a fixed or substantially fixed position during the bringing the substrate in contact with the cover plate. The bringing the substrate in contact with the cover plate may comprise moving the substrate and / or the cover plate towards one another by at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, or more millimeters. The bringing the substrate in contact with the cover plate may comprise moving the substrate and / or the cover plate towards one another by at most about 500, 480, 460, 440, 420, 400, 380, 360, 340, 320, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less millimeters. The bringing the substrate in contact with the cover plate may comprise moving the substrate and / or the cover plate towards one another in a range as defined by any two of the preceding values. For example, the bringing the substrate in contact with the cover plate may comprise moving the substrate and / or the cover plate towards one another by about 1 to 500, 10 to 500, 50 to 500, 10 to 250, 50 to 250, or 100 to 300 millimeters. One or more limit switches as described elsewhere herein may be used to detect a movement of the substrate and / or the cover plate via the one or more movable assemblies.

[0127] When coupled to one another, a gap between the substrate and the cover plate may be at least about 0.5, 1, 2, 3, 4, 5, or more millimeters. When coupled to one another, a gap between the substrate and the cover plate may be at most about 5, 4, 3, 2, 1, 0.5, or less millimeters. A small gap between the cover plate and the substrate may reduce reaction mixture volume changes.Atty Dkt No.: 61197-702601

[0128] After the bringing the substrate in contact with the cover plate, an alignment between the chamber and the thermal element corresponding to the chamber may have an error. For example, the thermal element may be offset from the chamber once the substrate is in place in the system. The offset may cause a reduced thermal transfer between the thermal element and the chamber, which can reduce system efficiency. The alignment may be within an offset as described elsewhere herein.

[0129] In some cases, the cover plate may comprise one or more features. The one or more features may permit access (e g., physical access, optical access) to the chamber of the substrate via, for example, providing an access hole, providing a tube in fluid communication with the chamber, providing an optically transparent portion, or a combination thereof. For example, the one or more features can provide an opening in the cover plate to access the chamber. In some cases, the one or more features can change a volume of the headspace above the chamber. The one or more features may provide an additional volume to the chamber of the substrate. The one or more features may form a volume over the chamber in gaseous communication with the chamber. For example, a feature comprising a raised indentation above the chamber can increase a headspace of the chamber. In some cases, the features may permit optical access to the one or more chambers. For example, the features can comprise an optically transparent or translucent portion that can permit optical interrogation of the one or more chambers. In this example, an optical read head can illuminate and detect a fluorescent signal from a chamber through the one or more features. The one or more features may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 16, 20, 24, 25, 30, 35, 40, 45, 48, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 96, 100, or more features. The one or more features may comprise at most about 100, 96, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 48, 45, 40, 35, 30, 25, 24, 20, 16, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 feature. The one or more features may comprise a number of features in a range as defined by any two of the preceding values. For example, the one or more features may comprise from about 1 to about 100, about 5 to about 100, about 5 to about 75, about 10 to about 100, about 10 to about 75, about 10 to about 50, about or 20 to about 40 features. The one or more features may comprise one or more holes, grooves, indentations, or tubes, or any combination thereof. A feature of the one or more features may correspond to the chamber of the substrate. Each feature of the one or more features may correspond with a different chamber of a plurality of chambers of the substrate. For example, each chamber of the substrate can have a corresponding feature of the cover plate configured to permit access to the chamber when the cover plate is engaged with the substrate.Atty Dkt No.: 61197-702601

[0130] FIG. 33 shows an example of cover plate 3303 interfacing with a top plate 3309 and a substrate 3308, according to some embodiments. The cover plate 3303 can be affixed to the top plate 3309 using one or more fasteners 3301 (e.g., screws). The one or more springs 3302 can be disposed between the cover plate 3303 and the top plate 3309 as described elsewhere herein. The cover plate 3303 may comprise a thermal element 3304 (e g., a heater). The thermal element can heat and / or cool the cover plate as described elsewhere herein. One or more air deflectors 3305 may be disposed around the cover plate 3303. The one or more air deflectors can deflect air around the cover plate 3303 and the substrate 3308, thereby reducing a change in a thermal environment of the cover plate 3303 and the substrate 3308. The one or more air deflectors 3305 may comprise one or more features 3306. For example, the pin features can break up a flow of the air around the one or more deflectors 3305. Control circuitry 3307 may be configured to control one or more sensors as described elsewhere herein (e.g., FIGs. 35A and 35B).

[0131] In the example of FIG. 33, spring loaded mounting and directed airflow control can be used. In the scenario of spring loaded mounting, consistent temperatures can be ensured across the chambers of the substrate (e.g., 96 wells of a 96 well plate), condensation can be minimized, and sample evaporation can be reduced for reliable amplification. The cover plate (e g., heated lid) can be mounted using at least about 4, 6, or 8 calibrated springs to apply a uniform pressure and maintain a consistent contact across the cover surface. In the scenario of directed airflow control, one or more air deflectors and the cover plate can guide airflow around the chambers, improving efficiency and reducing condensation.

[0132] FIG. 34 shows an example of a cover plate 3404, according to some embodiments. The top plate 3401 may be affixed to the cover plate 3404 via one or more fasteners 3405. The one or more air deflectors 3402 can be configured to deflect air from the cover plate 3404 as described elsewhere herein. A lead 3403 may be configured to supply electricity to a thermal element disposed within the cover plate 3404. The lead 3403 may be connected to one or more sensors in the cover plate 3404 (e g., temperature sensors). Features 3406 may be configured to aid in alignment of the cover plate 3404.

[0133] The cover plate may comprise one or more thermal elements. The one or more thermal elements may be one or more thermal elements as described elsewhere herein. The one or more thermal elements may correspond with one or more chambers. For example, a chamber can have a corresponding thermal element to adjust a temperature of the cover plate near the chamber. In some cases, thermal element of the cover plate may correspond to the chamber. For example, the thermal element may correspond to a single chamber. In other cases, a thermal element of the cover plate can correspond with a plurality of chambers of the substrate. For example, the coverAtty Dkt No.: 61197-702601 plate can comprise a thermal element that heats or cools the cover plate adjacent to the plurality of chambers. In other cases, a thermal element may correspond to a portion of the chambers of the substrate, but less than all of the chambers of the substrate.

[0134] In some cases, by adjusting the temperature of the cover plate near the chamber, the local environment around the chamber can be closer to the conditions within the chamber during the PCR, which can reduce the temperature error in the system and increase the efficiency of the PCR. Additionally, the adjusting the temperature can reduce evaporation and / or condensation, improving reaction efficiency. Each chamber of a plurality of chambers of the substrate may have a corresponding thermal element of the one or more thermal elements of the cover. A chamber of a plurality of chambers of the substrate may have a corresponding thermal element of the cover plate. In some cases, the cover plate may comprise a single thermal element. For example, the cover plate can comprise a single heater that adjusts a temperature of the cover plate above all of the chambers of the substrate. In this example, the cover plate can reduce the change in the volume of a plurality of reaction mixtures in the plurality of chambers.

[0135] During the PCR, a temperature of the cover plate may be maintained with an accuracy of at least about 0.01 , 0.05, 0.1 , 0.5, 1 , 2, 3, 4, 5, or more °C. The temperature of the cover plate may be maintained with an accuracy of at most about 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less °C. The temperature of the cover plate may be maintained with an accuracy in a range as defined by any two of the preceding values. For example, the accuracy can be from about 0.01 to about 5, about 0. 1 to about 5, about 1 to about 5, about 0. 1 to about 1, or about 0. 1 to about 2 °C. The accuracy may be a difference between a set temperature and an actual temperature of the cover plate during the PCR. For example, if the temperature of the cover plate is set to 90 °C and the actual temperature of the cover plate is 91 °C, the accuracy can be 1 °C. During the PCR, the temperature of the cover plate may be maintained at a temperature of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, or more °C. The temperature of the cover plate may be maintained at a temperature of at most about 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less °C. The temperature of the cover plate may be maintained at a temperature in a range as defined by any two of the preceding values. For example, the temperature can be from 5 to 100, 10 to 100, 20 to 100, 30 to 100, 10 to 90, 20 to 90, 30 to 90, 25 to 95, 25 to 110, 25 to 120, 100 to 120, or 50 to 95 °C.

[0136] The cover plate may comprise one or more air deflectors. The one or more air deflectors may reduce a circulation of air around the cover plate and / or the substrate. The reduction in air circulation can reduce temperature changes of the cover plate and / or the substrate, which canAtty Dkt No.: 61197-702601 result in reduced volume changes, as well as increased accuracy and / or efficiency of the PCR. The cover plate may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more air deflectors. The cover plate may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 air deflector. The cover plate may comprise a number of air deflectors in a range as defined by any two of the preceding values. The one or more air deflectors may comprise one or more features. The one or more features may comprise one or more of pins, ridges, channels, or slits, or any combination thereof. The one or more features may comprise at least about 1, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more features. The one or more features may comprise at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 5, or fewer features. The one or more features may comprise a number of features in a range as defined by any two of the preceding values. The one or more features may be configured to deflect the flow of the air. For example, a ridge feature can block a flow of air, while a channel feature can deflect the flow of the air into the channel.

[0137] The one or more air deflectors may be used to deflect air around the cover plate and / or the substrate. By deflecting the air around the cover plate and / or the substrate, the local temperature environment of the cover plate and / or the substrate can be more stable. The deflecting can reduce a variation in a thermal condition (e g., temperature) of the cover plate, substrate, and / or chamber by at least about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 99, or more percent. The deflecting can reduce a variation in a thermal condition of the cover plate, substrate, and / or chamber by at most about 99, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less percent. The deflecting can reduce a variation in a thermal condition of the cover plate, substrate, and / or chamber by an amount in a range as defined by any two of the preceding values. The one or more air deflectors can reduce contact with ambient air (e g., deflect ambient air from making contact with the substrate). This can reduce thermal loss from the substrate to the surrounding air, in turn reducing condensation on the substrate and reducing edge effects of chambers near an edge of the substrate.

[0138] In another operation 3630, the method 3600 may comprise, during the PCR, detecting one or more signals or signal changes from the reaction mixture. The detecting may comprise use of one or more optical systems as described elsewhere herein. For example, the detecting may comprise acquiring a fluorescent signal associated with the analyte or a PCR product thereof. The one or more signals or signal changes may be as described elsewhere herein.

[0139] In another operation 3640, the method 3600 may comprise determining the one or more properties of the analyte based at least in part on the one or more signals or signal changes. The determining may comprise comparing a signal intensity to the threshold (e g., relative threshold,Atty Dkt No.: 61197-702601 absolute value), fit to a predetermined function (e g., fit to a theoretical PCR curve), or using a comparison of a derivative of the signal intensity to a predetermined derivative value, or any combination thereof. The one or more properties of the analyte may comprise a presence of the analyte, an absence of the analyte, an identity of the analyte, a sequence of the analyte, a mutation status of the analyte, an identity of the sample the analyte was derived from, an identity of a subject the sample was derived from, or a presence or absence of a disease state in the sample and / or a subject the sample was derived from, or any combination thereof.

[0140] The one or more properties of the analyte may be determined with an accuracy, sensitivity, or specificity of at least about 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.5, 99.9, or more. The one or more properties of the analyte may be determined with an accuracy, sensitivity, or specificity of at most about 99.9, 99.5, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, or less. The one or more properties of the analyte may be determined with an accuracy, sensitivity, or specificity in a range as defined by any two of the preceding values. The determining may be performed in a time period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 70, 80, 90, 100, 110, 120, or more minutes. The determining may be performed in a time period of at most about 120, 110, 100, 90, 80, 70, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41 , 40, 39, 38, 37, 36, 35, 34, 33, 32, 31 , 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less minutes. The determining may be performed in a time period in a range a defined by any two of the preceding values. For example, the determining can be performed in a time from about 1 to about 120, about 10 to about 120, about 30 to about 130, about 60 to about 120, about 1 to about 60, about 10 to about 60, about 30 to about 60, or about 15 to about 45 minutes.

[0141] In another aspect, the present disclosure provides a device. The device may comprise a plurality of chambers configured to contain one or more samples. The device may comprise a plurality of elements disposed adjacent to the plurality of chambers. The plurality of elements may be configured to affect at least one condition within each chamber of the plurality of chambers. The device may comprise at least one additional element disposed adjacent to one or more elements of said plurality of elements. The additional element may be configured to affect at least one condition on the plurality of elements. The additional element may be, for example, an electrical control system as described elsewhere herein, a thermal element as described elsewhere herein, or a heatsink as described elsewhere herein. The device may comprise at least one optical system configured to measure a presence or absence of a signal from each chamberAtty Dkt No.: 61197-702601 of the plurality of chambers. The at least one optical system may be configured to measure a presence or absence of the signal from a subset of the plurality of chambers.

[0142] The device may comprise at least about 1, 2, 3, 4, 5, 10, 25, 50, 75, 96, 100, 150, 200, 250, 300, 350, 384, 400, 450, 500, or more chambers. The device may comprise at most about 500, 450, 400, 384, 350, 300, 250, 200, 150, 100, 96, 75, 50, 25, 10, 5, 4, 3, 2, or fewer chambers. The device may comprise a number of chambers as defined by any two of the proceeding values. A chamber may be configured to contain a volume of at least about 0.1, 0.5, 1, 5, 10, 50, 100, 500, 1,000, 5,000, 10,000, or more microliters. A chamber may be configured to contain a volume of at most about 10,000, 5,000, 1,000, 500, 100, 50, 10, 5, 1, 0.5, 0.1, or less microliters. Each chamber of the plurality of chambers may be of a same size. For example, an array of 96 chambers each with the same volume can be used. A subset of the plurality of chambers may be a same size. The chambers of the plurality of chambers may be different sizes. For example, a device can comprise a plurality of smaller chambers and a plurality of larger chambers. The plurality of chambers may in a 1 -dimensional, 2-dimensional, or 3-dimensional array. For example, the plurality of chambers may be configured similar to a 2-dimensional 96 well plate. In some cases, the plurality of chambers can be organized in a strip. For example, a substrate as described elsewhere herein can be a strip. The plurality of chambers may be configured as a series of individual tubes. In some cases, the chamber is at least a portion of a flow cell. For example, a flow cell can be configured such that a portion of the flow cell is adjacent to the element as described elsewhere herein. The chamber may be at least a portion of a chip. For example, a chip can comprise a plurality of chambers.

[0143] The plurality of chambers and / or a plurality of thermal elements may be disposed in an area of at least about 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6,3.7, 3.8, 3.9, 4.0, 4.1, 4.2, 4.3, 4.4, 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, or more square millimeters. The plurality of chambers and / or a plurality of thermal elements may be disposed in an area of at most about 5.0, 4.9, 4.8, 4.7, 4.6, 4.5, 4.4, 4.3, 4.2, 4.1, 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0,0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, or fewer square millimeters. The plurality of chambers and / or a plurality of thermal elements may be disposed in an area in a range as defined by any two of the preceding values.

[0144] The plurality of chambers may be configured to each contain at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more samples. For example, a single chamber can be configured with dividers to contain 2 samples within the chamber. The plurality of chambers may be configured to eachAtty Dkt No.: 61197-702601 contain at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 sample. The plurality of chambers may be collectively configured to contain at least about 1, 5, 10, 25, 50, 75, 100, or more samples. For example, an array of 96 chambers can be configured to contain 96 samples. The plurality of chambers may be collectively configured to contain at most about 100, 75, 50, 25, 10, 5, or less samples. Each chamber of the plurality of chambers may be configured to contain a different sample. A subset of the plurality of chambers may be configured to contain a different sample.

[0145] A chamber may be in the form of a well. For example, the chamber can be a cylinder with one open and one closed end. A chamber may comprise one or more of polymers (e g., plastics, polyethylene, polytetrafluoroethylene, etc.), metals (e.g., pure metals, alloys, etc.), oxides (e g., glasses, insulative oxides, etc ), semiconductors (e g., silicon, etc.), or the like, or any combination thereof. A chamber may be configured to not react with a material (e g., a sample, a reagent, etc.) deposited within the chamber. For example, an iron chamber can be lined with stainless steel to reduce a reaction with a water solvent. In another example, a polyethylene chamber can be lined with polytetrafluoroethylene to protect the chamber for harsh reagents.

[0146] The device may comprise a plurality of elements disposed adjacent to the plurality of chambers. The plurality of elements may be in contact with the plurality of chambers. The plurality of elements may be in thermal contact with the plurality of chambers. For example, the plurality of elements can be connected to the plurality of chambers via a plurality of thermal transfer pads.

[0147] The plurality of elements may be configured to affect at least one condition within each chamber of the plurality of chambers. The at least one condition may be a temperature, a magnetic field, an optical condition, or the like, or any combination thereof. The condition may be a condition of an individual chamber. For example, the temperature of a first chamber can be held at a different temperature from a second chamber. Each chamber of the plurality of chambers may be maintained at a temperature of at least about 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more degrees Celsius from a set temperature for each chamber. Each chamber of the plurality of chambers may be maintained at a temperature of at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less degrees Celsius from a set temperature for each chamber. Each chamber of the plurality of chambers may be maintained at a temperature range as defined by any two of the proceeding values. For example, each chamber can be maintained at a temperature in a range from 0.5 to 1 degree Celsius from a set point of each chamber. A portion of the plurality of chambers may be maintained at the temperature. Each set point may be the same for eachAtty Dkt No.: 61197-702601 chamber. For example, all chambers can be maintained at 50 degrees Celsius. Different chambers may have different set points. For example, a first set of chambers can be maintained at 95 degrees Celsius while a second set of chambers can be maintained at -15 degrees Celsius. The device may be configured to temperature cycle a first subset of the plurality of chambers while maintaining a second subset of the plurality of chambers at a constant temperature. For example, a PCR reaction can be performed in a plurality of chambers. In this example, once a fluorescence signal from a chamber reaches a predetermined level, the PCR reaction in that chamber can be halted by maintaining the chamber at a low temperature. In this example, other chambers can continue to be cycled to continue the PCR reactions occurring in those chambers.

[0148] The device may be configured to generate a condition gradient across the plurality of chambers. For example, the device can be configured to generate a temperature gradient across the plurality of chambers. In this example, the optimal temperature for a reaction can be determined by investigating the reaction progress across the temperature gradient. In another example, gradient of reagent concentrations can be generated. A melt-curve analysis may be performed by the device across the plurality of chambers.

[0149] The plurality of elements may comprise a plurality of heating and / or cooling elements (e.g., thermal elements). The heating and / or cooling elements may be separate elements. For example, a heating element and a cooling element can be adjacent to one another in contact with a chamber. The heating and / or cooling elements may be a same element. For example, a combined heating and cooling element can be disposed adjacent to the chamber. In this example, a thermoelectric element can be used as both a heating element and a cooling element. The heating element may comprise a resistive heater, an inductive heater, a thermoelectric heater, or the like, or any combination thereof. The cooling element may comprise an evaporative cooler, a compressive cooler, a thermoelectric cooler, or the like, or any combination thereof.

[0150] The at least one additional element may be disposed adjacent to one or more elements of the plurality of elements. The at least one additional element may be disposed adjacent to all of the plurality of elements. For example, the plurality of elements can each be in contact with the additional element. A portion of the plurality of elements can be in contact with the additional element. The additional element may comprise one or more additional heating and / or cooling elements. The one or more additional heating and / or cooling elements may be as described elsewhere herein. For example, the additional element may comprise a large thermoelectric element in thermal communication with a plurality of thermoelectric elements. The at least one condition on the plurality of elements may be a condition as described elsewhere herein. For example, the at least one additional element can be configured to affect a temperature of theAtty Dkt No.: 61197-702601 plurality of elements. In this example, a thermoelectric cooler can be used to reduce the hot side temperature and thereby increase the efficiency of a plurality of thermoelectric elements which in turn can individually control the temperature of the chambers. The at least one additional element may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more additional elements. The at least one additional element may comprise at most about 10, 9, 8, 7, 6, 5, 4, 2, or less additional elements.

[0151] The optical system may comprise one or more excitation sources. Examples of excitation sources include, but are not limited to, a laser (e.g., a single wavelength laser, a supercontinuum laser, etc.), an incoherent light source (e.g., a light emitting diode, an incandescent light source, etc.), or the like, or any combination thereof. In some cases, the light source can be a blue laser (e.g., with an emission maximum of at most about 460, 470, 480, 490, or more nanometers). The optical system may comprise one or more detectors. Examples of detectors include, but are not limited to, a zero-dimensional (0D) detectors (e.g., a photodiode), a silicon photomultiplier (SiPM), a one-dimensional (ID) detectors (e.g., a strip detector), a two-dimensional (2D) detectors (e.g., an array detector), a film detector (e.g., a detector using silver halide crystals on a film), a phosphor plate detector (e.g, a plate of downshifting or down-converting phosphor), a semiconductor detector (e.g., a semiconductor charge coupled device (CCD), a complementary metal oxide semiconductor (CMOS) device), or the like, or any combination thereof. The optical system may be configured to measure the presence or absence of the signal from each chamber of the plurality of chambers individually. For example, the optical system can collectively excite the plurality of chambers and individually read the signal from each chamber. In another example, the optical system can individually excite and read the signals from each chamber. The optical system can have a dynamic range of detection of at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more orders of magnitude. The optical system can have a dynamic range of detection of at most about 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, or less orders of magnitude. The optical system may have a sensitivity to detect at least about 1, 10, 100, 1,000, 10,000, 100,000, or more analyte molecules. The optical system may have a sensitivity to detect at most about 100,000, 10,000, 1,000, 100, 10, or 1 molecule. For example, the optical system can have single molecule detection sensitivity. The detectors, optics, and excitation sources selected can improve the sensitivity of the optical system.

[0152] The plurality of elements may comprise a plurality of thermoelectric heating, thermoelectric cooling, or combined thermoelectric heating and cooling elements. For example, a thermoelectric heating element can become a thermoelectric cooling element with a reversal of a current polarity across the thermoelectric element. The at least one additional element mayAtty Dkt No.: 61197-702601 comprise at least one thermoelectric heater, thermoelectric cooler, or combined thermoelectric heater and cooler. The device may comprise one or more heat pipes configured to reduce thermal crosstalk between the plurality of chambers. For example, the heat pipes can be placed in between the chambers to improve thermal isolation between the chambers. The heat pipes may be configured to, or be in thermal contact with an element configured to, dissipate excess heat and / or add heat to remove a heat deficit.

[0153] In another aspect, the present disclosure provides a method for performing a polymerase chain reaction (PCR) analysis of a sample containing or suspected of containing an analyte. The method may comprise providing the sample to a first chamber of a device comprising one or more chambers configured to contain the sample. The one or more chambers may be configured to have individually controllable conditions. A PCR reaction may be initiated using the sample. The PCR reaction may generate a signal indicative of a presence or absence of the analyte. The PCR reaction may be monitored using the signal. The PCR reaction may be stopped after a predetermined amount of signal is generated by the PCR reaction within the chamber.

[0154] FIG. l is a flow chart of a method 100 for performing a polymerase chain reaction (PCR) analysis of a sample or reaction mixture containing or suspected of containing an analyte, according to an embodiment of the present disclosure. The method may comprise providing the sample to a chamber of a substrate (110). The location may have a volume configured to retain the sample and permit the sample to be subjected to a condition for the PCR analysis. The device may be as described elsewhere herein. For example, the one or more chambers may be configured to have individually controllable conditions. In another example, the signal can comprise an optical signal.

[0155] A sample may be a material that may comprise an analyte. The sample may be suspected of comprising the analyte. A sample may be solid matter (e g., biological tissue) or may be a fluid (e g., a biological fluid). In general, a biological fluid can include any fluid associated with living organisms. Non-limiting examples of a samples include blood (or components of blood — e g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, breath, bone marrow, stool, semen, vaginal fluid, interstitial fluids derived from tumorous tissue, breast, pancreas, cerebral spinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, cavity fluids, sputum, pus, micropiota, meconium, breast milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric and digestive fluid, tears, ocular fluids, sweat, mucus, earwax, oil, glandular secretions, spinal fluid, hair, fingernails, skin cells, plasma,Atty Dkt No.: 61197-702601 nasal swab or nasopharyngeal wash, spinal fluid, cord blood, emphatic fluids, and / or other excretions or body tissues. A sample may be a cell-free sample. Such cell-free sample may include DNA and / or RNA. The analyte may be a nucleic acid molecule, a protein (e.g., an antibody), an antigen, a chemical (e.g., a toxin), a metal ion (e g., a heavy metal ion), or the like. For example, the analyte can be a nucleic acid molecule of a virus.

[0156] In some cases, a sample may be processed prior to use in the methods and systems of the present disclosure. Examples of processing include, but are not limited to, maceration, sonication, cell lysis, nucleic acid extraction, protein extraction, lipid extraction, enrichment (e.g., affinity enrichment, magnetic enrichment), purification, transcription, reverse transcription, or dilution, or any combination thereof. In some cases, a sample may be used in the methods and systems of the present disclosure without additional processing. For example, a blood sample can be mixed with reactants and analyzed in an absence of additional processing.

[0157] The method 100 may comprise using a first element (e g., a first thermal element) disposed adjacent to the location (e.g., chamber) to affect the condition in the location to thereby initiate the PCR reaction using the sample or reaction mixture (120). The methods of the present disclosure can comprise using the first element disposed adjacent to the chamber to affect the conditions in the chamber. The first element may be used to affect the conditions of a chamber to initiate and / or sustain a PCR reaction on a reaction mixture. The PCR reaction may generate a signal or signal change which is indicative of a property (e g., a presence, absence, concentration) of the analyte. The PCR reaction may generate a signal indicative of a presence or absence of the analyte. The PCR reaction may comprise Allele-specific PCR, Assembly PCR, Polymerase Cycling Assembly (PCA), Asymmetric PCR, Convective PCR, Dial-out PCR, Digital PCR (dPCR), Helicase-dependent amplification, Hot start PCR, In silico PCR, Intersequence-specific PCR, Inverse PCR, Ligation-mediated PCR, Methylation-specific PCR, Miniprimer PCR, Multiplex ligation-dependent probe amplification, MLP A, Multiplex-PCR, Nanoparticle- Assisted PCR (nanoPCR), Nested PCR, Overlap-extension PCR, Splicing by overlap extension (SOEing) , PAN-AC, quantitative PCR, Quantitative PCR, real-time, Reverse Complement PCR, Reverse Transcription PCR (RT-PCR), Rapid Amplification of cDNA Ends, RNase H-dependent PCR, Single Specific Primer-PCR, Solid Phase PCR, Suicide PCR, Thermal asymmetric interlaced PCR (TAIL-PCR), Touchdown PCR, Step-down PCR, Universal Fast Walking, or the like. In addition to PCR, the methods and systems of the present disclosure can perform qualitative analysis of PCR amplicons (e.g., melt curve analysis). The methods and systems of the present disclosure may perform one or more thermal shift assays. The signal may comprise an optical signal, an electrical signal, a physical signal, or the like, or any combinationAtty Dkt No.: 61197-702601 thereof. The optical signal may comprise an absorption signal (e.g., an absorption intensity, an absorption peak wavelength), a fluorescence signal (e.g., a fluorescence intensity, a fluorescence wavelength, a fluorescence lifetime), a plasmonic property, or the like, or any combination thereof. The electrical signal may comprise resistance, impedance, capacitance, or the like, a change thereof, or any combination thereof. The physical signal may comprise a physical state of the sample. For example, a physical signal can be a melting point of the sample.

[0158] The method 100 and methods of the present disclosure may comprise using a second element operably coupled to the first element to adjust an operating condition of the first element at least partially in response to the signal or signal change, to thereby affect the condition in the location (130) or the chamber. A second element may be operably coupled to the first element to adjust an operating condition of the first element at least partially in response to the signal or signal changes, to thereby affect the condition of the chamber. In some cases, the second element can comprise control circuitry for the first element, an additional thermal element, a sensor, or a thermally conductive element, or any combination thereof. Since the signal can be dependent on the amount of analyte within the sample, the device can use the amount of signal as a proxy for how far the reaction has progressed. As such, instead of using an arbitrary number of cycles to determine the reaction progress, the signal can instead be used as a quantitative measure. The monitoring may comprise real-time monitoring (e g., reading the signal in real-time as the reaction progresses), fixed-interval monitoring (e.g., reading the signal at predetermined times), or the like, or any combination thereof. The method 100 may comprise adjusting the operating condition of the first element to stop the PCR reaction upon said signal or signal change reaching a predetermined threshold (140). The methods of the present disclosure may comprise adjusting the operating condition of the first element to decrease a rate of the PCR reaction upon the one or more signal or signal changes reaching a predetermined threshold. For example, once a predetermined amount of fluorescent signal is generated in a chamber, the PCR reaction can be stopped by reducing the temperature. Examples of operating conditions include, but are not limited to, temperature, agitation (e.g., stirring, shaking, etc.), presence or absence of light, presence or absence of chemical compounds (e.g., additional reagents, gasses, etc.), or the like, or any combination thereof. For example, an operating condition can be a temperature of the reaction, and a change in the operating condition can be a change in the temperature. In another example, an operating condition can be a presence of light energy, and the change in the operating condition can be turning off the light.

[0159] A second chamber of the device may comprise a second sample. For example, the first chamber can contain a sample from a first subject, and the second chamber can comprise aAtty Dkt No.: 61197-702601 sample from a second subject. In another example, the first chamber can contain a first sample from a subject, and the second chamber can contain a second sample from the same subject. The second chamber may continue a second PCR reaction within the second chamber after the stopping of the PCR reaction in the first chamber. The conditions within the second chamber may be controlled separately from the conditions in the first chamber. As such, the reaction in the second chamber can be independent from the reaction in the first chamber. Since the reactions can be independently controlled and monitored, once a reaction is determined to be complete, it can be halted without impacting adjacent reactions. For example, when a reaction reaches a predetermined amount of signal indicative of a presence of an analyte, the reaction can be halted and kept under appropriate conditions to later retrieve the products of the reaction.

[0160] The method 100 may not comprise pre-normalization. In some cases, the methods and systems of the present disclosure may not use pre-normalization. For example, a reaction mixture may not be pre-normalized. The lack of pre-normalization may mean that normalization or quantification operations may not be performed prior to the methods of the present disclosure. For example, an amount of nucleic acid in a plurality of samples may not be normalized to a nominal value prior to quantification. By individually addressing different PCR reactions, the reaction parameters can be adjusted to perform the reaction to achieve a same output (e.g., same output amount of product, same level of signal or signal change) from reaction mixtures that have different amounts of analyte. By tuning the parameters and adjusting a number of cycles during each PCR, pre-normalization can be avoided. The method can be performed on a sample that does not comprise an internal standard. The method can be performed on a sample without quantifying a non-analyte target within the sample. For example, the method can be performed to quantify a viral nucleic acid analyte without also amplifying a human gene control. The method 100 may not comprise pre-quantification. The method may not comprise determining an amount of analyte within the sample. For example, the method can be performed on a sample with an unknown amount of nucleic acid molecules present in the sample. By performing the method on a sample that is not normalized and / or pre-quantified, the time taken to process the sample can be reduced. Additionally, the complexity and cost associated with processing the sample can be reduced as well. This may be contrary to other sample analysis techniques, which may require pre-normalization or pre-quantification to maintain high accuracy. The method 100 may maintain a high accuracy, sensitivity, and / or specificity without these operations. The one or more properties (e.g., presence, absence, concentration) of the analyte may be determined with a accuracy, sensitivity, and / or specificity of at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9% or more.Atty Dkt No.: 61197-702601The one or more properties of the analyte may be determined at an accuracy, sensitivity, and / or specificity of at most about 99.9%, 99.5%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 85%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, or less. In some cases, the PCR can be performed with an auto-normalization procedure. The auto-normalization procedure can normalize the amount of analyte (e.g., nucleic acid) in the reaction mixture. For example, a PCR can be performed to normalize an amount of nucleic acid in the reaction mixture to a predetermined value. The auto-normalization may occur without an input from a user. For example, the user can set up a threshold value, and the PCR can be performed until an amount of analyte within the reaction mixture reaches the threshold value. Examples of threshold values include, but are not limited to, an amount of signal or signal change related to the analyte, a concentration of the analyte within the reaction mixture, a yield of the PCR, or an amount of analyte within the reaction mixture, or any combination thereof.

[0161] A plurality of reaction conditions across the substrate can be in a gradient. For example, the temperatures of a plurality of chambers of a substrate can be arranged to provide a gradient of temperatures across the substrate. The plurality of reaction conditions across the substrate can be discrete reaction conditions. For example, the reaction conditions can be independently selected and / or assigned to the chambers of the substrate. In this example, the plurality of reaction conditions can be assigned according to, for example, a reaction profde, a multivariable experiment design, or a combination thereof. The discrete reaction conditions may be discontinuous reaction conditions. For example, the reaction conditions may not be along a continuous curve across the substrate. In this example, the reaction conditions can be selected to provide a different profde of the various reaction conditions (e.g., a step profde, a plurality of unrelated reaction conditions).

[0162] A sample of the present disclosure can be derived from a subject. A subject may be an animal, such as a mammal. A subject may be a human or non-human mammal. A subject may be a plant. A subject may be afflicted with a disease or suspected of being afflicted with or having a disease. The subject may not be suspected of being afflicted with or having the disease. The subject may be symptomatic. Alternatively, the subject may be asymptomatic. In some cases, the subject may be treated to alleviate the symptoms of the disease or cure the subject of the disease. A subject may be a patient undergoing treatment by a healthcare provider, such as a doctor. The subject may be a healthcare provider. The subject may be a student, a teacher, a long-term caregiver (e.g., a nursing home employee), a prison guard, or others who work and / or live in close proximity to others. Examples of diseases include, but are not limited to, acquired immunodeficiency syndrome, tuberculosis, hepatitis b, hepatitis c, human papillomavirus,Atty Dkt No.: 61197-702601 influenza, streptococcal infections (e.g., group a streptococcus), neisseria gonorrhoeae, chlamydia trachomatis, covid-19, dengue fever, zika virus, west nile virus, ebola virus disease, lyme disease, syphilis, pneumocystis pneumonia, cytomegalovirus infection, herpes simplex virus, respiratory syncytial virus, norovirus, Clostridium difficile infection, aspergillus, Candida infections, mycobacterium avium complex, toxoplasmosis, brucellosis, viral hemorrhagic fevers, sickle cell disease, cystic fibrosis, or cancer.

[0163] FIG. 2 is a schematic of a device 200, according to an embodiment. The device 200 may comprise one or more chambers 210. In this example, the chambers can be wells configured to be fluidically accessible from the top of the chamber. The chamber can additionally be configured to be optically accessible. For example, a camera positioned facing the top of the chamber can read an optical signal originating from the chamber. Each chamber may comprise insulation 220. The insulation may be configured to decrease thermal transfer between the chambers of the plurality of chambers. For example, when a first chamber is under heating conditions and a second chamber is under cooling conditions, the insulation can reduce the heat that leaks from the heated chamber into the cooled chamber, improving efficiency and accuracy. The insulation 220 may comprise one or more of a low thermal conductivity materials. The insulation may comprise mineral wools (e.g., fiberglass), natural fibers (e.g., cellulose), polymers (e.g., polystyrene, polyurethane, etc.), or any combination thereof. The insulation may comprise one or more heat pipes. The one or more heat pipes may be configured to provide transport of heat to or from the chambers. For example, the heat pipe can remove excess heat away from the chambers. The heat pipe may comprise a solid metal pipe (e.g., a copper wire), a phase change heat pipe (e.g., a copper tube filled with a phase change cooler), or the like, or any combination thereof. In some cases, each chamber can have an associated temperature reader (e.g., thermometer, thermocouple, etc ). The temperature reader can be configured to read a temperature of the chamber and provide the temperature to a central controller. This can enable individual, real-time control of the temperature of each chamber of the plurality of chambers.

[0164] The device may comprise one or more elements 230. The device may comprise one element for each chamber of the device as shown in FIG. 2. The device may comprise one element to at least a portion of the plurality of chambers. The device may comprise a plurality of elements for each chamber. The elements may be as described elsewhere herein. For example, the elements may be thermoelectric elements. The thermoelectric elements may be configured to individually control the temperature within each chamber of the device. For example, a thermoelectric element positioned below a chamber can heat and cool that chamber. The thermoelectric elements may be connected to an optional thermoelectric back plate 240Atty Dkt No.: 61197-702601 comprising an optional heater 270. The thermoelectric back plate may be configured to equalize the temperature for the elements 230 to improve efficiency. For example, if the chambers are being heated, the backs of the thermoelectric elements can get cold. In this example, the heater 270 can provide heat to the thermoelectric back plate to regulate the back side temperatures of the thermoelectric elements. The thermoelectric back plate can be in thermal contact with a heatsink 250 via an optional graphite pad 280. The graphite pad may be configured to aid in heat conduction between the thermoelectric back plate and the heatsink. For example, the graphite pad can fill defects in the thermoelectric back plate and the heatsink to provide a better thermal conduction. The heatsink may comprise a metal heatsink (e g., copper, aluminum, etc.), a polymer heatsink, a graphite heatsink, or the like, or any combination thereof. The heatsink may be cooled by a fan 260. The fan can circulate air around the heatsink to aid in the dissipation of heat from the device. In some cases, a plurality of thermal elements can share a same heatsink. For example, the heatsink can be configured to be in thermal contact with the plurality of thermal elements, thereby providing a thermal path for the thermal elements to discharge excess heat or cold.

[0165] The heatsink may have an area of at least about 0.0001, 0.001 , 0.01, 0.1, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25 or more square centimeters. The heatsink may have an area of at most about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 , 0.1 , 0.01 , 0.001 , 0.0001 , or fewer square centimeters. The heatsink may have a thickness of at least about 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more centimeters. The heatsink may have a thickness of at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, or less centimeters.

[0166] In some cases, the heatsink can comprise one or more features as described elsewhere herein. The heatsink can comprise the one or more features disposed on a reverse side of the heatsink from the substrate. In some cases, the heatsink can comprise a pillar design. FIGs. 15A- 15C are examples of a pillar design, according to some embodiments. The pillar design may be configured to facilitate heat transfer between the thermoelectric elements and a larger heatsink (e.g., a plurality of fins). The pillar design may be configured to reduce crosstalk between the individual chambers of the device. For example, the pillars can be configured to deflect heat through the pillars, but the space between the pillars can reduce crosstalk between the pillars. A chamber may be in thermal contact with a pillar. A pillar may be in contact with a portion of the plurality of chambers. Each chamber may be in thermal contact with a different pillar. In some cases, each pillar may not have a direct connection between the other pillars. In some cases, there may not be a direct connection between each of the chambers. For example, the chambers mayAtty Dkt No.: 61197-702601 be in communication (e.g., physical contact) with a thermally insulating material, but not in contact with a thermally conductive material placed between the chambers. In FIG. 15A, the device 1500 may comprise one or more elements 1510 (e.g., thermoelectric elements as described elsewhere herein) and one or more pillars (e g., insulating pillars) 1520. The pillars can be configured to thermally isolate the plurality of chambers while enabling movement of heat as described elsewhere herein. The device may further comprise one or more temperature sensors 1530 as described elsewhere herein. A retaining clip 1540 can be configured to retain the plurality of elements, while a retaining plate 1550 can be configured to retain the plurality of chambers.

[0167] FIG. 3 is a schematic of a device 300, according to an embodiment. The device 300 may be similar to the device 200 of FIG. 2 but for the addition of an additional element 310 disposed adjacent to the back plate 240 (e.g., thermoelectric back plate, heatsink, thermal conductor). In some cases, a plurality of back plates can be in operable communication with a plurality of chambers or thermal elements. The backplate may coupled (e g., affixed) to the additional element as described elsewhere herein. The backplate may be held in contact with the additional element by one or more fasteners. The backplate may be disposed in a holder. For example, the backplate can be held in place by grooves of a holder, and the additional element can be held in place by the holder. The backplate may have a maximum dimension of at least about 0.01, 0.05, 0.1, 0.5, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1, 12, or more centimeters. The backplate may have a maximum dimension of at most about 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less centimeters. The backplate may have a maximum dimension from about 0.01 to about 12, about 1 to about 10, about 6 to about 12, or about 1 to about 5 centimeters.

[0168] The additional element may be one or more additional thermoelectric elements, heatsinks, control circuitries, thermally conductive elements, or thermal sink, or thermal insulation elements, or any combination thereof. The one or more additional elements can be configured to control a condition (e.g., a temperature) of the one or more elements. For example, a second thermoelectric cooler can cool the hot sides of a plurality of thermoelectric coolers. Including the one or more additional elements can improve the performance of the one or more elements by removing waste heat from the one or more elements. Additionally, such additional elements can provide a dynamic control of the conditions for the one or more elements. For example, a first thermoelectric element can switch between heating and cooling a chamber. In this example, an additional thermoelectric element can switch between cooling and heating the first thermoelectric element. The additional element may be associated with a chamber. The additional element may be associated with a plurality of chambers. The additional element mayAtty Dkt No.: 61197-702601 be associated with a thermal element. The additional element may be associated with a plurality of thermal elements.

[0169] In another aspect, the present disclosure can provide a method for performing a PCR analysis of a sample containing or suspected of containing an analyte. The sample may be provided to a location on a substrate. The location can be operably coupled to a thermally conductive element disposed in at least a portion of a support. The location can have a volume sufficient to retain the sample. A thermal element can be disposed adjacent to the location to initiate the PCR reaction using the sample. The PCR reaction can generate one or more signals or signal changes which is indicative of a property associated with the analyte. The thermal element may be in thermal contact with the thermally conductive element.

[0170] FIG. 25 is a flow chart of a method 2500 of performing a PCR analysis of a sample containing or suspected of containing an analyte, according to some embodiments. In an operation 2510, the method may comprise providing the sample to a location on a support. The support can comprise a thermally conductive element disposed in at least a portion of the support. The location may have a volume sufficient to retain the sample.

[0171] The thermal element may be a thermoelectric element, resistive element, inductive element, light based heating element (e.g., microwave element, infrared element, etc.), or the like, as described elsewhere herein. In an example, a resistive element can be disposed adjacent to a well such that the resistive element provides heat to the well. In another example, a light based heating element can direct microwave radiation into a well, thereby heating the well. In another example, the inductive element can be positioned adjacent to a magnetically active element disposed in a well configured to receive energy from the inductive element and transmit the energy as heat in the well. The thermal element may be configured to adjust a temperature of the location. For example, the thermal element can be utilized to raise, lower, or maintain the temperature of the location depending on a predetermined temperature sequence. The thermoelectric element may have a maximum width of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more millimeters. The thermoelectric element may have a maximum width of at most about 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or less millimeters. The thermoelectric element may have a maximum length of at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 25, 30, or more millimeters. The thermoelectric element may have a maximum length of at most about 30, 25, 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, or less millimeters. The thermoelectric element may have a maximum dimension in a range as defined by any two of the preceding values. For example, the thermoelectric element may have a maximum dimension from about 1 to about 8 millimeters.Atty Dkt No.: 61197-702601The thermal element may be an element configured to adjust a temperature within the location. For example, the thermal element can be configured to heat the location to enable PCR as described elsewhere herein. A thermal element and a second thermal element may have a separation of at least about 0.01, 0.05, 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, or more millimeters. The thermal element and the second thermal element may have a separation of at most about 10, 9.5, 9, 8.5, 8, 7.5, 7, 6.5, 6, 5.5, 5, 4.5, 4, 3.5, 3, 2.5, 2, 1 .5, 1, 0.5, 0.1, 0.05, 0.01, or less millimeters. The thermal element and the second thermal element may have a separation from about 0.01 to about 10, about 0.01 and about 5, about 0.01 and about 1, about 0.1 and about 10, about 0.1 and about 5, about 0.1 to about 1, or about 0.1 and about 0.5 millimeters.

[0172] In some cases, the support is a printed circuit board. Examples of printed circuit boards include, but are not limited to, metal printed circuit boards, multi-layer metal printed circuit boards (e.g., comprising a plurality of metal layers within the circuit board), or the like, or any combination thereof. In some cases, the support is an engraved circuit board. For example, the support can be a circuit board engraved to generate the traces of the circuit board.

[0173] The at least a portion of the support the thermally conductive element is disposed in may comprise at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 99, or 100 percent of the support. For example, the thermally conductive element may be disposed through the entire support.

[0174] In some cases, the chamber and an additional chamber of the substrate can share a thermal element. In some cases, a chamber of the substrate can be associated with a first thermal element and an additional chamber of the substrate can be associated with a second thermal element. For example, the chamber and the additional chamber may not share a thermal element. In this way, the chamber and the additional chamber may have individually addressable temperatures. For example, the first thermal element can affect a temperature of the chamber while the second thermal element can affect a temperature of the additional chamber. The use of individual thermal elements may enable independent reactions to occur in the different chambers. The control circuitry may provide for individual electrical addressing of the chamber. For example, the control circuitry can individually address a thermal element in thermal communication with the chamber. The sensor may provide for individually addressable sensing of the chambers. For example, having the sensor detect a signal from the chamber can provide individually addressable sensing. In some cases, the chamber and an additional chamber may not share a sensor. For example, each chamber may have its own sensor.

[0175] In some cases, the chamber and the second chamber of the substrate can share the thermal element. For example, a plurality of chambers can be associated with a single thermalAtty Dkt No.: 61197-702601 element. In this example, the plurality of chambers can be a zone where the temperature of the plurality of chambers is affected by the single thermoelectric element. The use of a single thermal element for a plurality of chambers may enable parallelized processing of the reaction mixtures in the plurality of chambers, as well as reduced system complexity.

[0176] FIG. 35A shows an example of a plurality of chambers 3401 and a plurality of sensors 3503 operably coupled to a support 3507, according to some embodiments. A thermal element associated with a chamber of the plurality of chambers 3401 may be in thermal communication with the heatsink 3502. The plurality of sensors 3503 may be operably coupled to one or more control circuits 3504. The one or more control circuits may be configured to measure a temperature of a sensor corresponding to a chamber of the plurality of chambers 3501. The one or more control circuits may be operably coupled to a computer system as described elsewhere herein.

[0177] FIG. 35B shows an example of a detail of a plurality of chambers 3501 and a plurality of sensors 3503 operably coupled to a support 3507, according to some embodiments. The plurality of chambers 3501 may be operably coupled to the support 3507 via one or more fasteners 3506 (e.g., one or more screws). The support 3507 may comprise one or more thermal management elements 3505. The one or more thermal management elements may comprise one or more holes configured to reduce a heat flux through the support 3507 (e.g., reduce a thermal conductivity) as described elsewhere herein.

[0178] In the examples of FIGs. 35A and 35B, temperature sensor boards can be in communication with chambers. The temperature sensors can be mounted to achieve high quality temperature sensing in a compact design. The sensors may be mounted on a PCBA attached to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more chambers. Each temperature sensor can be thermally connected to a chamber via one or more thermally conductive materials (e g., indium foil, copper foil, graphite) to ensure minimum thermal resistance. The contact between the temperature sensor and the chamber can be reinforced using at least about 1, 2, 3, 4, or more fasteners (e.g., screws). The PCBA may be configured to minimize thermal bleeding between sensors (e.g., via one or more thermal management elements).

[0179] The chamber and / or the additional chamber be affixed to a same support (e.g., PCB support). For example, the chamber and the additional chamber can share a PCB. The chamber and an additional chamber of the substrate can share a PCB support. The PCB may operate as a support for the chamber and the additional chamber as described elsewhere herein. The PCB may be a portion of a PCB assembly (PCBA). The PCBA may comprise one or more resistors, capacitors, diodes, inductors, transistors, integrated circuits, switches, fuses, or heat sinks, or anyAtty Dkt No.: 61197-702601 combination thereof. The support may be configured to support one or more connections between the chamber or an associated element (e.g., thermal element, sensor) and a control system (e g., a computer system). The support can comprise one or more traces, wires, or contacts, or any combination thereof. For example, the PCB can comprise traces configured to conduct a signal from a sensor to a sensor control module. Placing a plurality of chambers on a single PCB can reduce the manufacturing complexity of the system, as well as improve a density of chambers within the system. For example, using common PCBs for a plurality of chambers can increase the density of chambers within the system, thereby reducing the footprint area of the system.

[0180] A support of the present disclosure may have a thickness of at least about 0.1, 0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, or more millimeters. The support may have a thickness of at most about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, 0.5, 0. 1, or less millimeters. The support may have a thickness in a range as defined by any two of the preceding values. For example, the support may have a thickness from about 0.1 to about 5, about 0.5 to about 5, about 1 to about 5, about 0.1 to about 2, about 1 to about 2, or about 1.5 to about 3.5 millimeters. The PCB may be a single sided PCB. For example, the PCB may have an insulating side and a conducting side. The PCB may be a multi-layer PCB. For example, the PCB may have a plurality of conducing and insulating layers. The PCB may have at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more conducting layers and / or insulating layers. The PCB may have at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 conducing layers and / or insulating layers. The PCB may have a number of layers in a range as defined by any two of the preceding values. The PCB may be a flexible PCB. For example, the PCB may be able to bend without breaking. The PCB may comprise one or more conducting layers. The one or more conducting layers may independently comprise one or more metals (e.g., copper, aluminum, indium). The one or more conducting layers may comprise one or more organic conductors (e g., graphite, graphene, conducting polymers). The PCB may comprise one or more insulating layers. The one or more insulating layers may comprise one or more polymer insulating layers. The one or more insulating layers may comprise fiberglass (e.g., FR-2, FR-4), epoxy resin, polyimide, Teflon, ceramic, or Bakelite, or any combination thereof.

[0181] In some cases, a support (e.g., PCB) may comprise one or more thermal management elements. The one or more thermal management elements can reduce a thermal conductivity along the support. For example, a thermal management element disposed between a fist chamber and a second chamber can reduce thermal crosstalk between the first chamber and the second chamber. Reducing thermal conductivity along the support can increase the ability of the system to individually address the chambers of the system, as well as improve the accuracy of the PCRAtty Dkt No.: 61197-702601 reactions of the chambers. The one or more thermal management elements may be disposed between the chamber and an additional chamber. A thermal management element may comprise one or more of a gap, or a thermal insulator, or a combination thereof. The gap may comprise a void within the support. For example, the gap can comprise a hole in the support. The gap may be at least about 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, or more millimeters in any dimension. The gap may be at most about 5, 4, 3, 2, 1 , 0.5, 0.1, 0.05, 0.01, or less millimeters in any dimension. The gap may be along an axis of the support separating the chamber and the additional chamber. In this way, the gap may provide increased thermal isolation between the chamber and the additional chamber. The thermal insulator may comprise a material with a low thermal conductivity. For example, the thermal insulator may comprise an aerogel, fiberglass, a foam, mineral wool, ceramic insulation, or gasses, or any combination thereof.

[0182] The thermal management element may have a thermal conductivity of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8. 0.9, 1, or more W / Km. The thermal management element may have a thermal conductivity of at most about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, 0.005, 0.001, or less W / Km. The thermal conductivity between the chamber and the additional chamber may be at least about 0.001 , 0.005, 0.01 , 0.05, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7. 0.8. 0.9, 1, or more W / Km. The thermal conductivity between the chamber and the additional chamber may be at most about 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01 , 0.005, 0.001 , or less W / Km. The thermal conductivity between the chamber and the additional chamber may be from about 0.1 to about 1, about 0.1 to about 0.5, about 0.5 to about 1, or about 0.3 to about 0.7 W / Km.

[0183] In some cases, the chamber can be operably coupled with a first sensor and the additional chamber can be operably coupled with a second sensor. The use of two sensors can permit individual recording of the conditions of the individual chambers. In some cases, each chamber of a plurality of chambers can be associated with a different sensor of a plurality of sensors. For example, the sensors may not be shared between the chambers. The use of the individual sensors can enable individual readouts and sensing of each chamber, which can provide individual addressability of the chambers and improved accuracy of the sensing of the conditions of the chamber. In some cases, a single sensor may be shared among a plurality of chambers. For example, a single temperature sensor can be used to measure an aggregate temperature for a plurality of chambers.

[0184] In another operation 2520, the method 2500 may comprise using a thermal element disposed adjacent to the location to initiate the PCR reaction using the sample. The PCR reactionAtty Dkt No.: 61197-702601 may generate a signal or signal change which is indicative of a property associated with the analyte. The thermal element may be in thermal contact with the thermally conductive element.

[0185] In an optional operation 2530, the method 2500 may comprise using a second element operably coupled to the thermal element to adjust an operating condition of the thermal element in response at least partially to the signal or signal change to thereby affect a condition in the location. The second element may comprise, for example, a second thermal element, a heat sink, controller circuitry, one or more sensors, or one or more thermally conductive elements, or any combination thereof as disclosed herein. In some cases, the support can comprise a second thermally conductive element disposed through at least a portion of the support. The second thermally conductive element can be configured to enhance flow of heat from a thermal element to the second element. The second element can be in thermal contact with the second thermally conductive element. The second element can be configured to aid in maintaining a stable temperature on a backside of the second thermal element. For example, for a thermoelectric second thermal element, the second thermally conductive element can remove excess heat or cold from the backside of the second thermal element and transmit that energy to or from the second element. For example, a plurality of thermal elements each corresponding to a different well of a well plate can be placed in thermal contact with a plurality of thermally conductive elements. In this example, the plurality of thermally conductive elements can each be attached to a same heatsink configured to provide thermal regulation of the plurality of thermal elements. The heatsink can be configured to regulate the temperatures of a plurality of thermal elements (e g., accept heat from some thermal elements and provide heat to some thermal elements). The second element may be a thermoelectric element, a heatsink, or the like, as described elsewhere herein.

[0186] The thermally conductive element may be electrically insulated from the thermal element. For example, if the thermally conductive element were not electrically insulated from the thermal element, the thermal element may short and cease functioning. In this example, a reaction in the location (e.g., well) of the substrate associated with the thermal element that ceases functioning may stop, causing errors to be introduced into the use of the system. The electrical insulation may be disposed between the thermal element and the thermally conductive element. For example, the electrical insulation can be disposed on an opposite side of the thermal element from the chamber. In some cases, the electrical insulation may be disposed between the thermal element and the chamber. In some cases, the electrical insulation can be disposed between a plurality of thermal elements and thermally conductive elements. For example, a plurality of thermal elements and a plurality of thermally conductive elements can share anAtty Dkt No.: 61197-702601 electrical insulation. The electrical insulation may be a thin film insulation (e g., a layer of a thin film insulator (e.g., a thin film oxide such as silicon oxide, or the like) with a thickness of at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or fewer nanometers), a polymer insulation (e.g., an insulating polymer (e g., polymer configured as an electrical insulator such as polyethylene, polypropylene, polystyrene, resin, polymethylmethacrylate, etc.)disposed between the thermal element and the thermally conductive element), or the like, or any combination thereof. The insulation may be thermally conductive while being electronically insulating. For example, the insulation can be thermally conductive to permit enhanced heat transfer from the thermal element to the thermally conductive element while maintaining the electrical insulation and isolation of the thermal element. In this example, the presence of the electrical isolation can reduce shorting for the thermal element and maintain operability of the thermal element.

[0187] In some cases, a heatsink is disposed on an opposite side of the support from the thermal element. The heatsink may be as described elsewhere herein. The heatsink may be configured to dissipate at least a portion of the heat generated from the thermal element. The presence of the heatsink can result in increased efficiency of the thermal element, as well as reduced wear by maintenance of the thermal environment of the backside of the thermal element. The heatsink may be configured to be in thermal communication with a plurality of thermal elements. The heatsink may be configured to be in thermal communication with a subset for the plurality of thermal elements. The heatsink may be in thermal communication with a thermal element. For example, a plurality of thermal elements individually be in thermal communication with a plurality of heatsinks. For example, a single heatsink can be configured to dissipate heat from a plurality of thermal elements. The thermal element can be in thermal communication with the heatsink at least partially through the thermally conductive element. For example, the thermally conductive element can be configured to transport heat from the thermal element to the heatsink. In this example, the thermally conductive element can provide enhanced thermal conductivity as compared to the support.

[0188] In some cases, a thermal element and a heatsink can be in thermal contact with a thermally conductive element. The thermal element and the heatsink can be in communication (e.g., physical contact (e g., physically touching)) the thermally conductive element. The thermally conductive element can be configured to reduce a thermal resistance between the thermal element and the heat sink. For example, the thermally conductive element can have a higher thermal conductivity than the environment or support around the thermally conductive element. In this example, the environment can comprise one or more of a gaseous atmosphere, a support (e g., a PCB), or an interior of a system of the present disclosure, or any combinationAtty Dkt No.: 61197-702601 thereof. In another example, the thermally conductive element can provide a path with high thermal conductivity. For example, the thermally conducive element can have a higher thermal conductivity than a PCB disposed between the thermal element and the heat sink. In another example, the thermally conductive element can be in closer thermal contact with the thermal element and the heatsink than an air atmosphere around the thermal element and the heat sink. In some cases, a chamber and a sensor associated with the chamber can be in thermal contact with a thermally conductive element. The chamber and the sensor can be in communication (e.g., physical contact) with the thermally conductive element. The thermally conductive element can be configured to reduce a thermal resistance between the chamber and the sensor. For example, the thermally conductive element can provide a path of increased thermal conductivity between the chamber and the sensor.

[0189] A distance between the thermally conductive element and the chamber can be at least about 0.01. 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more micrometers. The distance between the thermally conductive element and the chamber can be at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 , 0.05, 0.01 , or less micrometers. The distance between the thermally conductive element and the chamber may be in a range as defined by any two of the preceding values. For example, the distance may be from 1 to 100, 1 to 50, 1 to 10, 5 to 100, 5 to 50, or 5 to 10 micrometers. The thermally conductive element and the chamber can be in direct contact (e g., without an intervening gap). The thermally conductive element and the chamber can each be in contact with a thermally conductive element as described elsewhere herein.

[0190] A distance between the thermally conductive element and the thermal element can be at least about 0.01. 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more micrometers. The distance between the thermally conductive element and the thermal element can be at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less micrometers. The distance between the thermally conductive element and the thermal element may be in a range as defined by any two of the preceding values. For example, the distance may be from 1 to 100, 1 to 50, 1 to 10, 5 to 100, 5 to 50, or 5 to 10 micrometers. The thermally conductive element and the thermal element can be in direct contact (e g., without an intervening gap). The thermally conductive element and the thermal element can each be in contact with a thermally conductive element as described elsewhere herein.Atty Dkt No.: 61197-702601

[0191] A distance between the thermally conductive element and the heatsink can be at least about 0.01. 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more micrometers. The distance between the thermally conductive element and the heatsink can be at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, or less micrometers. The distance between the thermally conductive element and the heatsink may be in a range as defined by any two of the preceding values. For example, the distance may be from 1 to 100, 1 to 50, 1 to 10, 5 to 100, 5 to 50, or 5 to 10 micrometers. The thermally conductive element and the heatsink can be in direct contact (e.g., without an intervening gap). The thermally conductive element and the heatsink can each be in contact with a thermally conductive element as described elsewhere herein.

[0192] A distance between the thermally conductive element and the sensor can be at least about 0.01. 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, or more micrometers. The distance between the thermally conductive element and the sensor can be at most about 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 , 0.05, 0.01 , or less micrometers. The distance between the thermally conductive element and the sensor may be in a range as defined by any two of the preceding values. For example, the distance may be from 1 to 100, 1 to 50, 1 to 10, 5 to 100, 5 to 50, or 5 to 10 micrometers. The thermally conductive element and the sensor can be in direct contact (e.g., without an intervening gap). The thermally conductive element and the sensor can each be in contact with a thermally conductive element as described elsewhere herein.

[0193] A distance between the thermal element and the heat sink may be at least about 1, 2, 3, 4, 5, or more millimeters. A distance between the thermal element and the heat sink may be at most about 5, 4, 3, 2, 1, or less millimeters. The distance between the thermal element and the heat sink may be a thickness of a support (e.g., a PCB) disclosed elsewhere herein. A distance between the chamber and the sensor may be at least about 1, 2, 3, 4, 5, or more millimeters. A distance between the chamber and the sensor may be at most about 5, 4, 3, 2, 1, or less millimeters. The distance between the chamber and the sensor may be a thickness of a support (e.g., a PCB) disclosed elsewhere herein.

[0194] In some cases, a thermal conductor can be placed between the (i) thermal element and the thermally conductive element, (ii) the chamber and the thermally conductive element, (iii) the heatsink and the thermally conductive element, or (iv) the sensor and the thermally conductive element, or any combination thereof. The thermal conductor can reduce thermal resistanceAtty Dkt No.: 61197-702601 between the thermally conductive element and the other feature, enhancing thermal transfer and improving the operation of the thermally conductive element. The thermal conductor may improve heat transport in the system and enhance the efficiency of the thermally conductive element. The thermal conductor may comprise a thermal compound (e.g., a paste, liquid, powder, etc.), one or more thermal pads, one or more graphite sheets, or the like, or any combination thereof. The thermal conductor may be configured to fill a gap next to the thermally conductive element, thereby reducing a thermal resistance of the gap next to the thermally conductive element. The gap may be due to errors or tolerances of components of the system. For example, the errors in the size may be due to imperfect coupling of the thermal element and the thermally conductive elelement. The thermal conductor may have a same size as a thermally conductive element. A single thermal conductor may be operably connected to a plurality of thermally conductive elements. Each thermally conductive element of a plurality of thermally conductive elements may comprise a different thermal conductor. A subset of a plurality of thermally conductive elements may share a thermal conductor. The thermal conductor may have an area of at least about 0.0001, 0.001, 0.01, 0.1, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more square millimeters. The thermal conductor may have an area of at most about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.0001, or fewer square millimeters. The thermal conductor may have an area in a range as defined by any two of the preceding values. For example, the thermal conductor may have an area of about 0.0001 to 25, 0.0001 to 1, 0.0001 to 0.1, 0.01 to 1, or 0.1 to 5 square millimeters. The thermal conductor may have a thickness of least about 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120, 130, 140, 150, 200, 250, or more micrometers. The thermal conductor may have a thickness of at most about 250, 200, 150, 140, 130, 120, 110, 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, 1, or less micrometers. The thermal conductor may have a thickness from about 1 to about 250, about 10 to about 150, about 50 to about 150, about 75 to about 125, or about 100 to about 150 micrometers. The thermal conductor may be under at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or more percent compression. The thermal conductor may be under at most about 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less percent compression. The thermal conductor may be under an amount of compression from about 5 to about 95, about 25 to about 75, about 10 to about 50, or about 50 to about 95 percent. The thermal conductor may be held in place by friction, compression, shims (e.g., polymer shims, Kapton shims), of one or more fasteners, or any combination thereof.Atty Dkt No.: 61197-702601

[0195] The thermal conductor may have a thermal conductivity of at least about 1, 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110,120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300,310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490,500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900,2,000, or more watts / kelvin / meter (W / Km). The thermal conductor may have a thermal conductivity of at most about 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,200, 1,100, 1,000, 900, 800, 700, 600, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20, 19,18, 17, 16, 15, 14, 13, 12, 1 1 , 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less W / Km. The thermal conductor may have a thermal conductivity in a range as defined by any two of the preceding values. For example, the thermal conductor can have a thermal conductivity from about 50 to about 2,000, about 80 to about 1,000, about 100 to about 1,000, about 150 to about 350, or about 150 to about 250 W / Km.

[0196] The thermally conductive element may have a thermal conductivity of at least about 1 , 2, 3, 4, 5 ,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280,290, 300, 310, 320, 330, 340, 350, 360, 370, 380, 390, 400, 410, 420, 430, 440, 450, 460, 470,480, 490, 500, 600, 700, 800, 900, 1,000, 1,100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800,1,900, 2,000, or more watts / kelvin (W / Km). The thermally conductive element may have a thermal conductivity of at most about 2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,200, 1,100, 1,000, 900, 800, 700, 600, 500, 490, 480, 470, 460, 450, 440, 430, 420, 410, 400, 390, 380, 370, 360, 350, 340, 330, 320, 310, 300, 290, 280, 270, 260, 250, 240, 230, 220, 210, 200, 190, 180, 170, 160, 150, 140, 130, 120, 110, 100, 90, 80, 70, 60, 50, 45, 40, 35, 30, 25, 20,19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less W / Km. The thermally conductive element may have a thermal conductivity in a range as defined by any two of the preceding values. For example, the thermally conductive element can have a thermal conductivity from about 50 to about 2,000, about 80 to about 1,000, about 100 to about 1 ,000, about 150 to about 350, or about 150 to about 250 W / Km.

[0197] The thermally conductive element may have a thickness of at least about 1, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1, 100, 1,200, 1,300, 1,400, 1,500, 1,600, 1,700, 1,800, 1,900, 2,000, 2,100, 2,200, 2,300, 2,400, 2,500, 2,600, 2,700, 2,800, 2,900, 3,000, 3,100, 3,200, 3,300, 3,400, 3,500, 3,600, 3,700, 3,800, 3,900, 4,000, 4,100,Atty Dkt No.: 61197-7026014.200, 4,300, 4,400, 4,500, 4,600, 4,700, 4,800, 4,900, 5,000, 5, 100, 5,200, 5,300, 5,400, 5,500,5.600, 5,700, 5,800, 5,900, 6,000, 6, 100, 6,200, 6,300, 6,400, 6,500, 6,600, 6,700, 6,800, 6,900,7,000, 7,100, 7,200, 7,300, 7,400, 7,500, 7,600, 7,700, 7,800, 7,900, 8,000, 8,100, 8,200, 8,300,8.400, 8,500, 8,600, 8,700, 8,800, 8,900, 9,000, 9,100, 9,200, 9,300, 9,400, 9,500, 9,600, 9,700,9.800, 9,900, 10,000, or more micrometers. The thermally conductive element may have a thickness of at most about 10,000, 9,900, 9,800, 9,700, 9,600, 9,500, 9,400, 9,300, 9,200, 9,100, 9,000, 8,900, 8,800, 8,700, 8,600, 8,500, 8,400, 8,300, 8,200, 8, 100, 8,000, 7,900, 7,800, 7,700,7.600, 7,500, 7,400, 7,300, 7,200, 7,100, 7,000, 6,900, 6,800, 6,700, 6,600, 6,500, 6,400, 6,300,6.200, 6,100, 6,000, 5,900, 5,800, 5,700, 5,600, 5,500, 5,400, 5,300, 5,200, 5,100, 5,000, 4,900,4.800, 4,700, 4,600, 4,500, 4,400, 4,300, 4,200, 4,100, 4,000, 3,900, 3,800, 3,700, 3,600, 3,500,3.400, 3,300, 3,200, 3,100, 3,000, 2,900, 2,800, 2,700, 2,600, 2,500, 2,400, 2,300, 2,200, 2,100,2,000, 1,900, 1,800, 1,700, 1,600, 1,500, 1,400, 1,300, 1,200, 1, 100, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 1, or less micrometers. The thermally conductive element may have a range as defined by any two of the preceding values. For example, the thermally conductive element can have a thickness from about 1 to about 10,000, about 100 to about 1,000, about 200 to about 2,000, about 300 to about 3,000, about 400 to about 4,000, about 500 to about 5,000, or about 1,000 to about 5,000.

[0198] The thermally conductive element may have an area of at least about 0.0001, 0.001, 0.01, 0.1, 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, or more square millimeters. The thermally conductive element may have an area of at most about 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.1, 0.01, 0.001, 0.0001, or fewer square millimeters. The thermally conductive element may have an area in a range as defined by any two of the preceding values. The thermally conductive element may be smaller (e g., have a smaller area, volume, maximum dimension, minimum dimension) than the chamber, the sensor, or the heatsink, or any combination thereof. The thermally conductive element may be larger than the chamber, the sensor, or the heatsink, or any combination thereof.

[0199] The thermally conductive element may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, or more layers. The thermally conductive element may comprise at most about 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 layer. The thermally conductive element may comprise a number of layers in a range as defined by any two of the preceding values. The thermally conductive element may comprise a plurality of layers of a single material (e g., metal, graphite, paste). For example, the thermally conductive element may comprise a plurality of indium layers. The thermally conductive element may comprise a plurality of layers of different materials. ForAtty Dkt No.: 61197-702601 example, the thermally conductive element may comprise a plurality of copper layers and a plurality of graphite layers.

[0200] The thermally conductive element may comprise one or more thermally conductive materials. The thermally conductive element may comprise graphite. The thermally conductive element may comprise one or more metals. For example, the thermally conductive element may comprise one or more metals, mixtures of metals, or alloys thereof. Examples of metals include, for example, indium, copper, tin, aluminum, iron, silver, gold, platinum, nickel, chromium, or the like. The thermally conductive element may comprise metal to enhance the thermal conductivity of the thermally conductive element. For example, the use of metal can make the thermally conductive element more thermally conductive, thereby enhancing the transfer of heat between the thermal element and the second element.

[0201] In some cases, an isothermal amplification reaction (e.g., a PCR) as described elsewhere herein may be performed within the location (e.g., the chamber). For example, an amplification reaction can be performed in the location to generate a clonal nucleic acid population (e.g., amplicon) in the location. The clonal nucleic acid population can be further analyzed (e.g., sequenced, utilized as a reagent, etc ). In some cases, the methods and systems of the present disclosure can provide an amplicon. For example, a fluorescence measurement of a concentration of an amplicon in a reaction mixture can be measured during a PCR forming the amplicon.

[0202] In some cases, one or more nucleic acid molecules of the amplicon can be fragmented. The fragmenting may comprise enzymatic fragmentation. The fragmenting may comprise mechanical fragmentation. The one or more nucleic acids or fragments thereof may have end repair performed (e.g., filling sticky ends to generate blunt ends). One or more adapters can be ligated to one or more sides of the one or more nucleic acids or fragments thereof. The amplicon may be purified (e g., size purified, affinity purified, purified by centrifugation). The amplicon may be sequenced. The sequencing may comprise next-generation sequencing (NGS). The sequencing may comprise sequencing by synthesis.

[0203] In an aspect, the present disclosure provides a device (e.g., a device usable in method 2500). The device can comprise a thermoelectric element as described elsewhere herein operably coupled to a support. The support may comprise one or more thermally conductive elements disposed in at least a portion of the support. The thermoelectric element can be in thermal contact with the one or more thermally conductive elements. The device may comprise a power supply configured to provide power to the thermoelectric element. The power supply may comprise a current sinking converter in an electrical path of the thermoelectric element.Atty Dkt No.: 61197-702601

[0204] FIG. 26 is a flow chart of a method 2600, according to some embodiments. In an operation 2610, the method 2600 may comprise providing a device comprising a power supply comprising a circuit comprising a current sinking converter and a thermoelectric element in electrical communication with the power supply. The power supply comprising the current sinking convertor may provide an output with a difference between the heating and cooling output of the thermoelectric element as described elsewhere herein. In some cases, the heating output and the cooling output of the thermal element can be symmetrical (e.g., the same with opposite sign).

[0205] In some cases, the current sinking converter may be a buck-boost converter. The buckboost converter may be configured to symmetrize or otherwise adjust the output of the power supply. For example, the buck-boost converter can be configured to reduce and / or boost an output of the power supply to enable the power supply to provide a symmetrical range of power. For example, a power supply capable of providing 1.6 to -0.4 amps can, through use of a buckboost converter, provide 1.6 to -1.6 amps. The buck-boost converter may have a capacity to make symmetrical a heating output and a cooling output with a difference of at least about 0. 1, 0.2, 03, 0.4, 0.5, 0.6, 07, 0.8, 0.9, 1 , 1.1 , 1.2, 13, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2, 2.5, 3, 3.5, 4, 4.5, 5, or more amps. The buck-boost converter may have a capacity to make symmetrical a heating output and a cooling output with a difference of at most about 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1 , 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1 , or less amps. In some cases, alternative circuitry architectures can be used, for example, single-ended primary inductor converters, Cuk converters, flyback converters, or the like. The presence of the buck-boost converter can improve the efficiency of the system by making symmetric the power delivery to the thermal element, and can enable larger reaction temperature ranges within the system. For example, the buck-boost converter can enable high and low temperatures, which can enhance the range of chemistries operable within the system.

[0206] In another operation 2620, the method 2600 may comprise using the power supply to provide power to the thermoelectric element. When the power is supplied to the thermoelectric element, the current sinking converter can remove current from the circuit that is in excess of a current sufficient to operate the thermoelectric element. This can reduce over-current (e g., excess current) on the thermoelectric element, enhancing the longevity of the element and increasing the safety of the system. The excess current can be at least about 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, or more milliamps. The excess current can be at most about 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, or less milliamps.Atty Dkt No.: 61197-702601

[0207] In some cases, a polymerase chain reaction within a chamber in thermal contact with the thermoelectric element. For example, a PCR reaction as described elsewhere herein can be performed. In this example, the efficiency of the system performing the PCR reaction can be improved through use of the current sinking converter. In some cases, the chamber is a well as described elsewhere herein. The chamber may be a location as described elsewhere herein.

[0208] In some cases, another power supply can be used to provide power to another thermoelectric element. The another thermoelectric element may be of a same type as the thermoelectric element. For example, the thermoelectric element and the another thermoelectric element can both be the same size and have the same power specifications. The another thermoelectric element may be different from the thermoelectric element. The another power supply may comprise another current sinking converter. The another current sinking converter may be a same type of current sinking converter. For example, the another current sinking converter may be a buck-boost converter. In some cases, the temperature of another chamber can be adjusted using at least in part the another thermoelectric element. The adjusting the temperature can be as described elsewhere herein. For example, the adjusting the temperature can be a part of an isothermal amplification reaction. The temperature of the another chamber can be different from a temperature of a chamber in thermal contact with the thermoelectric element. For example, the chamber and the another chamber can comprise samples at different stages of a reaction using different temperatures. In some cases, the thermoelectric element and the another thermoelectric element are individually addressable. For example, the thermoelectric element and the another thermoelectric element can have independent amounts of current applied to each thermal element. A chamber and an additional chamber may be individually addressable. For example, a chamber can be interrogated by an optical system independently from the additional chamber. In another example, the chamber can have different reagents added to the chamber from the additional chamber. Multiple thermoelectric elements being powered by a single power supply can simplify the construction of a system and reduce the footprint of the system (e g., by reducing the number of power supplies in the system). Additionally, use of multiple thermoelectric elements can provide for individual addressability and reaction control as described elsewhere herein. For example, multiple PCR reactions can be operated in parallel with individual control using the individually addressable wells of the present disclosure.

[0209] In an optional operation 2630, the method 2600 may comprise adjusting a temperature of a well disposed in thermal contact with the thermoelectric element using at least in part the thermoelectric element. The adjusting the temperature may be as described elsewhere herein. In some cases, the adjusting the temperature may be a part of an amplification reaction. In someAtty Dkt No.: 61197-702601 cases, the adjusting the temperature of the well can comprise adjusting a power output of the power supply.

[0210] FIG. 27 is a flow chart of a method 2700, according to some embodiments. In an operation 2710, the method 2700 comprises providing a plurality of chambers, a thermoelectric plate comprising a plurality of thermoelectric elements, and a movable assembly in contact with the thermoelectric plate. A thermoelectric plate may be placed in contact with a substrate of the present disclosure. Each thermoelectric element of the plurality thermoelectric elements may correspond to a different chamber of the plurality of chambers. In some cases, the plurality of chambers is a plurality of wells as described elsewhere herein. For example, the plurality of wells can each house a different polymerase chain reaction. In some cases, the system comprises a lid positioned above the plurality of chambers. In some cases, the lid may be a cover plate as described elsewhere herein. In some cases, the lid comprises one or more heating elements. For example, the lid can comprise a single heating element configured to provide heat to all of the chambers. In another example, the lid can comprise a plurality of heating elements to provide granular heating zones of chambers. The heating zones can be different regions of the lid with different heating elements, and the granular control can be control over each region of the lid. The lid may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16, 20, 24, 30, 40, 48, 50, 60, 70, 80, 90, 96, or more zones. The lid may comprise at most about 96, 90, 80, 70, 60, 50, 48, 40, 30, 24, 20, 16, 10, 9, 8, 7, 6, 5, 4, 3, 2, or less zones. In another example, the lid can comprise a number of heating elements equal to the number of chambers, and each chamber can have a heating element disposed above the chamber. The heating elements may be heating elements as described elsewhere herein.

[0211] The movable assembly may be configured to raise and / or lower a substrate from a lid or cover plate. For example, the movable assembly can raise a substrate up to a cover plate. In some cases, the movable assembly can comprise one or more levers. The one or more levers can be configured to aid in the contacting of the thermoelectric plate to the lid. A lever can have a movement ratio of the lever to the movement of the movable assembly of at least about 10: 1, 9: 1, 8: 1, 7: 1, 6: 1, 5: 1, 4: 1, 3: 1, 2: 1, 1 : 1, 1 :2, 1 :3, 1 :4, 1 :5, 1 :6, 1 :7, 1 :8, 1 :9, 1 : 10, or more. The movable assembly may comprise one or more pivoting points. The one or more pivoting points may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more pivoting points. The one or more pivoting points may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or less pivoting points. The pivoting points may be locations where portions of the movable assembly pivots around while moving. The movable assembly may be positioned below the thermoelectric plate. For example,Atty Dkt No.: 61197-702601 the movable assembly can be positioned to raise the thermoelectric plate up to the bottoms of the plurality of chambers.

[0212] The movable assembly may comprise at least one screw mechanism operably coupled to a substrate. The at least one screw mechanism may rotate to move the movable assembly in a vertical direction and / or a horizontal direction. The at least one screw mechanism may comprise one or more screws (e g., ACME screws). The screw mechanism may comprise one or more nuts (e.g., ACME nuts) threaded on the one or more screws. The use of the ACME screws and / or ACME nuts may provide increased efficiency of power transmission to the movable assembly. The at least one screw mechanism may be oriented in a vertical orientation. The at least one screw mechanism may be oriented in a horizontal orientation. The at least one screw mechanism may be oriented in an orientation of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, or more degrees off vertical. The at least one screw mechanism may be oriented in an orientation of at most about 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less degrees off vertical. The at least one screw mechanism may be oriented in an orientation in a range as defined by any two of the preceding values.

[0213] The movable assembly may comprise one or more motors. The one or more motors can move the movable assembly (e g., between an open and a closed position). The one or more motors can be configured to move a substrate and / or a cover plate. The one or more motors may be connected to one or more levers, one or more screws, or one or more nuts, or any combination thereof. The one or more motors may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more motors. The one or more motors may comprise one or more stepper motors, brushed motors, brushless motors, servo motors, linear motors, induction motors, piezoelectric motors, or hydraulic motors, or any combination thereof.

[0214] The movable assembly may comprise one or more guides. The one or more guides may comprise one or more contactless or partially contactless guides. For example, a ring can be placed around a rod such that the rod and ring do not touch unless the movable assembly is out of alignment. The one or more guides may comprise one or more sliding guides. The one or more sliding guides may be configured to guide a movement of a substrate and / or a cover plate (e g., during an engagement of the cover plate and the substrate). The one or more sliding guides may provide constant guidance to the movable assembly by maintaining contact between the one or more sliding guides throughout the movement of the movable assembly. For example, using a sliding guide can keep contact with the guide throughout the movement of the movable assembly. In this way, the sliding guide can provide positive guidance to the movable assembly. The one or more sliding guides may comprise one or more metal sliding guides, one or moreAtty Dkt No.: 61197-702601 plastic sliding guides, or a combination thereof. The one or more plastic sliding guides may comprise Delrin, polytetrafluoroethylene, polyamide, polyethylene, or acetal, or any combination thereof.

[0215] The movable assembly may comprise one or more alignment members. The one or more alignment members may provide fine alignment between the substrate and the cover plate. For example, rough alignment between the substrate and the cover plate may be provided by one or more sliding guides while fine alignment can be provided by the one or more alignment members. The one or more alignment members may comprise one or more pins, holes, rings, rail guides, alignment rods, grooves, wheels, or latches, or any combination thereof. The one or more alignment members may be placed on the top plate, the cover plate, the substrate, or the movable assembly, or any combination thereof. The one or more alignment members may be configured to align the chamber and the thermal element. The one or more alignment members may be configured to interface with one another. For example, an alignment pin can slot into an alignment hole when the substrate is engaged with the cover plate. The one or more alignment members may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more alignment members. The one or more alignment members may comprise at most about 20, 19, 18, 17, 16, 15, 14, 13, 12, 1 1, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 alignment member. The alignment may be an automated alignment. For example, the alignment may be performed in an absence of an intervention by a user. The automated alignment may reduce an error in the alignment and reduce a time used to align the chambers and the thermal elements.

[0216] In some cases, the system or movable assembly may comprise one or more limit switches. A limit switch can detect a position of the movable assembly (e g., upon activation of the limit switch). For example, the limit switch can detect a position of the movable assembly while stationary or in motion. In this example, the limit switch can detect a position of the substrate as a portion of the movable assembly. The limit switch may be configured to detect when the movable assembly has reached a far end of its movement range. For example, a limit switch can detect when the movable assembly has fully opened or closed. The movable assembly may be as described elsewhere herein (e.g., comprise a plurality of thermoelectric elements configured to be disposed adjacent to a plurality of locations). The movable assembly can be configured to permit loading of a substrate (e g., well plate) and subsequently permit the close contact of the plurality of thermal elements coupled to the support. For example, the movable assembly can move to open the system and permit loading of a new well plate comprising a plurality of samples. In this example, the movable assembly can permit use of disposable well plates which can enhance throughput of the system. Examples of limit switches include, but areAtty Dkt No.: 61197-702601 not limited to, optical limit switches, magnetic limit switches, inductive limit switches, mechanical limit switches, capacitive limit switches, ultrasonic limit switches, or the like, or any combination thereof. The one or more limit switches may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more limit switches. The one or more limit switches may comprise at most about 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 limit switch.

[0217] In another operation 2720, the method 2700 may comprise moving, via the movable assembly, the thermoelectric plate, thereby contacting the plurality of thermoelectric elements to the plurality of chambers. A chamber of the plurality of chambers can be aligned to a thermoelectric element of the plurality of thermoelectric elements or a hole or access feature within the cover plate with an offset of at most about 1, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1 ,000, or more micrometers. The offset may be at least about 1, 5, 10, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1,000, or more micrometers. The offset may be an error in the alignment. The offset can be a distance difference between the predetermined location of the thermoelectric elements and the actual location of the thermoelectric element. For example, the offset can be an offset distance between where the plurality of thermoelectric elements were intended to be placed and where they were actually placed. The offset may be a maximum offset for a plurality of chambers and a plurality of thermal elements. For example, the offset may be the largest offset between any chamber and the corresponding thermal element. The plurality of chambers and the plurality of thermal elements may be as described elsewhere herein. For example, there may be 96 thermal elements and 96 chambers.

[0218] The contacting the plurality of thermoelectric elements with the plurality of chambers can be an automated contacting. For example, the contacting can be performed without the intervention of a user. The thermoelectric plate can be contacted to the plurality of chambers with at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, or more pounds of force. The thermoelectric plate can be contacted to the plurality of chambers with at most about 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less pounds of force. The thermoelectric plate can be contacted to the plurality of chambers with a clamping force in a range as defined by any two of the preceding values. The clamping may comprise the use of a spring loading system. For example, the system can comprise one or more springs configured to adjust a force applied by the loading system. In this example, the springs can adjust the force of the loading system.

[0219] The contacting the plurality of thermoelectric elements with the plurality of chambers may comprise automated contacting. The automated contacting may not use intervention from a user. For example, the automated contacting may occur in an absence of input from a user. TheAtty Dkt No.: 61197-702601 contacting may be semi-automated (e.g., using some interaction from a user) or manual (e.g., the user performs the contacting). The device (e.g., the system comprising the substrate) may have a footprint (e.g., an external area the device occupies) of at most about 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 590, 580, 570, 560, 550, 540, 530, 520, 510, 500, 450, 400, or fewer square centimeters. The device may have a footprint of at least about 400, 450, 500, 510, 520, 530, 540, 550, 560, 570, 580, 590, 600, 700, 800, 900, 1 ,000, 1,500, 2,000, 2,500, 3,000, 3,500, 4,000, 4,500, 5,000, or more square centimeters. The device may have a footprint in a range as defined by any two of the preceding values. The reduced footprint can provide enhanced usability for the system and decreased bench space requirements. Within the footprint, the device may comprise one or more substrates, one or more cover plates, one or more optical systems, one or more thermal elements, one or more fluidic reservoirs, or one or more computer processors, or any combination thereof.

[0220] The device may have weight of at least about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75 or more pounds. The device may have a weight of at most about 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, 5, or less pounds. The device may have a weight in a range as defined by any two of the preceding values. The device may have a maximum dimension of at least about 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 120, 140, 160, 180, 200, 220, 240, 260, 280, 300, 320, 340, 360, 380, 400, 420, 440, 460, 480, 500, or more centimeters. The device may have a maximum dimension of at most about 500, 480, 460, 440, 420, 400, 380, 360, 340, 320, 300, 280, 260, 240, 220, 200, 180, 160, 140, 120, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less centimeters. The device may have a maximum dimension in a range as defined by any two of the preceding values. The system may have a volume of at least about 0.01, 0.05, 0.1, 0.15, 0.2, 0.25, 0.3, 0.35, 0.4, 0.45, 0.5, 0.55, 0.6, 0.65, 0.7, 0.75, 0.8, 0.85, 0.9, 0.95, 1, or more cubic meters. The system may have a volume of at most about 1.0, 0.95, 0.9, 0.85, 0.8, 0.75, 0.7, 0.65, 0.6, 0.55, 0.5, 0.45, 0.4, 0.35, 0.3, 0.25, 0.2, 0.15, 0.1, 0.05, 0.01, or less cubic meters. The system may have a volume in a range as defined by any two of the preceding values.

[0221] In some cases, the method 2700 may comprise performing an isothermal amplification reaction within a chamber of the plurality of chambers. Examples of isothermal amplification reactions include, but are not limited to, loop-mediated isothermal amplification (LAMP), rolling circle amplification (RCA), helicase-dependent amplification (HD A), recombinase polymerase amplification (RPA), transcription mediated amplification (TMA), or the like. In some cases, the method 2700 may comprise performing a non -isothermal amplification reaction within a chamber of the plurality of chambers. Examples of non-isothermal amplification reactions include, but are not limited to, polymerase chain reaction (PCR), quantitative PCR, reverseAtty Dkt No.: 61197-702601 transcription PCR, ligase chain reaction, strand displacement amplification, or the like. In some cases, an independent isothermal amplification reaction can be performed within each chamber of the plurality of chambers. Amplicons generated from the methods and systems of the present disclosure can be used for, for example, nucleic acid sequencing, next-generation nucleic acid sequencing, metagenomics, diagnostic applications, environmental monitoring, forensics, or the like, or any combination thereof.

[0222] A system of the present disclosure may comprise, for example, a tray configured to accept one or more substrates such as well plates. The tray may be configured to align the substrates such that, upon insertion of the tray into the system, the substrate is located adjacent to a plurality of thermoelectric elements. The plurality of thermoelectric elements can be connected to a plate that is in turn connected to an arm such that, upon movement of the arm, the plurality of thermoelectric elements are contacted to the substrate and are available for modulating a reaction condition in various locations (e.g., wells) of the substrate. Other element may be present in the system, for example, optical assemblies including optics heads, reagent reservoirs, fluid handling systems, other temperature control systems, excitation sources, microprocessors, controllers, electrical power delivery circuitry, computer processors, or the like, or any combination thereof as described elsewhere herein. The reagent reservoirs may be configured to contain one or more reagents as described elsewhere herein. For example, the reagent reservoirs may comprise PCR reagents. The system may comprise from about 0 to about 10 reagent reservoirs. The fluid handling systems may comprise from about 0 to about 10 fluid handling systems. The fluid handling systems may be configured to provide one or more fluids (e g., reagents, samples, reaction mixtures) to the substrate. The other temperature control systems may control a temperature of an interior of the device (e.g., via one or more fans), or of circuitry of the system, or any combination thereof. The electrical power delivery circuitry may be configured to provide power to one or more components of the system. For example, the electrical power delivery can take in power from a wall outlet and supply the power to the electrical control circuitry and to a computer processor of the system.

[0223] Methods of the present disclosure may comprise dispensing the reaction mixture into the chamber. The dispensing may occur within a same system as the substrate. In some cases, a fluid handling system may be configured to dispense a reaction mixture and / or a sample into a chamber. For example, the fluid handling system can be configured to dispense fluid (e.g., reagents, solvents, samples) into one or more chambers of the system. The dispensing may occur outside of a system comprising the substrate. In some cases, the reaction mixture can be dispensed into the chamber by a user. For example, a user can dispense the reaction mixture priorAtty Dkt No.: 61197-702601 to loading the substrate into the system. In this example, the user can load a plurality of chambers with a plurality of rection mixtures, and then load the substrate comprising the plurality of chambers into the system. The dispensing may use an automated system. In some cases, the fluid handling system can be an automated fluid handling system. For example, the fluid handling system can dispense the fluid without an intervention from a user. The use of an automated fluid handling system can enable higher throughput and reduced error. The automated fluid handling system may comprise an automated pipetting system (e.g., a system configured to use pipettes to dispense the fluid). The automated fluid handling system may comprise a pumping fluidic system (e.g., a system that utilizes pumps and fluid lines to dispense the fluid).

[0224] FIGs. 16A-16B show different views of an example of a device 1600, according to some embodiments. The device may be as described elsewhere herein. In some cases, a microwell plate 1601 can be placed within a plate holder 1602. The plate holder may be configured to move the microwell plate into the device (e.g., into contact with a thermal elements 1603). The movement of the plate holder may be automated (e.g., actuated by motors within the device). The movement of the plate holder may be manual (e g., a user can slide the plate holder into the device). In some cases, the microwell can, once inserted into the device, be held in a fixed position while a thermal element can be moved to engage with the well plate. For example, a heat pump can be moved vertically to engage with the bottom of the microwell plate. In some cases, the microwell plate can be moved once inserted into the device to engage the microwell plate with a thermal element. For example, the microwell plate can be pressed down to engage with a heat pump. The movement within the device may be affected by, for example, a linear motor, a rotary motor, or the like, or any combination thereof.

[0225] FIG. 17 shows an example of an optics assembly 1700, according to some embodiments. The optics assembly may be configured to provide optical excitation and / or receive optical emissions from one or more chambers (e.g., wells) of a plate. For example, the optics assembly can be configured to provide an excitation beam from a first optical port 1701 into the optics assembly. In some cases, the first optical port can comprise a laser (e.g., a diode laser). In this way, the excitation beam can be generated from within the optics assembly. In some cases, the optics assembly can comprise a dichroic mirror 1702 configured to direct the excitation beam from the first optical port to a chamber. The excitation beam can excite a fluorophore within the chamber, and the resultant emission can travel back into the optics assembly, through the dichroic mirror, and be directed to a second optical port and / or a detector module 1703. In some cases, the emission can be removed from the optics assembly by, for example, coupling the emission light into a fiber optic. The removed emission light can then be directed towards aAtty Dkt No.: 61197-702601 detector. In some cases, the optics assembly can comprise a detector configured to detect the emission light without the emission light being removed from the optics assembly. FIG. 18 shows an example of a configuration of a plurality of optical assemblies 1800, according to some embodiments. In this example, 8 optics assemblies have formed as a single unit, which can be configured to monitor the optical signal from 8 chambers. The plurality of optics assemblies can be configured into a unit comprising at least about 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, or more optics assemblies. The plurality of optics assemblies can be configured into a unit comprising at most about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer optics assemblies. For example, FIG. 20 shows an example unit comprising 8 optics assemblies 1800, according to some embodiments. In another example, FIG. 21 shows an example unit comprising 16 optics assemblies in a single unit, according to some embodiments. In some cases, the optics assemblies can be configured on different sides of a unit. In some cases, the optics assemblies can be configured on a same side of a unit (e.g., stacked). The addition of additional optics units may not substantially increase the footprint of the unit while reducing the length of time for a device to collect fluorescent intensities from the chambers of the device. FIG. 19 shows an example of a unit comprising a plurality of optics assemblies 1800 positioned adjacent to a plurality of chambers 1901, according to some embodiments. The unit can be configured to move across the plurality of chambers by use of, for example, a motor (e.g., a stepper motor) and a belt (e.g., a timing belt) 1902.

[0226] FIG. 4A is a schematic of a thermoelectric control architecture 400, according to an embodiment, while FIG. 4B is a schematic of a thermoelectric control architecture 410 with a current sink capacity, according to an embodiment. The thermoelectric control architecture can be configured to control thermoelectric elements as described elsewhere herein. The thermoelectric control architecture can be based on a converter. The thermoelectric control architecture can be based on a DC / DC controller (e.g., a buck controller, a buck-boost controller). The thermoelectric control architecture can be based on a DC / DC converter. The DC / DC convertor can be configured to covert a DC input (e.g., first voltage, first amperage) to a DC output (e.g., second voltage, second amperage). For example, the DC / DC convertor can boost or reduce a voltage and / or amperage of a DC input (e.g., from a power source) to a DC output (e g., to a thermal element).

[0227] The architecture may be configured to fit within a footprint of a device described elsewhere herein. For example, the architecture can be miniaturized to fit within a microwell based device. The thermoelectric control architecture may be addressable by a microcontroller. The microcontroller may be a computer system as described elsewhere herein. TheAtty Dkt No.: 61197-702601 thermoelectric control architecture may be configured to switch one or more thermoelectric elements between heating and cooling modes using a digital switching of two opposed transistors. The switching may occur over a period of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1,000, 1,500, 2,000,2.500, 3,000, 3,500, 4,000, 4,500, 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000,9.500, 10,000, or more microseconds. The switching may occur over a period of at most about 10,000, 9,500, 9,000, 8,500, 8,000, 7,500, 7,000, 6,500, 6,000, 5,500, 5,000, 4,500, 4,000, 3,500, 3,000, 2,500, 2,000, 1,500, 1,000, 900, 800, 700, 600, 500, 400, 300, 200, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, or less microseconds. The switching may enable electrical flow within at least about 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 75, or more percent of a predetermined target. The switching may enable electrical flow within at most about 75, 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1 or less percent of a predetermined target. The thermoelectric control architecture may be configured to control each element (e.g., control conditions within each well) using a feedback loop. The thermoelectric control architecture may not comprise use of pulse width modulation (PWM). Not using PWM may reduce interference effects (e g., electromagnetic noise) and improve the functioning of the control architecture. The control architecture may comprise a continuous switching. For example, the continuous switching may comprise providing a continuously adjustable power to the thermal element. The control circuitry may comprise use of one or more proportional-integral-derivative (PID) controllers.

[0228] An efficiency of power delivery to the one or more thermal elements (e g., a ratio of input power to the controller to power delivered to the thermal elements of the system) may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more percent. An efficiency of the power delivery to the one or more thermal elements may be at most about 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10, or less percent. Having a high power delivery efficiency can provide robust temperature control over a plurality of chambers while maintaining a power budget that can be supplied by a wall outlet.

[0229] FIGs. 12A-12B show example schematics of control architectures (e g., control circuits), according to some embodiments. The control architecture can comprise a computer processor 1203. The computer processor may be within the device. The computer processor may be external to the device. The control architectures may provide communication between the daughterboards, master control circuitry, thermal elements, and / or sensors. A daughterboard may be operably coupled to a sensor and be configured to receive a signal from the sensor. TheAtty Dkt No.: 61197-702601 master controller can be operably coupled to one or more daughterboards and provide a set value for a condition within a chamber. For example, the daughterboard can receive the set value from the mater controller and transmit a signal to a thermal element associated with the chamber to affect a temperature condition within the chamber. The control architecture may be configured to receive one or more signals from the sensor, interpret the one or more signals to determine a property (e.g., a temperature) of a chamber operably coupled to the sensor, provide power to a thermal element operably coupled to the chamber, or change a condition (e.g., a temperature) of the chamber, or any combination thereof. Each daughterboard may comprise a master microcontroller 1201 and at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more sub-controllers 1202. The master microcontroller and the sub-controllers may be connected via a communication line 1205. Each sub-controller may be connected to a set of sensor boards configured to measure and / or control the temperature of at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more reaction chambers. Each sensor board can comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more temperature sensors (e g., digital temperature sensors). The temperature sensors may be interfaced through one or more slave controllers 1204. The temperature sensors can be calibrated (e.g., calibrated by a National Institute of Science and Technology (NIST) tracible sensor). The temperature sensor may be configured to read a temperature with an accuracy of at least about + / - 0.01, 0.05, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, or more degrees Celsius. The temperature sensor may be configured to read a temperature with an accuracy of at most about + / - 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, 0.05, 0.01, or less degrees Celsius. The measurements of the temperature sensor may be digital measurements. The digital measurements may be configured to avoid the effects of electrical noise (e.g., electromagnetic interference), which can improve the functioning of the temperature sensors and can enable the individual control of the reaction chambers.

[0230] Other sensors include, but are not limited to, optical sensors, electrical sensors, magnetic field sensors, viscosity sensors, or pH sensors, or any combination thereof. The optical sensors may comprise fluorescence sensors, absorption sensors, spectrographic sensors, or refractive index sensors. The electrical sensors may comprise voltage sensors, resistance sensors, conductivity sensors, or capacitance sensors.

[0231] The use of the daughterboard architecture may be modular. For example, the daughterboards may be interchangeable. The daughterboards may have a same function to one another. For example, the daughterboards can control one or more thermal elements in a same way. A daughterboard may be configured to control any thermal elements of the one or more thermal elements. The modular architecture can provide for efficient replacement of damagedAtty Dkt No.: 61197-702601 parts (e ., a damaged daughterboard can be replaced by another daughterboard). The modular architecture may allow for swapping daughterboards in a way that does not affect the communications protocol with or functioning of the temperature sensors or elements. The daughterboards may have a same communication protocol. The overall communication can be controlled by the master controller (e.g., master microcontroller). Examples of communication protocol buses include, but are not limited to, Serial Peripheral Interface (SPI), Inter-Integrated Circuit (I2C), Universal Asynchronous Receiver / Transmitter (UART), or the like. The communication protocol bus may be in contact with the daughterboard’s master controller. Commands and / or data can flow through the bus to the breakout board master controller and then to the main onboard processing unit. The communication and data collection architecture can provide modularity and can be used to address a plurality (e g., 96) of reaction chambers.

[0232] FIG. 5 is a schematic of an addressable control array 500, according to an embodiment. The addressable control may be configured to control a plurality of elements situated as an array. The control array may comprise connecting one or more elements to a single microcontroller. The elements may be connected via a serial bus (e g., serial peripheral interface (SPI), I2C, etc.). The array may comprise a plurality of microcontrollers each connected to an additional controller. For example, a series of micro controllers can be connected to another microcontroller. The additional controller and / or the plurality of microcontrollers can be connected to a master controller. For example, a first microcontroller can control a first plurality of elements and a second microcontroller can control a second plurality of elements. In this example, the two microcontrollers can in turn be connected to the master controller. In another example, a first and second microcontroller can be connected to a third microcontroller while a fourth and fifth microcontroller are connected to a sixth microcontroller. In this example, the third and sixth microcontrollers can be connected to the master controller. The master controller may comprise a computer system as described elsewhere herein. For example, the master controller can comprise a microcontroller. In another example, the master controller can comprise a field programmable gate array (FPGA). At least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more elements can be connected to a single microcontroller. At most about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 element can be connected to a single microcontroller. At least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or more microcontrollers can be connected to another microcontroller. At most about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 microcontroller can be connected to another microcontroller. At least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, or moreAtty Dkt No.: 61197-702601 microcontrollers can be connected to the master controller. At most about 50, 45, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 microcontroller can be connected to the master controller.

[0233] FIG. 9 is an example of a plurality of views of a controller daughterboard 900, according to some embodiments. The controller daughterboard may be configured to perform the methods described elsewhere herein. For example, the daughterboard can be configured to control at least a temperature in a single well of a multi-well plate. The controller daughterboard can comprise controller circuitry 910. The controller circuitry may be configured to control a temperature of a single well. For example, the daughterboard 900 can comprise 8 sets of controller circuitry as shown. A controller daughterboard may comprise at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 30, 40, 50, 60, 70, 80, 90, 96, or more sets of control circuitry 910. A controller daughterboard may comprise at most about 96, 90, 80, 70, 60, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or fewer sets of control circuitry. The controller daughterboard may comprise an interface 901. The interface may be configured to permit addressing of the various components of the daughterboard from a device the daughterboard is interfaced with. For example, a plurality of contacts can be positions on the interface to permit communication between a system and a daughterboard. The daughterboard may comprise one or more microprocessors 902. The one or more microprocessors may be configured as intermediaries between the control circuitry and the system. For example, a microprocessor can be configured to interpret signals from the system to provide output electrical signals to the control circuitry.

[0234] In some cases, the control circuitry may comprise one or more switch buck regulators. The switch buck regulators may have an efficiency of at least about 50, 60, 70, 80, 90, 95, 96, 97, 98, 99, or more percent. The switch buck regulators may have an efficiency sufficient to enable the individual addressability of the thermal elements as described elsewhere herein while maintaining a power budget that can be supplied by, for example, a wall plug. The devices described elsewhere herein may be configured to provide individual thermal control via thermoelectric elements to at least about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 16, 24, 25, 48, 50, 75, 96, 100, 150, 200, 250, 300, 350, 384, 400, 450, 500, or more wells using a single wall plug. The individual thermal control may comprise individual thermal elements, individual sensors, thermal management elements disposed between the thermoelectric elements, or individual thermally conductive elements, or any combination thereof. The control circuitry may comprise two or more switching buck converters controlled externally by one or more microcontrollers. Such a design may not use an H-bridge. Not using an H-bridge may provide improved efficiency (e.g., improved due to the lower loss profile of not having the transistors of the H-bridge). The design may use of one or more microcontrollers and / or one or more DC / DC converters in placeAtty Dkt No.: 61197-702601 of an H-bridge. For example, the properties and conditions of the thermal element can be controlled by the control circuitry comprising the one or more microcontrollers and / or the one or more DC / DC converters. Use of an external microcontroller may enable reduced losses by turning off the buck converters prior to turning off the transistors. The buck converters can be controlled by an external microcontroller. The buck converters can be powered up or powered down by the external microcontroller. In some cases, the daughterboard can comprise 8 sets of controller circuitry in an area of at most about 10,000, 9,500, 9,000, 8,500, 8,154, 8,000, 7,500, 7,000, 6,500, 6,000, 5,500, 5,000, 4,500, 4,000, 3,000, 2,000, 1,000 or fewer centimeters squared.

[0235] FIG. 10 is an example of a control circuitry 910, according to some embodiments. The TEC may be a thermal element as described elsewhere herein (e g., a thermoelectric element). The control circuitry may be configured to control a temperature of a single thermal element. For example, each thermal element of a system may have a corresponding set of control circuitry. The control circuitry may be configured to take an input signal from, for example, one or more microprocessors and convert the input signal into a temperature change in the thermal element. The control circuitry may be temperature control circuitry. The control circuitry may be control circuitry for another property (e.g., gas conditions, stirring, etc ). The control circuitry may utilize digital control of the power delivered to the one or more thermal elements. The control circuitry may not use exclusively analog control of the one or more thermal elements. The current and / or voltage output of the control circuitry may not be fixed (e g., may be dynamic). The current and / or voltage may be controlled at least in part by software.

[0236] The DAC set (e g., DAC set Chi, DAC set Ch2) may be a signal from a digital analog convertor (DAC). For example, a DAC can receive a digital signal from a processor (e g., a microcontroller), convert it to an analog signal, and provide the analog signal to the DC / DC convertor (DC / DC). From the DC / DC convertor, voltages Vol and Vo2 can be applied to the TEC. Chi and Ch2 can be control signals from the DC / DC converter configured to direct a flow of electricity through the thermal element. For example, when a signal is provided from Ch2, the thermal element can have a flow of electricity if a first direction, while a signal from Chi can have the flow of electricity in an opposite second direction. The DC / DC converter of FIG. 10 may control both directions of current flow through the thermal element. For example, instead of using a plurality of DC / DC converters as in FIG. 4A, a single DC / DC converter can control the current through the thermal element.

[0237] The control circuitry can comprise one or more of a DC / DC convertor, a microcontroller (e g., a microcontroller comprising embedded control software), a temperature probe, or the like,Atty Dkt No.: 61197-702601 or any combination thereof. The control circuitry may have a latency of controlling the current and / or voltage provided to a thermal element of at most about 100, 50, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, 0.05, 0.01, 0.005, 0.001, or fewer milliseconds. The control circuitry may have a latency of controlling the current and / or voltage provided to a thermal element of at least about 0.001, 0.005, 0.01, 0.05, 0.1, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 100, or more milliseconds. The fast control of the power delivered to the thermal element can enable fast reactions to changes in the thermal conditions within, for example, a well adjacent to the thermal element. For example, a well that is starting to experience temperature drift can undergo a quick correction that can, in turn, keep the temperature of the well close to the set temperature. The low latency may be related to a use of a digital controller (e.g., microprocessor). While other systems may employ fixed feedback loops, the direct control of the power delivery to the thermal element can enable fast and efficient control of the thermal element. The control circuitry may provide power to the thermal element with an efficiency of at least about 50, 60, 70, 80, 90, 95, 99, or more percent. Each thermal element of a plurality of thermal elements in the system can have different control parameters, which can enable use of a wider variety of thermoelectric elements and increase overall system efficiency.

[0238] The control circuitry may enable gradient control of the power delivery to the thermal element. For example, the gradient control can comprise tuning the power delivery to provide the predetermined thermal conditions from the thermal element along a continuous spectrum. The power delivery to the thermal element may be continuous. For example, the power delivery can be any value within a range of powers. The power delivery to the thermal element may be discrete. For example, the power delivery can be on or off. In this example, instead of achieving thermal control by modulating an on / off fraction of the thermal element, the modulating can be achieved by controlling the amount of power provided to the thermal element. The amount of power delivered to the thermal element can be controlled by the microcontroller. Such continuous control can provide fast control of the thermal elements as well as finer temperature control. The control circuitry may have an electrical noise of at most about 20, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.5, 0.1, or less percent of the amount of power delivered. The control circuitry may have an electrical noise of at least about 0.1 , 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 20, or more percent of the amount of power delivered.

[0239] FIG. 11 is a cut away (e.g., showing an interior) example of a system 1100 as described elsewhere herein, according to some embodiments. For example, the system can be configured to perform the methods described elsewhere herein. The system may comprise a well plate 1101. The well plate may be as described elsewhere herein. For example, the well plate may be a 96Atty Dkt No.: 61197-702601 well plate. Each chamber of the substrate 1101 may be configured to have an individually controlled temperature as described elsewhere herein. The individual control of the temperature may be achieved at least in part through use of one or more controller daughterboards 900. The controller daughterboards may be interfaced into the system via ports 1103. The ports may be configured to operably couple to the controller daughterboards to provide electrical contact between the thermal elements of the systems and the control circuitry of the daughterboards. The use of ports may enable switching the controller daughterboards (e.g., for maintenance, for different functionalities, etc ). The use of ports may enable changes to the system configuration over time (e.g., addition or subtraction of control channels, etc.). The system may be configured with a number of daughterboards such that each well of the well plate is operably coupled to control circuitry. For example, the system can be configured with daughterboards comprising control circuitry such that there is a control circuit for every thermal element of the system.

[0240] The system may comprise a heatsink 1102. The heatsink may be configured as described elsewhere herein. For example, the heatsink can be configured to aid in the thermal equilibrium (e.g., heating and / or cooling) of a thermoelectric element. The system may comprise one or more fans 1 104. The one or more fans may be configured to provide cooling for the daughterboards, the heatsink, or a combination thereof. For example, the fans can be configured to both cool the control circuitry of the daughterboards as well as the heatsink.Computer systems

[0241] The present disclosure provides computer systems that are programmed to implement methods of the disclosure. FIG. 6 shows a computer system 601 that is programmed or otherwise configured to implement the methods and / or interface with the devices of the present disclosure. The computer system 601 can regulate various aspects of the present disclosure, such as, for example, regulate conditions within a chamber, monitor signals from the chamber, etc. The computer system 601 can be an electronic device of a user or a computer system that is remotely located with respect to the electronic device. The electronic device can be a mobile electronic device.

[0242] The computer system 601 includes a central processing unit (CPU, also “processor” and “computer processor” herein) 605, which can be a single core or multi core processor, or a plurality of processors for parallel processing. The computer system 601 also includes memory or memory location 610 (e.g., random-access memory, read-only memory, flash memory), electronic storage unit 615 (e.g., hard disk), communication interface 620 (e.g., network adapter) for communicating with one or more other systems, and peripheral devices 625, such as cache, other memory, data storage and / or electronic display adapters. The memory 610, storage unitAtty Dkt No.: 61197-702601615, interface 620 and peripheral devices 625 are in communication with the CPU 605 through a communication bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit (or data repository) for storing data. The computer system 601 can be operatively coupled to a computer network (“network”) 630 with the aid of the communication interface 620. The network 630 can be the Internet, an internet and / or extranet, or an intranet and / or extranet that is in communication with the Internet. The network 630 in some cases is a telecommunication and / or data network. The network 630 can include one or more computer servers, which can enable distributed computing, such as cloud computing. The network 630, in some cases with the aid of the computer system 601, can implement a peer-to-peer network, which may enable devices coupled to the computer system 601 to behave as a client or a server.

[0243] The CPU 605 can execute a sequence of machine-readable instructions, which can be embodied in a program or software. The instructions may be stored in a memory location, such as the memory 610. The instructions can be directed to the CPU 605, which can subsequently program or otherwise configure the CPU 605 to implement methods of the present disclosure. Examples of operations performed by the CPU 605 can include fetch, decode, execute, and writeback.

[0244] The CPU 605 can be part of a circuit, such as an integrated circuit. One or more other components of the system 601 can be included in the circuit. In some cases, the circuit is an application specific integrated circuit (ASIC).

[0245] The storage unit 615 can store files, such as drivers, libraries and saved programs. The storage unit 615 can store user data, e g., user preferences and user programs. The computer system 601 in some cases can include one or more additional data storage units that are external to the computer system 601, such as located on a remote server that is in communication with the computer system 601 through an intranet or the Internet.

[0246] The computer system 601 can communicate with one or more remote computer systems through the network 630. For instance, the computer system 601 can communicate with a remote computer system of a user. Examples of remote computer systems include personal computers (e g., portable PC), slate or tablet PC’s (e.g., Apple® iPad, Samsung® Galaxy Tab), telephones, Smart phones (e g., Apple® iPhone, Android-enabled device, Blackberry®), or personal digital assistants. The user can access the computer system 601 via the network 630.

[0247] Methods as described herein can be implemented by way of machine (e g., computer processor) executable code stored on an electronic storage location of the computer system 601, such as, for example, on the memory 610 or electronic storage unit 615. The machine executable or machine readable code can be provided in the form of software. During use, the code can beAtty Dkt No.: 61197-702601 executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610.

[0248] The code can be pre-compiled and configured for use with a machine having a processer adapted to execute the code, or can be compiled during runtime. The code can be supplied in a programming language that can be selected to enable the code to execute in a pre-compiled or as- compiled fashion.

[0249] Aspects of the systems and methods provided herein, such as the computer system 601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and / or associated data that is carried on or embodied in a type of machine readable medium. Machine-executable code can be stored on an electronic storage unit, such as memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk.“Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless links, optical links or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

[0250] Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, a tangible storage medium, a carrier wave medium or physical transmission medium. Non-volatile storage media include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such as may be used to implement the databases, etc. shown in the drawings. Volatile storage media include dynamic memory, such as main memory of such a computer platform. Tangible transmissionAtty Dkt No.: 61197-702601 media include coaxial cables; copper wire and fiber optics, including the wires that comprise a bus within a computer system. Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD or DVD-ROM, any other optical medium, punch cards paper tape, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables or links transporting such a carrier wave, or any other medium from which a computer may read programming code and / or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

[0251] The computer system 601 can include or be in communication with an electronic display 635 that comprises a user interface (UI) 640 for providing, for example, results of a PCR reaction from a chamber. Examples of UI’s include, without limitation, a graphical user interface (GUI) and web-based user interface.

[0252] Methods and systems of the present disclosure can be implemented by way of one or more algorithms. An algorithm can be implemented by way of software upon execution by the central processing unit 605. The algorithm can, for example, determine that a PCR reaction has completed and halt the reaction within a single chamber.EXAMPLES

[0253] The following examples are included for illustrative purposes only and are not intended to limit the scope of the invention.Example 1 - normalization of a sample using a fluorescence endpoint

[0254] FIG. 7 is a flow chart of control logic 700 for a chamber, according to an embodiment. The logic may begin with loading a temperature cycling profile { ti,Ti]p. The temperature cycling profile may comprise p pairs of time and temperature points {ti,T;} . The temperature cycling profile may be configured to enable a performance of a PCR reaction as described elsewhere herein. For example, the temperature cycling profile, when performed, can result in a cycle of PCR being performed. Next, a new cycle can be performed with the loaded profile. Upon or before completion of the cycle, the fluorescence signal can be acquired. For example, the signal can be acquired at a quantification operation {tq,Tq}. The fluorescence signal can be acquired as described elsewhere herein. Upon receipt of the fluorescence signal, one or more computer processors as described elsewhere herein can determine if a threshold condition (e g., aAtty Dkt No.: 61197-702601 predetermined normalization threshold, a predetermined level of fluorescent intensity, etc ). If the fluorescence signal has not reacted the threshold condition, the number of cycles can be incremented and the process repeated. If the fluorescence signal meets the threshold condition, the logic can stop the PCR reaction in the chamber by setting the temperature to < 20 °C. Since, as described elsewhere herein, different chambers can be controlled independently, the completion of one reaction does not mean that other reactions will not continue as described above. For example, a first chamber can meet the threshold condition while a second chamber does not. In this example, the reaction in the first chamber can be halted by reducing the temperature while the reaction in the second chamber can be unaffected.

[0255] FIG. 8 is an example of a normalization based on a fluorescence endpoint, according to an embodiment. The plot 800 may be a plot of relative fluorescent intensity on the y-axis against the number of PCR cycles performed on the x-axis. The number of cycles to reach an endpoint may be different for different chambers (e.g., chambers Chx, Chy, and Chz). In this example, Chxcan reach a predetermined fluorescence signal threshold after 9 cycles, while Chytakes 10 and Chztakes 11. For each chamber, once the chamber reaches the predetermined normalization threshold, the chamber can be deactivated using the individual temperature control for each chamber. For example, each chamber can be cooled to halt a PCR reaction after the signal from the chamber exceeds the normalization threshold.Example 2 - thermal performance of adjacent wells

[0256] FIG. 13 is an example of the thermal performance of a plurality of adjacent individually controlled wells, according to some embodiments. In initial time points (e.g., at early times on the graph), the three wells can be performing the same temperature cycling protocol (e g., cycling between 95 and 60 degrees Celsius). The different wells may show similar performance when under the same protocol, as evidenced by the similar temperature curves. After 2 cycles, the third well may no longer be under the same temperature protocol. For example, the third well can be set to dwell at 60 degrees Celsius while the other two wells continue cycling. The data of FIG. 13 may show that the wells of the plate are individually addressable, with each well being able to be put at a predetermined temperature independent of the other wells. This may be further evidenced by the performance of wells 2 and 1 , which are cycled for 7 and 17 cycles, respectively.

[0257] The lower plot of FIG. 13 may show an expanded temperature and time range for the three wells. After being held at 60 degrees Celsius, well 3 can be cooled to 17 degrees (e.g., below ambient temperature) while the other two wells continue the elevated temperature cycling. Despite being adjacent (e g., < 10 millimeters distant) to the elevated temperature wells, well 3Atty Dkt No.: 61197-702601 can be maintained at a lower temperature due to the thermal isolation of the well. Such a capacity can provide the ability to individually control the reaction conditions of a given well, which can permit customization of the reaction to the given well. After a time, the temperature control of well 3 can be turned off, which can result in the observed temperature drift of well 3. Wells 1 and 2 can be kept at similar temperatures (e.g., about 0.2 degrees Celsius difference) until well 2 is cooled and subsequently allowed to drift.

[0258] FIG. 14 shows the performance comparison of individually controlled wells versus non- individually controlled wells, according to some embodiments. In some cases, un-normalized PCR reactions can generate different amounts of signal depending on a number of factors, such as, for example, reaction conditions, reagent loading, initial loading of the target nucleic acid, etc. Such inconsistencies can generate curves such as those present in the upper plot of FIG. 14. In these plots, the reactions occurring in the various wells can provide different amounts of signal after different numbers of cycles. In some cases, individual control of the wells can enable normalization of the wells to provide a similar amount of signal from different wells, as shown in the lower plot of FIG. 14. In the lower plot, the wells can be normalized (e.g., by controlling the individual conditions of the wells) to provide a similar amount of signal for a given number of cycles.Example 3 - optimization of annealing temperature

[0259] FIG. 22 shows an example of an annealing temperature optimization experiment, according to some embodiments. In some cases, performing an amplification at an optimal annealing temperature can improve the signal collected from the amplification reaction, as well as improve the speed at which the reaction is performed. An optimal annealing temperature can be determined on a per-reaction basis, where a test reaction can be performed, and subsequent reactions can utilize the optimal temperature determined by the test reaction. In this way, the optimization of the annealing temperature may be performed once for a plurality of reactions.

[0260] The annealing temperature optimization may be enabled by use of real-time monitoring of the well the reaction is being performed in, as well as control of the reaction conditions within the well. A system described elsewhere herein may be configured to perform a plurality of optimization reactions at a same time. For example, each well of a well plate can comprise a different optimization reaction.

[0261] In some cases, an optimization reaction can comprise beginning an amplification reaction at a first temperature (e.g., the 60 degrees Celsius of FIG. 22). The amplification reaction can be repeated for a plurality of cycles until a signal is detected from the amplification reaction (e g., cycle 10 of FIG. 22). Once the signal is detected from the amplification reaction, the temperatureAtty Dkt No.: 61197-702601 of the reaction can be increased with each subsequent cycle. As the temperature is increased, the rate of increase of the signal (e.g., fluorescent signal) may increase. At some point (e.g., cycle 18 of FIG. 22), the signal increase may stop. This may be due to the temperature of the reaction being set too high, and thus the optimal temperature may be determined (e.g., the temperature one cycle lower than the temperature that caused the signal to stop). The optimal temperature may then be applied for subsequent cycles, as well as for the processing of subsequent reactions.Example 4 - multi-well temperature gradients

[0262] FIG. 23 shows a plot of temperature profiles obtained within 24 wells executing a protocol with a 65 degree Celsius (C) linear gradient, according to some embodiments. In this example, 24 wells were set to temperatures according to FIGs. 24A - 24B. For example, FIG. 24B can show a temperature gradient over 24 wells across 3 columns. In each case, the wells were all set to 95 degrees Celsius at first and all set to 72 degrees Celsius to finish. The wells were set to a gradient according to FIG. 24B between the 95 degree Celsius and 72 degree Celsius settings spanning 30 to 80 degrees Celsius. As observed in FIG. 23, no cross-talk between the wells is seen, demonstrating individual control of the thermal properties of each of the wells.Example 5 - support comprising thermally conductive elements

[0263] FIG. 28 shows an example of a support comprising one or more thermally conductive elements 2801 , according to some embodiments. The support 2800 may be a support as described elsewhere herein (e.g., a support for use in method 2500 of FIG. 25, etc.). The support can comprise a plurality of thermal elements 2810 disposed above one or more thermally conductive elements 2801 between thermal elements (e g., thermoelectric elements) 2820 and the heatsink 2830. Thermal conductors 2802 may decrease a gap between the thermally conductive element and the thermal element or the heatsink.

[0264] Instead of connecting a thermoelectric element or thermal element directly to a heatsink, the heat from the thermal element can be sunk into the heatsink through a metal PCB. The main breakout board (e.g., daughterboard, support) can be a multi-layered metal PCB, and the thermal element can be disposed on top of the PCB (e g., to simplify an assembly process). The metal PCB can be a breakout board bringing signal and / or power to the thermal elements while also providing an intermediate heatsink to the heatsink. Thermally conductive elements (e.g., copper pads, aluminum pads, indium pads) can be disposed where the thermal element comes in contact with the PCB to improve heat sinking capacity. To further improve thermal contact between all of the elements, a thermal conductor (e.g., graphite sheets, indium foils, thermal pastes) can be inserted between the thermal element, support, and heatsink.Atty Dkt No.: 61197-702601Example 6 - current sinking power supplies

[0265] FIGs. 29A and 29B show examples of power supplies configured to provide power to a thermoelectric element, according to some embodiments. The power supply can comprise a buck-boost converter as described elsewhere herein. The power sinking converter can comprise a feedback signal loop configured to adjust power provided by the power supply to the thermoelectric element. For example, feedback from the thermoelectric element can be provided to the current sinking converter, and the information from the feedback can influence the power delivery of the power supply. The presence of the power sinking converter can make a power range (e.g., maximum power deliverable by the power supply) symmetrical. For example, without use of a power sinking converter, the power supply can provide between about 1.6 and - 0.4 amps of power. In this example, the current sinking converter can make the power delivery range symmetrical (e.g., between 0.4 and -0.4 amps).

[0266] In some cases, a buck-boost mode convertor can be used. The buck-boost converter can have a thermal element (e.g., thermoelectric element) connected between a voltage input and a voltage output of the buck-boost device. The buck-boost converter can have a current sink capacity. The convertor and the microprocessor may enable high frequency control of the thermal element. For example, the control may have a frequency of at least about 0. 1, 0.5, 1, 2, 3, 4, 5, 10, 15, or more megahertz (MHz). In another example, the control may have a frequency of at most about 15, 10, 5, 4, 3, 2, 1 , 0.5, 0.1 , or less MHz.

[0267] Vin can be a voltage input (e.g., form a power source). The power source can be, for example, an output of a DC / DC converter, an output of an AC / DC converter, or a combination thereof. Vout can be an output voltage (e.g., a regulated output voltage) of the buck-boost converter. Vcti can be a control voltage. The control voltage can be generated by a digital to analog converter (DAC). For example, a control signal can be generated by control circuitry and / or a computer processor, and the control signal can be converted to an analog control voltage by the DAC. The control voltage can be used to regulate the output voltage of the buck-boost converter. A resister bridge comprising resistors Rl, R2, and R3 can convert the value of the Vcti into a feedback signal to scale the output voltage of the buck-boost converter from its minimum to its maximum voltage. The value of Vcti may be at least about 0, 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9,3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, or more volts. The value of Vcti may be at most about 4.0, 3.9, 3.8, 3.7, 3.6, 3.5, 3.4, 3.3, 3.2, 3.1, 3.0, 2.9, 2.8, 2.7, 2.6, 2.5, 2.4, 2.3, 2.2, 2.1, 2.0, 1.9, 1.8, 1.7, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, 0.1, or less volts.Cl and C2 may be capacitors that stabilize the voltage input and output, and L can be an inductorAtty Dkt No.: 61197-702601 that further stabilizes the voltage (e.g., during high frequency switchin). En can be an on / off signal (e.g., an enabling signal) for the control circuitry. For example, when En is applied to the converter, the converter can be switched on.

[0268] In the example of FIGs. 29 A and 29B, for a Vinof 3 V the output voltage can be varied from 1 V to 5 V. A thermoelectric element can be set to heat or cool depending on the voltage different between V;nand Vout. A buck-boost converter can source more current than it has the capacity to sink, which can result in an asymmetric net current passing through the thermoelectric element and can result in a different between the cooling and heating power of the thermoelectric element. A transistor (e.g., a field effect transistor) can be coupled to the design such that, when the current sinking of the buck-boost converter is exceeded, the buck-boost converter can be disabled and the thermoelectric element can be grounded, thereby permitting more current to pass and revert to the original output asymmetry. The thermal element can be in place between a plurality of buck-boost converters, which can make the output of the thermal element symmetrical with regard to current source and sink.

[0269] The power supply may have an input voltage between Vout and Vin. The current used by a plurality of controllers may not scale (e g., the current used by the plurality of controllers may not be additive).Example 7 - systems and movable assemblies

[0270] FIGs. 30A and 30B show an example of a system comprising a thermoelectric plate and a plurality of chambers, according to some embodiments. The system 3000 can comprise a plurality of chambers 3010, a thermoelectric plate 3020, and a movable assembly 3030 as described elsewhere herein. The system can be configured to use a lifting bracket 3040 to move the thermoelectric plate 3020 into contact with the bottoms of the plurality of chambers 3010. The use of the lifting bracket, along with the linear motion of the thermoelectric plate can enable precise alignment of the thermoelectric elements of the thermoelectric plate with the plurality of chambers.

[0271] The system can comprise a drawer 3060 configured to hold one or more substrates (e g., well plates) as described elsewhere herein. The drawer can be configured to hold at least about 1, 2, 3, 4, 5, 6, 7, 8, or more well plates. The drawer may provide access to insert and remove the well plates into and out of the system. For example, the drawer may enable insertion of a well plate without opening the rest of the system, reducing the chance of contamination or damage to the system. Once the drawer with the well plate is inserted into the system, the lever 3050 can be actuated to lift the lifting bracket 3040 and the thermoelectric elements in the thermoelectric plate 3020 up and into contact with the substrate. The contact may be as described elsewhereAtty Dkt No.: 61197-702601 herein (e.g., with a precise positioning of the thermoelectric elements to the wells of a well plate). Once in position, the optics head 3070 (e.g., an optics head as shown in FIG. 20) can be moved over the substrate to image the substrate (e.g., the wells of the substrate). The imaging may be as part of an amplification reaction as described elsewhere herein.

[0272] FIG. 42A shows an example of a system 4201 with a movable assembly 4203 and substrate 4204 in an up position, according to some embodiments. The up position may place the substrate into contact with the cover plate 4205. When in the up position, the system may be ready to perform a PCR reaction. FIG. 42B shows an example of a system 4202 with a movable assembly and substrate in a down position, according to some embodiments. The down position may permit a loading and / or unloading of the substrate from the system. FIG. 42C shows an example of the system 4202 with the movable assembly in a down position and the substrate ready to be removed from the system via door 4206, according to some embodiments. FIG. 43A shows another angle of the system 4201 in a closed position, according to some embodiments. The motor 4301 can drive screw 4302 to move nut 4303 to the up position, thereby closing the substrate to the cover plate. FIG. 43B shows another angle of system 4202 in an open position, according to some embodiments. Arm 4305 can be attached to the nut 4303 to aid in the movement of the movable assembly. In this example, the motor 4301 can drive the screw 4303 down via nut 4303, lowering the movable assembly and permitting access to the substrate. A pivot 4304 can enable movement (e.g., rocking) of the motor. This may provide a compact conversion of rocking motion (e.g., of the motor and the screw) into linear motion of the movable assembly. Interface 4306 can provide, for example, switches, of power cable interfaces, data interfaces, or any combination thereof. FIG. 46 shows an example of a system 4601 (e.g., a system of the present disclosure) and associated computer system 4602 (e.g., a computer system of the present disclosure), according to some embodiments.

[0273] In this example, the movable assembly (e.g., comprising a plurality of thermal elements and a heatsink) can be lowered using a rocking motor (e g., stepper motor). The well plate drawer 4206 can be opened using a drawer motor (e.g., stepper motor) to permit loading of the substrate comprising a reaction mixture. The movable assembly can be raised via the motor to enclose the chambers of the substrate between the plurality of thermal elements and the cover plate. The vertical movement of the movable assembly can be guided by one or more sliding guides 4207 within the system. Pivots 4208 can convert semi-arc movement of the motor into linear movement of the movable assembly. Motor 4209 can be configured to open or close a door (e.g., a door that permits outside access to the substrate).Atty Dkt No.: 61197-702601

[0274] FIG. 44 shows an example of an alignment feature, according to some embodiments. Alignment feature 4401 (e.g., alignment pin) can be configured to couple with alignment feature 4402 (e.g., alignment hole), thereby aligning the cover plate 4403 with the substrate 4404. Limit switch 4405 can detect a placement of the substrate 4404 and prevent overtravel. In order to assist in precise positioning of the substrate, the drawer mechanism can use a limit switch (e.g., optical limit switch) and two or more features (e g., alignment features). When the drawer is travelling into and close to its final position, it can actuate the limit switch which, in turn, stops the movement of the motor driving the drawer. This can ensure that the substrate is roughly aligned with its final position. During the movement of the movable assembly, the final precise positioning can be ensured by the one or more alignment features engaging with one another.

[0275] FIG. 45 shows an example of a system of the present disclosure 4500, according to some embodiments. The system can comprise a drawer 4501 configured to permit access to the substrate. Baseplate 4502 can provide a foundation for the other components of the system. Movable assembly 4503 can hinge on pivot 4504. Data connection (e g., ethemet connection 4505 can provide data access to the system, while power connector 4506 can provide power into the system. Motor 4507 can be supported by bracket 4508 (e g., a rocking bracket) and be configured to drive screw 4509. Screw 4509 can interface with nut 4510 to move the movable assembly 4503. Control circuitry 4511 can be configured to operate the various subsystems of the present disclosure (e.g., sensing systems, thermal elements, optical systems, etc ). Motors 4512 can be configured to provide two dimensional movement of optical assembly 4513.Example 8 - Next-generation sequencing (NGS) library normalization

[0276] A lack of normalization in a DNA library for next-generation sequencing (NGS) can lead to inconsistencies in data quality. For example, some libraries with high concentration can be overrepresented while libraries with low concentration can be underrepresented, which can lead to inaccurate or incomplete data for analysis. Thus, NGS library normalization is important and highlights the need for accurate and efficient method of normalizing NGS libraries. This example provides an example of how a plurality of samples can be normalized using the methods and systems of the present disclosure in preparation for use in a next generation sequencing operation.

[0277] The number of cycles (e g., PCR cycles) in preparing a library for NGS (e g., via the methods and systems of the present disclosure) may be minimized. The library generation may comprise complimentary DNA (cDNA) amplification, generating library indexes, or a combination thereof. The library generation may comprise generating DNA without overamplification. Setting a maximum number of cycles may reduce over-amplification. TheAtty Dkt No.: 61197-702601 maximum number of cycles may be within at most about 5, 4, 3, 2, or 1 cycles of a manufacturer recommended maximum number of cycles.

[0278] A level of fluorescence in a PCR reaction may be correlated with an amount of double started DNA present in the reaction mixture. For some applications, larger amounts of DNA are used. For applications that use a larger amount of DNA, the slopes auto-normalization mode described elsewhere herein, which may stop a reaction at a higher end of an amplification curve. Cycles can be added to increase the amount of DNA generated. Samples may be amplified to similar levels and be able to be pooled. The pooling may provide higher amounts of DNA for downstream applications. The normalized pools may be generated in an absence of additional purification (e.g., bead purification), DNA quantification, or manual normalization, or any combination thereof. In an example, if the product of 96 reaction mixtures is to be pooled and sequenced, the amount of DNA from each reaction mixture can be lower than if 8 reaction mixtures are to be processed.

[0279] As an example of the methods and systems of the present disclosure’s ability to automate normalization across different DNA concentrations, 1 ng / ul of DNA template (e g., pUC19) was serial diluted by 4-fold to yield DNA concentrations of 0.25 ng / ul, 0.06250 ng / ul, 0.1563 ng / ul, 0.00391 ng / ul, 0.00098 ng / ul, 0.00024 ng / ul, and 0.00006 ng / ul. Next, PCR master mix was prepared with water, universal qPCR Master Mix (e g., Luna Universal qPCR Master Mix), forward primer, and reverse primer. The final PCR master mix was made up of IX qPCR Master Mix, 0.4 uM of forward primer, and 0.4 uM of reverse primer. 45 ul of the final PCR master mix were distributed into each well of two 8 well strip. For both 8 well strips, 5 ul of various concentrations of DNA template were added into each well of the 8-well strip, wherein each well had 1 ng / ul, 0.25 ng / ul, 0.06250 ng / ul, 0.1563 ng / ul, 0.00391 ng / ul, 0.00098 ng / ul, 0.00024 ng / ul, or 0.00006 ng / ul of the DNA template. One of the 8-well strip was put into a thermocycler with the thermocycling conditions shown in Table 1. The second 8-well strip was put into the device described herein.Table 1. Thermocycling conditionsAtty Dkt No.: 61197-702601

[0280] As shown in FIG. 32A, when using a reference method with a fixed number of cycles set in a thermocycler, many samples were under amplified, as shown by the low relative fluorescence unit (RFU). The samples are samples A-H, corresponding to samples numbered 1-8 (# column) of FIG. 31. Samples 1-8 have the listed input concentrations of nucleic acids (in the range from 1 to 0.00006 nanograms per microliter), representing a 16,384 fold range. After amplification, the output concentrations of the various samples can be found in the ‘n6 output’ column, also in nanograms per microliter. Despite the wide range of input concentrations, the outputs are shown to be in a 1 .21 fold range, showing the ability of the methods and systems of the present disclosure to normalize disparate ranges of nucleic acid concentrations. Using a fixed number of cycles (e g., not a dynamic normalization) provides the results shown in the ‘Current method’ column. These results show the over-amplification of the more concentrated samples and the under-amplification of the samples with lower concentration. This shows the improvement of the methods and systems of the present disclosure by providing consistent amplification reactions that are not achieved by other methods.

[0281] However, as shown in FIG. 32B, when the same samples were amplified using the device described herein (e g., individually controlled wells, dynamic numbers of cycles, etc ), each sample reached ideal amount of amplification, wherein all samples reached the same RFU. The concentration was calculated for each sample. As shown in FIG. 29, individual control of the wells using the device described herein resulted in less than 1 .5 fold range of concentration with simultaneous amplification of the 8 samples with various input concentrations. With the reference method, on the other hand, samples with low input concentration resulted in little to no amplification. Collectively, these data show that NGS libraries can be normalized with the device described herein without human intervention or decision making at ideal amount of amplification. Individual conditions of the wells can be controlled with the device described herein to produce similar amount of signal (e g., RFU) for a given number of cycles.Example 9 - Software based experimental design

[0282] The methods and systems of the present disclosure can be controlled via a user interface as described elsewhere herein. FIGs. 37A - 37B show an example of a control interface 3700, according to some embodiments. Panel 3710 can provide a plurality of inputs 371 1 - 3717. The inputs can provide parameters for a reaction (e.g., a PCR reaction) such as, for example, temperature, time, number of cycles, or number of repetitions, or any combination thereof. In the example of FIG. 37A, 3711 can provide a temperature of 98 °C for 30 s, 3712 can provide a temperature of 98 °C for 10 s, 3713 can provide a temperature of 60 °C for 30 s, 3714 can provide a temperature of 72 °C for 30 s, 3715 can provide repeating operations 3712 - 3714 12Atty Dkt No.: 61197-702601 times (e.g., going to operation 3712 12 times), 3716 can provide a temperature of 72 °C for 1 minute, and 3717 can hold the reaction at 12 °C to terminate the reaction. Profile 3702 can provide a graphical representation of the operations of panel 3710 (e.g., inputs 3711 - 3717). Curve 3703 can provide a theoretical amplification curve and a graphical representation of a threshold target fluorescence 3731. Parameters 3704 may be parameters of the rection. For example, a variable number of operations can be selected (e g., the reaction may not have a predetermined number of cycles to perform). An auto-normalization toggle can be selected to auto-normalize the reaction mixture (e g., perform operations until the reaction achieves a given slope of the fluorescence curve, fluorescence value, or difference in the fluorescence value from the baseline, or any combination thereof). After the completion of the auto-normalization, an additional number of cycles can be added.

[0283] Map 3705 can show a graphical representation of a condition of each chamber of a plurality of chambers. In the example of FIG. 37B, the temperature of the well can be displayed in the map. The map may be generated by an input of a start and end point, and the intermediary points can be interpolated (e.g., linearly interpolated) to generate the map 3705. Variables 3706 can provide control over the generation of the values of the map 3705. For example, the gradient of the map can be across the whole substrate, over individual columns, individual rows, columns, or rows, or any combination thereof. In some cases, Cqcan be a quantification cycle (e.g., number of cycles at which the amplification curve passes a predetermined threshold). A protocol can be an ensemble of one or more of temperatures, times, cycles, or data capture element, or any combination thereof used for a specific run. Auto-normalization can be a function whereby the system can automatically stop a reaction mixture’s cycling based on the passing of a predetermined threshold.Example 10 - Example system parameters

[0284] Table 2 shows an example of input parameters of a system, according to some embodiments. Table 3 shows an example of environmental condition considerations of a system, according to some embodiments.Table 2Atty Dkt No.: 61197-702601Table 3

[0285] FIGs. 38A - 38C show a front view (FIG. 38A), an isometric view (FIG. 38B), and a rear view (FIG. 38C) of a system, according to some embodiments. Table 4 shows an example of operating parameters of a system, according to some embodiments.Table 4Atty Dkt No.: 61197-702601

[0286] Examples of substrate (e.g., plate) consumables can include, but are not limited to BioRad HSS9665, Eppendorf twin-tec real-time PCR plate 96, Thermo Scientific AB-2400 / W, or USA Scientific TcmpAssurc 0.2 m PCR 8-tubc Strips, Att. Optical Caps. In some cases, clear substrates can be used. In some cases, translucent or opaque substrates can be used (e.g., white well plates). Translucent or opaque substrates may provide improved results to clear substrates. Examples of seals include, but are not limited to, Bio-Rad microseal B or applied biosystems microamp optical adhesive film. Examples of fluorescent molecules (e g., optical dyes) that can be used with the methods and systems of the present disclosure can include, but are not limited to, Invitrogen SYBR green I, Biotium EvaGreen Dye 20x, or Biotium EvaGreen Plus Dye 20x.

[0287] The system can be placed on a firm, flat, and level surface to provide a stable foundation for the system. Avoiding installing the system on a slippery or prone to vibration surface can improve the operating accuracy of the system. An ambient temperature from about 10 to about 30, about 15 to about 25, or at least about 20 °C can provide improved operating conditions. The shipping screw may be removed prior to operation of the system.Example 11- Operation of a system

[0288] Prior to operation of the system, the system may be correctly connected (e g., connected to a power supply or control computer), the shipping screw may be removed, the system may be free from damage, and air may be able to circulate freely through and around ventilation slots of the system. The power switch may be turned on. The LED indicator at the front of the system may alternate through a plurality of colors (e.g., blue, red, green), and return to an operation color (e g., blue). A user interface program may be started on a computer system (e.g., laptop) operably coupled to the system.

[0289] FIG. 39A shows an example of a data tab of a user interface, according to some embodiments. Existing data can be retrieved from a file shown in the data tab. Files can be sorted (e.g., by start time, end time, name). The fdes can be searched. The file comprising the existing data can be selected and imported. FIG. 39B shows an example of a protocol tab of a user interface, according to some embodiments. A previously used protocol can be selected from a protocol list. A pre-programmed template protocol can be used. A new (e.g., partially or entirely user generated) protocol can be used. FIG. 39C shows an example of a default protocol, according to some embodiments. An operation of the protocol can be added or deleted. For example, step 3 can be deleted as shown in FIG. 39D, an additional fluorescence capture operation (e.g., fluorescent read of one or more chambers) can be added as shown in FIG. 39E, a number of cycles for each chamber of a plurality of chambers can be adjusted as in FIG. 39F, orAtty Dkt No.: 61197-702601 a variable number of cycles can be programmed (e.g., by rows, columns, entire substrate) as shown in FIG. 39G, or any combination thereof.

[0290] An auto-normalization operation can be initiated by selecting an auto-normalization toggle. Different auto-normalization modes can be selected (e.g., slope, baseline, target fluorescence). The slope mode (e.g., FIG. 40A) may monitor a rate of an increase of a signal (e g., a fluorescence signal. Once the slope has reached a maximum slope (e.g., as determined by a first derivative of the fluorescence signal), the system can halt cycling on that chamber. This can be confirmed by one or more cycles of slope decrease (e g., stopping at least Slopemax + 1, 2, 3, 4, 5, 6, or more). The baseline mode (e.g., FIG. 40B) can halt cycling once a chamber has reached a certain factor of the baseline (e g., a ...

Claims

Atty Dkt No.: 61197-702601CLAIMSWHAT IS CLAIMED IS:

1. A method, comprising:(a) providing (i) a first reaction mixture comprising a first analyte to a first chamber of a substrate and (ii) a second reaction mixture comprising a second analyte to a second chamber of the substrate, wherein the first chamber and the second chamber are each individually addressable; and(b) subjecting (i) the first reaction mixture to first conditions sufficient to perform a first polymerase chain reaction (PCR) on the first analyte and (ii) the second reaction mixture to second conditions sufficient to perform a second PCR on the second analyte, wherein (1 ) a coefficient of variation of a yield of the first PCR and the second PCR is less than about 35% or (2) a difference of a temperature between the first chamber and the second chamber is at least about 30 °C.

2. The method of claim 1, wherein the coefficient of variation of a yield of the first PCR and the second PCR is less than about 35%.

3. The method of claim 2, wherein the coefficient of variation of a yield of the first PCR and the second PCR is less than about 35% in an absence of depletion of a reagent of the first reaction mixture or the second reaction mixture.

4. The method of any one of claims 1 - 3, wherein the difference of a temperature between the first chamber and the second chamber is at least about 30 °C.

5. The method of any one of claims 1 - 4, further comprising, (i) during the first PCR, detecting one or more first signals or first signal changes from the first reaction mixture or (ii) during the second PCR, detecting one or more second signals or second signal changes from the second reaction mixture.

6. The method of any one of claims 1 - 5, wherein a first thermal element is in operable communication with the first chamber.

7. The method of claim 6, wherein the first thermal element is a thermoelectric element.

8. The method of any one of claims claim 6 - 7, wherein the first thermal element has a length of at most or a width of at most about 9 millimeters.

9. The method of any one of claims claim 6 - 8, wherein a second thermal element is in operable communication with the second chamber.

10. The method of claim 9, wherein the first thermal element and the second thermal element are different thermal elements.Atty Dkt No.: 61197-70260111. The method of any one of claims 1 - 10, further comprising, subsequent to (b), extracting, at least a portion of the first reaction mixture from the first chamber or at least a portion of the second reaction mixture from the second reaction chamber.

12. The method of claim 11, wherein the at least the portion of the first reaction mixture or the at least the portion of the second reaction mixture are used for a next-generation sequencing application13. The method of any one of claims 1 - 12, wherein the first reaction mixture or the second reaction mixture are not pre-normalized.

14. The method of any one of claims 1 - 13, further comprising adjusting the first conditions to decrease a rate of or halt the first PCR.

15. The method of claim 14, wherein the second PCR continues after the rate of the first PCR is decreased or halted.

16. The method of any one of claims 14 - 15, wherein the adjusting is in response to one or more signals from the first reaction mixture.

17. The method of any one of claims 1 - 16, wherein, during the subjecting of (b), (i) a thermal conductivity between a first thermal element associated with the first chamber and a heat sink in thermal contact with the first thermal element is at least about 5 W / Km, or (ii) a thermal conductivity between the first chamber and a first sensor associated with the first chamber is at least about 5 W / Km.

18. The method of claim 17, wherein the thermal conductivity between the first thermal element associated with the first chamber and the heat sink in thermal contact with the first thermal element is at least about 5 W / Km.

19. The method of any one of claims 17 - 18, wherein the thermal conductivity between the first chamber and the first sensor associated with the first chamber is at least about 5 W / Km.

20. The method of claim 17, further comprising measuring, using the first sensor, a property of the first chamber.

21. The method of claim 20, wherein the property of the first chamber comprises a temperature of the first chamber.

22. The method of any one of claims 17 - 21, wherein the first thermal element, the heat sink, the first chamber, or the first sensor are in contact with a thermally conductive element.

23. The method of claim 22, wherein the thermally conductive element has a thermal conductivity of at least about 5 W / Km.Atty Dkt No.: 61197-70260124. The method of any one of claims 22 - 23, further comprising transferring heat through the thermally conductive element.

25. The method of any one of claims 22 - 24, wherein the thermally conductive element comprises at least about 2 layers.

26. The method of any one of claims 17 - 25, wherein a difference in a temperature between the first chamber and a temperature of the first sensor is at most about 0. 1 °C.

27. The method of any one of claims 17 - 26, wherein, during the subjecting of (b), (i) a thermal conductivity between a second thermal element associated with the second chamber and a heat sink in thermal contact with the second thermal element is at least about 5 W / Krn; or (ii) a thermal conductivity between the second chamber and a second sensor associated with the second chamber is at least about 5 W / Km.

28. The method of any one of claims 1 - 27, wherein, during (b), (i) a first volume of the first reaction mixture or (ii) a second volume of the second reaction mixture changes by less than about 10%.

29. The method of claim 28, wherein the first volume and the second volume stay within about 10% of one another during the subjecting of (b).

30. The method of any one of claims 28 - 29, further comprising, prior to (b), bringing the substrate in contact with a cover plate.31 . The method of claim 30, wherein the cover plate comprises a first feature corresponding to the first chamber and a second feature corresponding to the second chamber.

32. The method of claim 31, wherein the feature comprises a first hole disposed above the first chamber and a second hole disposed above the second chamber.

33. The method of any one of claims 30 - 32, wherein the cover plate comprises a thermal element.

34. The method of any one of claims 30 - 33, wherein the cover plate comprises one or more air deflectors.

35. The method of any one of claims 30 - 34, wherein the cover plate is connected to a top plate.

36. The method of claim 35, wherein one or more springs are disposed between the cover plate and the top plate.

37. The method of any one of claims 1 - 36, wherein a contact force between the substrate and a cover plate disposed above the substrate during the subjecting of (b) is at least about 30 pounds.Atty Dkt No.: 61197-70260138. The method of any one of claims 1 - 37, wherein (i) a first thermal element used to affect a first temperature condition of the first conditions and (ii) a second thermal element used to affect a second temperature condition of the second conditions each have a heating output and a cooling output that differ by at most about 0.5 W.

39. The method of any one of claims 1 - 38, wherein a system comprising the substrate has an area of at most about 2,500 square centimeters (cm2).

40. The method of any one of claims 1 - 39, wherein a thermal conductivity between the first chamber and the second chamber is at most about 1 W / Km.

41. The method of claim 40, wherein one or more thermal management elements are disposed between the first chamber and the second chamber.

42. The method of claim 41 , wherein the one or more thermal management elements comprise a thermal insulator.

43. The method of any one of claims 41 - 42, wherein the one or more thermal management elements comprise a gap.

44. The method of claim 1, wherein the individual addressability comprises use of control circuitry.

45. The method of claim 44, wherein the control circuitry comprise a converter.

46. The method of any one of claims 44 - 45, wherein the converter comprises a buck-boost converter or a buck converter.

47. The method of claim 46, wherein the buck-boost converter is a current sinking buckboost converter.

48. A method for determining a property of an analyte, comprising:(a) providing a reaction mixture comprising an analyte to a chamber of a substrate, wherein the chamber is individually addressable;(b) subjecting the reaction mixture to conditions sufficient to perform a polymerase chain reaction (PCR) on the analyte;(c) during the PCR, detecting one or more signals or signal changes from said reaction mixture, and(d) determining a property of the analyte with an accuracy of at least 90% within at most 60 min based at least in part on the one or more signals or signal changes.

49. The method of claim 48, further comprising, during the PCR, detecting one or more signals or signal changes from the reaction mixture.Atty Dkt No.: 61197-70260150. The method of any one of claims 48 - 49, wherein a thermal element of the system has a thermal element with a maximum length or maximum width of at most about 9 millimeters.51 . The method of any one of claims 48 - 50, wherein the reaction mixture is not prenormalized.

52. The method of any one of claims 48 - 51, further comprising, in (b), using a first element disposed adjacent to the chamber to affect the conditions in the chamber to thereby initiate the PCR reaction.

53. The method of any one of claims 48 - 52, further comprising using a second element operably coupled to a first element to adjust an operating condition of the first element at least partially in response to the signal or signal changes, to thereby affect the condition of the chamber.

54. The method of any one of claims 48 - 53, wherein the chamber comprises a first thermal element and an additional chamber of the substrate comprises a second thermal element.

55. The method of any one of claims 48 - 54, wherein the chamber is operably coupled to a first sensor and an additional chamber is operably coupled to a second sensor.

56. The method of any one of claims 48 - 55, wherein a thermal element and an additional thermal element share a heatsink.

57. The method of any one of claims 48 - 56, wherein, during the subjecting of (b), (i) a thermal conductivity between a thermal element associated with the chamber and a heat sink in thermal contact with the thermal element is at least about 5 watts / kelvin / meter (W / Km); or (ii) a thermal conductivity between the chamber and a sensor associated with the chamber is at least about 5 watts / kelvin / meter (W / Km).

58. The method of any one of claims 48 - 57, wherein a volume of the reaction mixture within the chamber changes by at most about 10% during the PCR.

59. The method of any one of claims 48 - 58, wherein a contact force between the substrate and a cover plate disposed above the substrate during the subjecting of (b) is at least about 30 pounds.

60. The method of any one of claims 48 - 59, wherein a thermal element used to affect a temperature condition of the conditions has a heating output and cooling output that differ by at most about 0.5 W.61 . The method of any one of claims 48 - 60, wherein an alignment between the chamber and a feature of a cover plate corresponding to the chamber has an error of at most about 250 micrometers.Atty Dkt No.: 61197-70260162. The method of any one of claims 48 - 61, wherein an alignment between the chamber and a thermal element corresponding to the chamber has an error of at most about 250 micrometers.

63. The method of any one of claims 48 - 62, wherein a system comprising the substrate has an area of at most about 2,500 square centimeters (cm2).

64. The method of any one of claims 48 - 63, wherein an additional chamber of the substrate comprises an additional reaction mixture.

65. The method of claim 64, wherein an additional PCR reaction continues within the additional chamber after the PCR reaction in the first chamber is stopped.

66. The method of any one of claims 64 - 65, wherein the chamber and the additional chamber do not share a sensor.

67. A method for performing a polymerase chain reaction (PCR), comprising:(a) providing a reaction mixture comprising an analyte to a chamber on a substrate, wherein the chamber is individually addressable;(b) subjecting the reaction mixture to conditions sufficient to perform the PCR on the analyte;(c) during the PCR, having a volume change of the reaction mixture of at most about 10%.

68. The method of claim 67, wherein the volume change of the reaction mixture is at most about 1 microliter.

69. The method of any one of claims 67 - 68, further comprising, in (b), using a first element disposed adjacent to the chamber to affect the conditions in the chamber to thereby initiate the PCR reaction.

70. The method of any one of claims 67 - 69, further comprising using a second element operably coupled to a first element to adjust an operating condition of the first element at least partially in response to a signal or signal changes from the PCR, to thereby affect the condition of the chamber.

71. The method of any one of claims 67 - 70, further comprising adjusting the operating condition of a first element to decrease a rate of the PCR reaction upon one or more signal or signal changes from the PCR reaching a predetermined threshold.

72. The method of any one of claims 67 - 71, wherein the chamber and an additional chamber of the substrate share a printed circuit board (PCB) support.

73. The method of any one of claims 67 - 72, wherein a thermal element and an additional thermal element share a heatsink.Atty Dkt No.: 61197-70260174. The method of any one of claims 67 - 73, wherein a cover plate in contact with the substrate comprises a feature corresponding to the chamber.

75. The method of claim 74, wherein the feature comprises a hole disposed above the chamber.

76. The method of any one of claims 67 - 75, wherein a cover plate in contact with the substrate comprises a thermal element.

77. The method of any one of claims 67 - 76, wherein a cover plate in contact with the substrate comprises one or more air deflectors.

78. The method of any one of claims 67 - 77, wherein a cover plate in contact with the substrate is connected to a top plate.

79. The method of claim 78, wherein one or more springs are disposed between the cover plate and the top plate.

80. The method of any one of claims 67 - 79, wherein a contact force between a cover plate in contact with the substrate and the substrate during the subjecting of (b) is at least about 30 pounds.81 . A system for performing a polymerase chain reaction (PCR), comprising: a substrate comprising a chamber; a cover plate disposed above the substrate, wherein the cover plate is configured to, when engaged with the substrate, cover the chamber, wherein the cover plate is configured to maintain a volume of a reaction mixture within the reaction chamber within about 10% of a starting volume of the reaction mixture.

82. The system of claim 81, wherein the volume change of the reaction mixture is at most about 1 microliter.

83. The method of any one of claims 81 - 82, wherein the cover plate is disposed against a top plate configured to hold the cover plate above the substrate.

84. The method of claim 83, further comprising one or more springs disposed between the cover plate and the top plate.

85. The method of claim 84, wherein the one or more springs provide a contact force between the cover plate and the substrate.

86. The method of any one of claims 84 - 85, wherein the one or more springs comprise one or more calibrated springs.

87. The method of any one of claims 81 - 86, wherein the cover plate comprises a thermal element.Atty Dkt No.: 61197-70260188. The method of claim 87, wherein the thermal element comprises a heater.

89. The method of any one of claims 87 - 88, wherein the thermal element is configured to control a temperature of the cover plate.

90. The method of any one of claims 81 - 89, wherein the cover plate comprises one or more air deflectors.91 . The method of any one of claims 81 - 90, wherein the substrate comprises a plurality of chambers.

92. The method of claim 91, wherein the plurality of chambers comprises at least about 96 chambers.

93. The method of any one of claims 81 - 92, wherein said cover plate comprises a feature.

94. The method of claim 93, wherein the cover plate comprises a plurality of features95. The method of claim 94, wherein, when said substrate and said cover plate are engaged, said plurality of features align with a plurality of chambers comprising the chamber.

96. The method of any one of claims 94 - 95, wherein, said plurality of features comprise a plurality of access holes through said cover plate.

97. The method of any one of claims 81 - 96, further comprising a thermoelectric element in thermal communication with the chamber.

98. The method of claim 97, further comprising a plurality of thermoelectric elements in thermal communication with a plurality of chambers comprising the chamber.

99. The method of any one of claims 81 - 98, wherein a sample evaporation rate of the PCR is less than about 1 microliters / hour.

100. The method of any one of claims 81 - 99, wherein a temperature of the chamber may be at most about 0.2 °C from a set temperature of the chamber.

101. The method of any one of claims 81 - 100, further comprising one or more air deflectors configured to deflect air around or away from the cover plate and the substrate.

102. A method for performing a polymerase chain reaction (PCR) analysis of a sample containing or suspected of containing an analyte, comprising:(a) providing said sample to a location on a substrate, wherein said location is operably coupled to a thermally conductive element disposed in at least a portion of a support, wherein said location has a volume sufficient to retain said sample; and(b) using a thermal element disposed adjacent to said location to initiate a PCR reaction using said sample, wherein said PCR reaction generates one or more signals or signal changes which is indicative of a property associated with said analyte, andAtty Dkt No.: 61197-702601 wherein said thermal element is in thermal contact with said thermally conductive element.

103. The method of claim 102, wherein said thermal element is a thermoelectric element.

104. The method of any one of claims 102 - 103, further comprising (c) using a second element operably coupled to said thermal element to adjust an operating condition of said thermal element in response at least partially to said signal or signal change, to thereby affect a condition in said location.

105. The method of claim 104, wherein said support comprises a second thermally conductive element disposed through at least a portion of said support, and wherein said second element is in thermal contact with said second thermally conductive element.

106. The method of any one of claims 102 - 105, wherein said thermally conductive element is electrically insulated from said thermal element.

107. The method of any one of claims 102 - 106, wherein the support is a printed circuit board.

108. The method of claim 107, wherein said printed circuit board is a multi-layered metal printed circuit board.

109. The method of any one of claims 102 - 108, wherein said at least a portion of said support is an entire thickness of said support.

110. The method of any one of claims 102 - 109, further comprising a heatsink disposed on an opposite side of said support from said thermal element.

111. The method of claim 110, wherein said thermal element is in thermal communication with said heatsink at least partially through said thermally conductive element.

112. The method of any one of claims 102 - 111, further comprising a thermal conductor placed between said thermal element and said thermally conductive element.

113. The method of claim 112, wherein thermal conductor is a graphite sheet or a thermal compound.1 14. The method of any one of claims 102 - 1 13, wherein said thermally conductive element comprises one or more metals.

115. The method of claim 114, wherein said one or more metals comprises copper.

116. The method of any one of claims 114 - 115, wherein said one or more metals comprises aluminum.Atty Dkt No.: 61197-702601117. The method of any one of claims 102 - 116, further comprising performing an isothermal amplification reaction within said location.

118. A method, comprising:(a) providing a device comprising(i) a power supply comprising a circuit comprising a current sinking converter, and(ii) a thermoelectric element in electrical communication with said power supply; and(b) using said power supply to provide power to said thermoelectric element, wherein when power is supplied to said thermoelectric element, said current sinking converter removes current from said circuit that is in excess of a current sufficient to operate said thermoelectric element.

119. The method of claim 118, further comprising (c) adjusting a temperature of a chamber disposed in thermal contact with said thermoelectric element using at least in part said thermoelectric element.

120. The method of claim 1 19, wherein said adjusting said temperature of said chamber comprises adjusting a power output of said power supply.

121. The method of any one of claims 118 - 120, wherein said current sinking converter is a current sinking buck-booster converter.

122. The method of any one of claims 118 - 121, further comprising performing a polymerase chain reaction within a chamber in thermal contact with said thermoelectric element.

123. The method of any one of claims 118 - 122, wherein said chamber is a well.

124. The method of any one of claims 118 - 123, further comprising using another power supply to provide power to another thermoelectric element.

125. The method of claim 124, wherein said another power supply comprises another current sinking converter.

126. The method of claim 125, wherein said another current sinking converter is a current sinking buck-boost converter.

127. The method of any one of claims 124 - 126, further comprising adjusting a temperature of another chamber using at least in part said second thermoelectric element.

128. The method of claim 127, wherein said temperature of said another chamber is different from a temperature of a chamber in thermal contact with said thermoelectric element.Atty Dkt No.: 61197-702601129. The method of any one of claims 124 - 128, wherein said thermoelectric element and said another thermoelectric element are individually addressable.

130. The method of any one of claims 118 - 129, further comprising performing an isothermal amplification reaction within a chamber in thermal contact with said thermoelectric element.

131. A method, comprising:(a) providing(i) a plurality of chambers,(ii) a thermoelectric plate comprising a plurality of thermoelectric elements, wherein each thermoelectric element of said plurality of thermoelectric elements corresponds to a different chamber of said plurality of chambers(iii) a movable assembly in contact with said thermoelectric plate; and(b) moving, via said movable assembly, said thermoelectric plate, thereby contacting said plurality of thermoelectric elements to said plurality of chambers, wherein a chamber of said plurality of chambers is aligned to a thermoelectric element of said plurality of thermoelectric elements with an offset of at most about 250 micrometers.

132. The method of claim 131, wherein said plurality of chambers are a plurality of wells.

133. The method of any one of claims 131 - 132, wherein said contacting said plurality of thermoelectric elements with said plurality of chambers is an automated contacting.

134. The method of any one of claims 131 - 133, wherein said thermoelectric plate is contacted to said plurality of chambers with at least about 30 pounds of force.

135. The method of claim 134, wherein said thermoelectric plate is contacted to said plurality of chambers with at most about 80 pounds of force.

136. The method of any one of claims 131 - 135, wherein said device has a footprint of at most about 540 square millimeters (mm2).

137. The method of any one of claims 131 - 136, further comprising a lid positioned above said plurality of chambers.

138. The method of claim 137, wherein said lid comprises a heating element.

139. The method of any one of claims 131 - 137, wherein said movable assembly comprises a lever.

140. The method of claim 139, wherein said lever has a movement ratio of said lever to said movable assembly of about 2: 1.Atty Dkt No.: 61197-702601141. The method of any one of claims 131 - 140, wherein said movable assembly comprises a plurality of pivoting points.

142. The method of claim 141, wherein said movable assembly comprises at least three pivoting points.

143. The method of any one of claims 131 - 142, further comprising a limit switch configured to detect a position of said movable assembly.

144. The method of claim 143, wherein said limit switch is an optical limit switch.

145. The method of any one of claims 143 - 144, wherein said limit switch is a mechanical limit switch.

146. The method of any one of claims 131 - 145, wherein said movable assembly is positioned below said thermoelectric plate.

147. The method of any one of claims 131 - 146, further comprising performing an isothermal amplification reaction within a chamber of said plurality of chambers.

148. The method of claim 147, further comprising performing an independent isothermal amplification reaction within each chamber of said plurality of chambers.

149. A device, comprising: a thermoelectric element operably coupled to a support, wherein said support comprises one or more thermally conductive elements, which one or more thermally conductive elements are disposed in at least a portion of said support, wherein said thermoelectric element is in thermal contact with said one or more thermally conductive elements.

150. The device of claim 149, wherein said one or more thermally conductive elements are electrically insulated from said thermoelectric element.

151. The device of any one of claims 149 - 150, wherein said support is a printed circuit board.

152. The device of claim 151, wherein said printed circuit board is a multi-layered metal printed circuit board.

153. The device of any one of claims 149 - 152, wherein said one or more thermally conductive elements are metallic.

154. The device of any one of claims 149 - 153, wherein said at least a portion of said support is an entire thickness of said support.

155. The device of any one of claims 149 - 154, further comprising a heatsink disposed on an opposite side of said support from said thermoelectric element.Atty Dkt No.: 61197-702601156. The device of claim 155, wherein said thermoelectric element is in thermal communication with said heatsink.

157. The device of any one of claims 149 - 156, further comprising a thermal conductor placed between said thermoelectric element and said one or more thermally conductive elements.

158. The device of claim 157, wherein said thermal conductor is a graphite sheet or a thermal compound.

159. The device of any one of claims 149 - 158, wherein said one or more thermally conductive elements are metal.

160. The device of claim 159, wherein said metal is copper.

161. The device of any one of claims 159 - 160, wherein said metal is aluminum.

162. A device, comprising: a thermoelectric element; and a power supply configured to provide power to said thermoelectric element, wherein said power supply comprises a current sinking converter in an electrical path of said thermoelectric element.

163. The device of claim 162, wherein said current sinking converter is a current sinking buck-booster converter.

164. The device of any one of claims 162 - 163, wherein said current sinking converter comprises a feedback signal loop configured to adjust power provided by said power supply to said thermoelectric element.

165. The device of any one of claims 162 - 164, wherein said current sinking converter makes a power range supplied from said power supply to said thermoelectric element symmetrical.

166. A device, comprising: a plurality of chambers; a thermoelectric plate comprising a plurality of thermoelectric elements, wherein each thermoelectric element of said plurality of thermoelectric elements corresponds to a different chamber of said plurality of chambers; and a movable assembly in contact with said thermoelectric plate, wherein said movable assembly is configured to, through movement, contact said plurality of thermoelectricAtty Dkt No.: 61197-702601 elements to said plurality of chambers, wherein a chamber of said plurality of chambers is aligned to a thermoelectric element of said plurality of thermoelectric elements with an offset of at most about 250 micrometers.

167. The device of claim 166, wherein said plurality of chambers are a plurality of wells.

168. The device of any one of claims 166 - 167, wherein said contact is an automated contact.

169. The device of any one of claims 166 - 168, wherein said thermoelectric plate is contacted to said plurality of chambers with at least about 30 pounds of force.

170. The device of claim 169, wherein said thermoelectric plate is contacted to said plurality of chambers with at most about 80 pounds of force.

171. The device of any one of claims 166 - 170, wherein said device has a footprint of at most about 540 square millimeters (mm2).

172. The device of any one of claims 166 - 171, further comprising a lid positioned above said plurality of chambers.

173. The device of claim 172, wherein said lid comprises a heating element.

174. The device of any one of claims 166 - 173, wherein said movable assembly comprises a lever.

175. The device of claim 174, wherein said lever has a movement ratio of said lever to said movable assembly of about 2: 1.

176. The device of any one of claims 166 - 175, wherein said movable assembly comprises a plurality of pivoting points.

177. The device of claim 176, wherein said movable assembly comprises at least three pivoting points.

178. The device of any one of claims 166 - 177, further comprising a limit switch configured to detect a position of said movable assembly.

179. The device of claim 178, wherein said limit switch is an optical limit switch.

180. The device of any one of claims 178 - 179, wherein said limit switch is a mechanical limit switch.

181. The device of any one of claims 166 - 180, wherein said movable assembly is positioned below said thermoelectric plate.

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