Thermal adapter for automated thermal cycling

Abstract

A system and method for processing samples in a multi-well reaction vessel can include inserting the multi-well reaction vessel into a heating chamber of a thermal cycler, enclosing the multi-well reaction vessel in the heating chamber, and compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert. The compliant thermally conductive insert can be positioned between the multi-well reaction vessel and a heating element of the thermal cycler, and heat flux from the heating element passes through the compliant thermally conductive insert to the reaction vessel. The compliant thermally conductive insert can include an elastically deformable creped graphite sheet that can reversibly deform according to different compression profiles depending on the topography or flexure of the reaction vessel and / or the heating element.

Description

[Technical field]

[0001] (CROSS REFERENCE TO RELATED APPLICATIONS) This application claims the benefit of and priority to U.S. Provisional Patent Application No. 63 / 072,838, filed August 31, 2020, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] (background) The general commercial system for amplifying DNA is typically based on polymerase chain reaction (PCR), which is based on an enzyme that is activated above a certain temperature and then inactivated below a certain temperature. In many applications, this cycle is repeated many times. For example, in the PCR method for quantifying the amount of DNA, called qPCR, the cycle between the upper and lower temperature limits can occur more than 40 times. Therefore, shortening the time to achieve the desired activation and inactivation temperatures is an important factor in the productivity of this method.

[0003] It is also desirable to process many samples at once in a manner compatible with automation. Many commercial PCR reactions are performed in parallel using multiple reaction vessels combined in a regular array, such as a 96-well microplate. Higher density microplates with 1,536 wells for use in commercial PCR thermal cyclers are less common. One conventional strategy to reduce the time to cycle between upper and lower temperature limits for a microplate involves the use of a customized heating block with an "egg box" shape to match the similarly shaped bottom surface of a specialized multi-well microplate. This approach improves heat transfer to the plate and within the plate itself, but requires strict specifications to maintain physical thermal contact by matching the shape of a thermally conductive and non-compliant metal heating block (often aluminum) with a plastic injection molded microplate or an expensive composite plate. However, such plates are difficult to mold to precise dimensions, are machined from non-identical mold cavities, and can be prone to distortion due to stresses from different material responses to temperature (either after release from the mold or after PCR thermal cycling).

[0004] One disadvantage of multi-well plates designed for PCR thermal cycling is that they are designed with the goal of maximizing heat transfer, and they generally fail to meet the requirements for use with acoustic ejection or interrogation, both of which are highly desirable attributes for process automation. Sample transport methods using acoustic ejection (i.e., acoustic pressure waves) are described, for example, in U.S. Pat. No. 10,156,499 (incorporated herein by reference). However, acoustically compatible plates generally fail to meet the physical requirements (e.g., surface area, thinness, resistance to heat-induced deformation) needed to provide the fast temperature changes of PCR thermal cycling. Because these plates deviate from flatness due to bending across their wells of about several hundred microns, they do not provide uniform thermal coupling to PCR heating blocks. Furthermore, the plates are not sufficiently compliant to allow uniform thermal coupling even with large compressive forces. Thus, there is interest in providing solutions that allow both manual and automated (robotic) manipulation of microplates into systems that can provide fast thermal cycling as well as automated sample handling. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 10,156,499 Summary of the Invention [Means for solving the problem]

[0006] (summary) The terms "invention," "the invention," "this invention," and "the present invention" as used in this patent are intended to broadly refer to all of the subject matter and claims of this patent that follow. Statements containing these terms should be understood not to limit the subject matter described herein or to limit the meaning or scope of the claims that follow. The embodiments of the invention covered by this patent are defined by the claims that follow, not this Summary. This Summary is a broad overview of various aspects of the invention and introduces some of the concepts that are further described in the Detailed Description section that follows. This Summary is not intended to identify key features or essential features of the claimed subject matter, nor is it intended to be used in isolation to determine the scope of the claimed subject matter. The subject matter should be understood by reference to the entire specification of this patent, any or all drawings, and appropriate portions of each claim.

[0007] According to certain embodiments of the present disclosure, a system for sample handling for a multi-well reaction vessel can include a robotic sample handler configured to hold and move the multi-well reaction vessel and a thermal cycler for performing a thermal cycling operation on a sample contained within the reaction vessel. The thermal cycler includes a heating chamber shaped to receive the multi-well reaction vessel containing a heating element, a compliant thermally conductive insert positioned adjacent to the heating element, and a closing mechanism that, when closed, presses the multi-well reaction vessel toward the compliant thermally conductive insert and the heating element. According to at least one embodiment, the compliant thermally conductive insert can be formed from an elastically deformable creped graphite sheet or an assembly of any suitable number of parallel graphite sheets that have high thermal conductivity and are reversibly deformable.

[0008] The compliant thermally conductive insert can accommodate mismatches between the geometry of the bottom surface of the multiwell reaction vessel and the top surface of the heating element by reversibly deforming when compressed between the two. This effect can compensate for non-parallel flat or curved surfaces, for surface imperfections, or for changes in surface profile caused by thermal deformation during thermal cycling. In the context of an automated system using a controller, the system can have a robotic sample handler insert a multiwell reaction vessel into a thermal cycler, enclose the multiwell reaction vessel in a heating chamber, and compress the bottom surface of the multiwell reaction vessel into the compliant thermally conductive insert by a closing mechanism. The system can then have the thermal cycler automatically cycle the multiwell reaction vessel in the heating chamber by the heating element by applying a controlled heat flux from the heating element through the compliant thermally conductive insert to the multiwell reaction vessel.

[0009] The thermal cycling process step can be performed with a multi-well reaction vessel designed for automated acoustic sample handling, and the system can therefore utilize acoustic sample transport and acoustic sample interrogation techniques to inject samples from a source vessel into the multi-well reaction vessel by acoustic ejection, or to analyze aspects of the sample by acoustic interrogation before or after thermal cycling. Additionally, the system can utilize acoustic sample transport techniques to transport samples from the multi-well reaction vessel to a sample analyzer after thermal cycling.

[0010] According to an embodiment of the present disclosure, a method of sample handling for a multi-well reaction vessel can include inserting the multi-well reaction vessel into a heating chamber of a thermal cycler by placing the multi-well reaction vessel on a compliant thermally conductive insert on a heating element of the thermal cycler. The compliant thermally conductive insert can include one or more elastically deformable creped graphite sheets, placed alone or in parallel, that have high thermal conductivity and are reversibly deformable. The compliant thermally conductive insert can accommodate mismatches between the geometry of the bottom surface of the multi-well reaction vessel and the top surface of the heating element by reversibly deforming when compressed between the two. This effect can be compensated for non-parallel flat or curved surfaces, for surface defects, or for changes in surface profile caused by thermal deformation during thermal cycling.

[0011] The methods described herein may further include compressing the bottom surface of the multiwell reaction vessel into a compliant thermally conductive insert to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert, and thermally cycling the multiwell reaction vessel in a heating chamber by applying a controlled heat flux to the multiwell reaction vessel by a heating element through the compliant thermally conductive insert. The compliant thermally conductive insert can maintain the increased contact area throughout the thermal cycling operation even when the multiwell reaction vessel or the heating element deforms during the repeated heating and cooling stages of the cycle. Optionally, a compliant thermally conductive insert may be used between the top surface of the multiwell reaction vessel and the upper surface of the heating chamber if the multiwell reaction vessel requires improved thermal contact between these surfaces.

[0012] According to certain embodiments of the present disclosure, a thermal cycler assembly for use in sample handling for a multi-well reaction vessel can include a heating chamber, a heating element contained in the heating chamber, and a closing mechanism that when closed presses on the multi-well reaction vessel to enclose the multi-well reaction vessel within the heating chamber and compress it into a compliant thermally conductive insert positioned within the heating chamber in contact with the heating element. According to various embodiments, the compliant thermally conductive insert comprises an elastically deformable creped graphite sheet or an assembly of a plurality of deformable creped graphite sheets having high thermal conductivity and being partially or fully reversibly deformable.

[0013] According to an embodiment of the present disclosure, a method of sample handling for multi-well reaction vessels can include inserting a first multi-well reaction vessel into a heating chamber of a thermocycler by placing the multi-well reaction vessel on a compliant thermally conductive insert on a heating element of the thermocycler, removing the first multi-well reaction vessel, and subsequently inserting a second multi-well reaction vessel into the heating chamber of the thermocycler by placing the second multi-well reaction vessel on the compliant thermally conductive insert. The compliant thermally conductive insert can include an elastically deformable creped graphite sheet or an assembly of elastically deformable creped graphite sheets with high thermal conductivity. When the first multi-well reaction vessel is compressed into the compliant thermally conductive insert, pressure between the first bottom surface and the heating element causes a reversible deformation of the creped graphite sheet according to a first compression profile. Similarly, when a second multi-well reaction vessel is compressed into the compliant thermally conductive insert, pressure between the second bottom surface and the heating element causes a reversible deformation of the creped graphite sheet according to a second compression profile that is different from the first compression profile. In response to the pressure and the reversible deformation, the compliant thermally conductive insert returns to an uncompressed state from the first compressed profile and from the second compressed profile without permanently deforming or "flowing." The present specification also provides, for example, the following items: (Item 1) 1. A system comprising: a robotic sample handler configured to hold and move multi-well reaction vessels; a thermal cycler comprising a heating chamber containing a heating element, a compliant, thermally conductive insert comprising an elastically deformable creped graphite sheet positioned adjacent to the heating element, and a closing mechanism, the heating chamber shaped to receive the multi-well reaction vessel, the closing mechanism configured to press the multi-well reaction vessel against the compliant, thermally conductive insert and the heating element; a controller operatively connected to the robotic sample handler and the thermal cycler; Equipped with The controller comprises at least one processor and a memory containing executable instructions that, when executed by the at least one processor, causing the robotic sample handler to insert the multi-well reaction vessel into the thermal cycler; causing the closure mechanism to enclose the multi-well reaction vessel within the heating chamber; compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert with the closure mechanism; thermally cycling said multi-well reaction vessel within said heating chamber with said heating element by applying a controlled heat flux from said heating element to said multi-well reaction vessel through said compliant thermally conductive insert; and configuring the controller to: (Item 2) compressing the bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert causes a reversible deformation of the creped graphite sheet according to a first compression profile; compressing a second bottom surface of a second multi-well reaction vessel into the compliant thermally conductive insert causes reversible deformation of the creped graphite sheet according to a second compression profile different from the first compression profile; Item 1 , the system of item 1 , wherein the compliant thermally conductive insert returns to an uncompressed state from the first compressed profile and from the second compressed profile without permanently deforming. (Item 3) a source vessel containing a reagent or sample; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and a source vessel with wells of the multi-well reaction vessel; Further equipped with The executable instructions, when executed by the at least one processor, causing the actuator to selectively align the transducer and source vessel with one of the wells of the multi-well reaction vessel; causing the acoustic ejector to eject one or more droplets from the source vessel into the well by applying the focused acoustic radiation to a sample contained within the source vessel; 2. The system of claim 1, further comprising: (Item 4) a multi-well receiving vessel; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and the multi-well reaction vessel with wells of the multi-well receiving vessel; Further equipped with The executable instructions, when executed by the at least one processor, causing the actuator to selectively align the transducer and multi-well reaction vessel with one or more of the wells of the multi-well receiving vessel; causing the acoustic ejector to eject one or more droplets from the multiwell reaction vessel into the wells of the multiwell receiving vessel by applying the focused acoustic radiation to a sample contained within the multiwell reaction vessel; 2. The system of claim 1, further comprising: (Item 5) an analyzer having a sample inlet; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and the multi-well reaction vessel with the sample inlet of the analyzer; Further equipped with The executable instructions, when executed by the at least one processor, causing the actuator to selectively align the transducer and multi-well reaction vessel with the sample inlet of the analyzer; causing the acoustic ejector to eject one or more droplets from the multi-well reaction vessel into the sample inlet by applying the focused acoustic radiation to a sample contained within the multi-well reaction vessel; 2. The system of claim 1, further comprising: (Item 6) 1. A method comprising: inserting a multi-well reaction vessel into a heating chamber of a thermocycler by placing the multi-well reaction vessel on a compliant, thermally conductive insert on a heating element of the thermocycler, the compliant, thermally conductive insert comprising an elastically deformable creped graphite sheet; enclosing the multi-well reaction vessel within the heating chamber; compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert; thermally cycling the multi-well reaction vessel within the heating chamber by applying a controlled heat flux to the multi-well reaction vessel by the heating element through the compliant thermally conductive insert; A method comprising: (Item 7) 7. The method of claim 6, wherein the compliant thermally conductive insert has an uncompressed thickness in the range of 250 microns to 2,000 microns. (Item 8) 7. The method of claim 6, wherein the compliant thermally conductive insert has an in-plane thermal conductivity of at least 200 W / mK, preferably at least 700 W / mK. (Item 9) 7. The method of claim 6, wherein the compliant thermally conductive insert has a through-plane thermal conductivity that increases non-linearly with compressive stress, the through-plane thermal conductivity ranging from 1-5 W / mK at 100 kPa compressive stress to 10-30 W / mK at 700 kPa compressive stress. (Item 10) 7. The method of claim 6, wherein the compliant thermally conductive insert reversibly compresses to less than 60% of its original thickness when subjected to a compressive stress of 700 kPa. (Item 11) The multi-well reaction vessel comprises a microplate comprising an array of wells having at least one flat bottom surface configured to allow acoustic interrogation of samples contained in the array of wells through the flat bottom surface, and the method further comprises: emitting an interrogation tone burst from an acoustic emitter through said flat bottom surface; detecting an acoustic echo caused by said interrogation tone burst; determining parameters of the sample from the detected acoustic echoes; The method according to any one of items 6 to 10, comprising: (Item 12) 11. The method of any one of items 6-10, wherein thermally cycling the multi-well reaction vessel in the heating chamber comprises sequentially heating and cooling the sample contained in the multi-well reaction vessel according to a PCR thermal cycling program at a heating or cooling rate of at least 1° C. / sec, preferably at least 1.5° C. / sec, preferably at least 2° C. / sec. (Item 13) aligning one or more wells of the multi-well reaction vessel with a source well and an acoustic ejector positioned to acoustically eject fluid droplets from the source well; ejecting one or more droplets from the source wells into the wells of the multi-well reaction vessel by applying focused acoustic radiation from the acoustic ejector to a sample contained in the source well; The method according to any one of items 6 to 10, further comprising: (Item 14) aligning one or more wells of the multiwell reaction vessel with an acoustic ejector positioned to acoustically eject fluid droplets from the multiwell reaction vessel and with a multiwell receiving vessel; ejecting one or more droplets from the wells of the multiwell reaction vessel into the wells of the multiwell receiving vessel by applying focused acoustic radiation from the acoustic ejector to samples contained in the wells of the multiwell reaction vessel; The method according to any one of items 6 to 10, further comprising: (Item 15) aligning wells of the multi-well reaction vessel with an acoustic ejector positioned to acoustically eject fluid droplets from the multi-well reaction vessel and with a sample inlet of an analytical device; ejecting one or more droplets from the wells of the multi-well reaction vessel into the sample inlet by applying focused acoustic radiation from the acoustic ejector to the sample contained in the wells; The method according to any one of items 6 to 10, further comprising: (Item 16) 1. A thermal cycler assembly comprising: A heating chamber; a heating element contained in the heating chamber; and a closure mechanism configured to enclose the heating chamber and press onto the multi-well reaction vessel when the multi-well reaction vessel is received in the heating chamber; a compliant thermally conductive insert positioned within the heating chamber in contact with the heating element, the compliant thermally conductive insert comprising an elastically deformable creped graphite sheet; A thermal cycler assembly comprising: (Item 17) Item 17. The thermal cycler assembly of item 16, wherein the compliant thermally conductive insert comprises a layered, elastically deformable creped graphite sheet. (Item 18) Item 17. The thermal cycler assembly of item 16, wherein the compliant thermally conductive insert comprises an interface frame connected with the elastically deformable creped graphite sheet that is removably insertable into the heating chamber and shaped to align the heating element and the multiwell reaction vessel with the elastically deformable creped graphite sheet when the multiwell reaction vessel is inserted into the heating chamber. (Item 19) Item 17. The thermal cycler assembly of item 16, wherein the compliant thermally conductive insert has an uncompressed thickness in the range of 250 microns to 2,000 microns and an in-plane thermal conductivity of at least 200 W / mK, preferably at least 700 W / mK. (Item 20) Item 17. The thermal cycler assembly of item 16, wherein the compliant thermally conductive insert has a through-plane thermal conductivity that increases non-linearly with compressive stress, the through-plane thermal conductivity ranging from 1-5 W / mK at 100 kPa compressive stress to 10-30 W / mK at 700 kPa compressive stress, and the compliant thermally conductive insert is reversibly compressible to less than 60% of its original thickness in response to a 700 kPa compressive stress. (Item 21) 1. A method comprising: inserting a first multi-well reaction vessel into a heating chamber of a thermal cycler by placing the multi-well reaction vessel on a compliant, thermally conductive insert on a heating element of the thermal cycler, the compliant, thermally conductive insert comprising an elastically deformable creped graphite sheet; compressing a first bottom surface of the first multi-well reaction vessel into the compliant thermally conductive insert such that pressure between the first bottom surface and the heating element causes a reversible deformation of the creped graphite sheet according to a first compression profile; inserting a second multiwell reaction vessel into the heating chamber of the thermal cycler by placing the second multiwell reaction vessel on the compliant thermally conductive insert; compressing the second bottom surface into the compliant thermally conductive insert such that pressure between the second bottom surface of the second multi-well reaction vessel and the heating element causes a reversible deformation of the creped graphite sheet according to a second compression profile different from the first compression profile, the compliant thermally conductive insert returning to an uncompressed state from the first compressed profile and from the second compressed profile without permanent deformation; A method comprising: (Item 22) 22. The method of claim 21, further comprising thermally cycling the multi-well reaction vessel within the heating chamber by applying a controlled heat flux to the multi-well reaction vessel by the heating element through the compliant thermally conductive insert. (Item 23) aligning wells of the first or second multiwell reaction vessel with an acoustic emitter positioned to apply focused acoustic radiation from the acoustic emitter to samples contained in the wells of the multiwell reaction vessel; emitting an interrogation tone burst from said acoustic emitter through a flat bottom surface of said first or second multi-well reaction vessel; detecting an acoustic echo caused by said interrogation tone burst; determining a parameter of the sample in the well from the detected acoustic echoes; 23. The method according to any one of items 21-22, further comprising: (Item 24) 23. The method of any one of items 21-22, further comprising applying a heat flux from the heating element through the compliant thermally conductive insert to the first or second multi-well reaction vessel sufficient to cause a temperature change of a sample in the first or second multi-well reaction vessel of at least 1° C. / sec, preferably at least 1.5° C. / sec, preferably at least 2° C. / sec. (Item 25) aligning wells of the first or second multiwell reaction vessel with an acoustic ejector positioned to apply focused acoustic radiation from the acoustic ejector to samples contained in the wells of the multiwell reaction vessel; ejecting droplets from said wells by emitting an ejection tone burst from said acoustic ejector through a flat bottom surface of said first or second multi-well reaction vessel; 23. The method according to any one of items 21-22, further comprising: [Brief description of the drawings]

[0014] Various embodiments according to the present disclosure will now be described with reference to the drawings.

[0015] [Figure 1] FIG. 1 is a simplified block diagram illustrating an exemplary system for sample handling for multi-well reaction vessels according to various embodiments of the present disclosure.

[0016] [Diagram 2] FIG. 2 is a simplified cross-sectional side schematic illustrating a thermal cycler assembly compatible with the system of FIG. 1 for receiving a multi-well reaction vessel and incorporating a compliant thermally conductive insert.

[0017] [Diagram 3] 3 is a detailed perspective view illustrating a side of the compliant thermally conductive insert of FIG. 2. FIG.

[0018] [Figure 4-1] 4A-4E are simplified cross-sectional side schematics illustrating various deformation profiles of the compliant thermally conductive insert as shown in FIGS. 2 and 3. FIG. [Figure 4-2] 4A-4E are simplified cross-sectional side schematics illustrating various deformation profiles of the compliant thermally conductive insert as shown in FIGS. 2 and 3. FIG.

[0019] [Diagram 5] FIG. 5 is a graph illustrating the comparative ramp rates of sample-containing multi-well reaction vessels in a thermal cycler with compliant thermally conductive inserts and alternative materials.

[0020] [Figure 6] FIG. 6 is a process flow diagram illustrating a first embodiment of a process for sample handling and thermal cycling of samples contained within a multi-well reaction vessel using a compliant thermally conductive insert.

[0021] [Figure 7] FIG. 7 is a process flow diagram illustrating a second embodiment of a process for sample handling and thermal cycling of samples contained within a multi-well reaction vessel using a compliant thermally conductive insert.

[0022] [Figure 8] FIG. 8 is a process flow diagram illustrating a third embodiment of a process for sample handling and thermal cycling of samples contained within a multi-well reaction vessel using a compliant thermally conductive insert. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] (Detailed Description) In the following description, various embodiments will be described. For purposes of explanation, specific configurations and details are set forth in order to provide a thorough understanding of the present embodiments. However, it will be apparent to one skilled in the art that the embodiments may be practiced in other configurations or without the specific details. Additionally, well-known features may be omitted or simplified so as not to obscure the embodiments being described.

[0024] In a thermal cycler, non-uniform thermal coupling between sample-containing multi-well reaction vessels and heating elements is most often associated with air between the heating block surface and the well bottom surface on the microplate. When non-uniform thermal coupling occurs, this means that some wells with voids will not experience heat flux at the same level as adjacent wells where voids exist. Heat flux through traditional microplate materials (such as polymers like polypropylene, cycloolefins, and the like) cannot provide sufficient lateral heat flux through the microplate to rapidly diffuse heat within the microplate and support fast thermal cycling for wells that do not contact the heating block. Flattening the plate for thermal contact generally fails to alleviate the void problem.

[0025] Automation of the thermal cycling process can be greatly improved by utilizing acoustic sample handling. However, acoustic interrogation and acoustic sample ejection pose engineering challenges in the context of the common "egg-crate" multi-well reaction vessel for PCR and other procedures requiring thermal cycling. The use of flat-bottom acoustically compatible multi-well reaction vessels with flat heating element blocks has been considered, but is also ineffective. Producing plates that are acoustically compatible with "perfectly flat" bottom surfaces, or that are "reproducibly flat" to within the range of compliance when compressed against the flat block of a conventional PCR system, is cost-prohibitive. Greater flexibility in plastic materials is also likely to have a detrimental effect on acoustic microplates, since material stiffness is correlated with acoustic performance. In particular, more compliant materials tend to have greater acoustic attenuation. Also, more compliant materials often undergo greater deformation to stresses from thermal cycling, potentially leading to failure when handled by automated plate handling robots due to deflection of the microplate.

[0026] Illustrated embodiments of the present invention describe systems and methods relating to sample handling for multi-well reaction vessels. According to various embodiments, such systems can include a robotic sample handler that holds and moves a multi-well reaction vessel for automated insertion and / or removal into and from a thermal cycler for performing thermal cycling operations on samples contained in the reaction vessel, such as, but not limited to, PCR. A thermal cycler for use with such a system can include a heating chamber shaped to receive a multi-well reaction vessel containing a heating element, a compliant thermally conductive insert positioned adjacent to the heating element, and a closing mechanism that, when closed, presses the multi-well reaction vessel towards the compliant thermally conductive insert and the heating element. According to at least one embodiment, the compliant thermally conductive insert is a deformable solid with a lateral thermal conductivity that exceeds that of the surface of the heating element. According to specific embodiments, the compliant thermally conductive insert can be formed from an elastically deformable creped graphite sheet or an assembly of any suitable number of parallel graphite sheets that have high thermal conductivity and are reversibly deformable.

[0027] Turning to the figures in which like reference numbers indicate associated elements, FIG. 1 is a simplified block diagram illustrating an exemplary system 100 for sample handling for multi-well reaction vessels in accordance with various embodiments of the present disclosure.

[0028] The system 100 includes a controller 101, which may be a computer system operating from one or more processors 103 and a non-transitory memory device 105 containing executable instructions that control tasks automated by the system. Note that the controller 101 may be distributed or centralized, may be cloud-based, or may operate from one or more of the on-board controllers of the various assemblies described herein. Additionally, certain portions of the system 100 described below may be manually operated or otherwise separate from the automated system, including any suitable subset of the assemblies described herein. Control by the controller 101 of the system elements may be effected over a network 107, which may be a wired or wireless network.

[0029] According to at least one embodiment, system 100 includes one or more of an acoustic sample handler 120, a thermal cycler 140, and an analyzer assembly 160. These system elements may be controlled automatically, for example, by a controller 101, or may be controlled locally, either autonomously or semi-autonomously with user input. A multi-well reaction vessel 119 may be transported between the system elements, for example, by hand or by an automated robotic system 110 under the control of the controller 101. The automated robotic system 110 may include any suitable assembly of actuators for effecting sample transport between the system elements, but according to one embodiment includes at least a rotary actuator 111 that rotates a robotic arm 113 between the different system elements with an end 115 having a manipulator 117 thereon that can grip the multi-well reaction vessel 119 and manipulate its orientation in space to insert or remove the multi-well reaction vessel into or from the various system elements.

[0030] The acoustic sample handler 120 can acoustically transport samples between multi-well reaction vessels, individual sample vessels, or the like. The acoustic sample handler 120 can include an on-board processor 121 and memory device 123 to control acoustic interrogation and / or ejection, an acoustic ejector 125, and any suitable number of actuators 127 to hold and move one or more of the acoustic ejector, a source vessel 129, a receiving vessel 131, which may be a multi-well reaction vessel 119, or may be another vessel. For example, according to some embodiments, a single source vessel can be used to inject samples into a multi-well reaction vessel, or a first multi-well reaction vessel can inject samples into a second multi-well reaction vessel by acoustic ejection from a well in one into a well in the other via the acoustic ejector 125. According to some embodiments, the acoustic ejector 125 can be used to acoustically interrogate a well within the source vessel 129, for example, by emitting acoustic energy into the source vessel, detecting echoes of the emitted acoustic energy, and determining parameters of the interrogated well from the echoes. Such parameters can include, but are not limited to, meniscus height, viscosity, acoustic impedance, and the like.

[0031] The thermal cycler 140 can receive a multi-well reaction vessel 119 in a heating chamber 151 and perform thermal cycling on samples in the reaction vessel under the control of a local processor 141 and a memory device 143, which can contain program instructions for a thermal process under the control of the processor. The thermal cycler includes an insulating body 145 and a closure 147 connected at a hinge 149, which together define a heating chamber 151. The heating chamber 151 contains a heating element 153, such as a resistive heating filament or equivalent, generally enclosed within a thermally conductive heating element block 155 that protects the heating element. The heating element 153 and heating element block 155 are collectively referred to throughout as the heating element.

[0032] A compliant thermally conductive insert 157 can be placed within the heating chamber 151 on the heating element 153 in a position for thermal contact with the heating element (or heating element block 155) and for direct contact with the multiwell reaction vessel 119 when the vessel is inserted into the heating chamber 151. The heating chamber 151 is sized such that when the multiwell reaction vessel 119 is inserted therein and the lid 159 is secured, the occluder compresses the compliant thermally conductive insert 157 against the multiwell reaction vessel such that the insert deforms to adopt a compressed profile that increases the thermal contact area of ​​the compliant thermally conductive insert with both the heating element 153 and the multiwell reaction vessel.

[0033] The compliant thermally conductive insert 157 is reversibly deformable under pressure such that it may adopt a variety of different compressed profiles in response to compression between multi-well reaction vessels 119 having different specific topographies or between heating elements in different thermal cyclers having different specific topographies. According to some embodiments, the compliant thermally conductive insert includes any suitable number of graphite layers arranged in a compressible configuration. The graphitic thermally conductive insert can be comprised of, for example, a creped graphite layer or an assembly of multiple creped graphite layers. Suitable creped graphite layers can be formed by microscopically deforming a planar graphite sheet to introduce a multitude of microfolds that adopt an accordion-like microstructure, with a sheet thickness of about 10 to 2,000 microns or greater. According to various other embodiments, the compliant thermally conductive insert can be a thermally conductive and compliant elastic polymer or polymer composite.

[0034] One suitable creped graphite material is produced by NeoGraf Solutions (LLC, OH, USA) and described in PCT Patent Publication No. WO2019 / 142082A2, entitled "A GRAPHITE ARTICLE AND METHOD OF MAKING SAME," which is incorporated herein by reference for all purposes. A specific product has an in-plane thermal conductivity of over 400 W / mK, exceeding that of aluminum (below 250 W / mK), but exhibits compliance for a thickness range of over 250 microns at 100 kPa for a 500 micron sheet. By way of comparison, a thermal cycler can generate pressure on a microplate during cycling to maintain a significant percentage of hermetic integrity of at least 100 kPa. Of note, elastically deformable graphite has never been adapted for use in a thermal cycler, but instead has been hypothesized as a solution for permanent placement within electronic equipment, for example, permanently crimped between a processor and a heat sink.

[0035] According to various embodiments, the compliant thermally conductive insert can have an uncompressed thickness in the range of 250 microns to 2,000 microns, or 250 microns to 1,000 microns, or 250 microns to 750 microns. The compliant thermally conductive insert may have an in-plane thermal conductivity of at least 200 W / mK, preferably at least 700 W / mK, or greater. The compliant thermally conductive insert can have a through-plane thermal conductivity that increases non-linearly with compressive stress when placed between a heating element and a multi-well reaction vessel. The through-plane thermal conductivity can range from 1-5 W / mK at 100 kPa compressive stress to 10-30 W / mK at 700 kPa compressive stress, or higher. Alternatively, the through-plane thermal conductivity of the compliant thermally conductive insert may be sufficient to allow sufficient heat flux from the heating element to the sample in the flat-bottomed acoustically-enabled plate to produce sample heating of at least 0.5° C., or 1° C. / sec, preferably at least 1.5° C. / sec, more preferably at least 2° C. / sec. The compliant thermally conductive insert may also be reversibly deformable. For example, when subjected to a compressive stress of 700 kPa, the compliant thermally conductive insert may be reversibly compressed to less than 60% of its original (unloaded) thickness and may return to at least 70% of its original thickness, preferably at least 80% of its original thickness, more preferably at least 90% of its original thickness. When repeatedly compressed, the compliant thermally conductive insert may return to the same uncompressed thickness after subsequent compression. According to some embodiments, the compliant thermally conductive insert may also be configured for placement above the multi-well reaction vessel to facilitate flexing of the multi-well reaction vessel within the heating chamber and / or to provide improved lateral heat conduction across the reaction vessel via the compliant thermally conductive insert.

[0036] The analyzer assembly 160 can accept a multi-well reaction vessel 119 to effect automated sample analysis via acoustic ejection under the control of a local processor 161 and a memory device 163, which may contain program instructions for controlling the analyzer 169, according to various embodiments of the present disclosure. A variety of specific automated analyzers may be shown for use with the described automation system 100. For example, according to some embodiments, the analyzer 169 can be a DNA scanner, a flow cytometer, a gas chromatograph and / or a mass spectrometer, a high pressure liquid chromatograph, or other suitable analyzer for receiving and processing liquid samples. The multi-well reaction vessel 119 can be inserted into the analyzer assembly 160 at a sample handling stage 167. Individual samples from the multi-well reaction vessel 119 can be ejected from the vessel into an inlet port 171 of the analyzer 169 via an acoustic ejector 165. The sample handling stage 167 can include any suitable actuator for moving the multi-well reaction vessel 119 relative to the inlet port 171, for moving the acoustic ejector 165 to effect ejection, or both. According to some embodiments, the acoustic ejector 165 can also function as an emitter for acoustic interrogation of sample wells within the multi-well reaction vessel 119, for example, to test depth and / or acoustic impedance prior to ejection or other suitable interrogation.

[0037] In combination, the system 100 can provide partially or fully automated sample handling and transport between source containers and multi-well reaction vessels 119 via the acoustic sample handler 120, for example by the robotic system 110, to and from the thermal cycler 140, and to the analyzer assembly 160, according to various embodiments. The thermal cycler 140 has the technical advantage of being able to handle acoustically compatible microplates that are generally rigid and flat-bottomed, by improving the thermal contact between the inserted multi-well reaction vessels 119 and the heating element 153 via the compliant thermally conductive insert 157. The use of a reversibly deformable and inert material in the compliant thermally conductive insert also prevents the accumulation of residues on the bottom surface of the multi-well reaction vessel, allowing it to remain sufficiently clean throughout the entire automated sample handling procedure for repeated acoustic sample transport. This approach is in contrast to conventional approaches in which specialized plate geometries are employed to enhance dry heat transfer between a heating element and a reaction vessel, or partial immersion in which a working fluid can be used to enhance heat transfer.

[0038] 2 is a simplified cross-sectional side schematic illustrating in greater detail a thermal cycler 140 compatible with the FIG. 1 system 100 for receiving a multi-well reaction vessel 119 and incorporating a compliant thermally conductive insert 157. The compliant thermally conductive insert 157 can be layered between a heating element 153 and a heating element block 155 that contains the multi-well reaction vessel 119. Closing of the lid 159 compresses the compliant thermally conductive insert 157 between the bottom surface 118 of the multi-well reaction vessel 119 and the heating element 153. The compression deforms the compliant thermally conductive insert 157 to increase the contact area of ​​the insert along the bottom surface 118 of the multi-well reaction vessel 119, preventing the formation of air pockets under any particular well 116 and facilitating the application of heat to the sample 114 contained therein. The compliant thermally conductive inserts are at least sufficiently compliant to maintain physical contact between a conventional "flat" PCR heat transfer block and the surface beneath the wells of a "flat-bottom" microplate. Depending on the thickness of the compliant thermally conductive inserts, the number of sheets used, and the pressure applied, the non-flatness between the heating element block 155 and the bottom surface 118 of the multi-well reaction vessel plate that may be filled by the inserts may be characterized by a "gap" of 100, 200, or even 500 microns (i.e., a topographical difference between adjacent highest and lowest points of up to 100, up to 200, or up to 500 microns).

[0039] According to various embodiments, the compliant thermally conductive insert 157 can include a creped graphite material 156 supported in a frame 158 that is sized and shaped to fit the insert within the heating chamber 151 to match the bottom surface of the multi-well reaction vessel 119 and the heating element 153, or both. According to some embodiments, the frame 158 can be compatible with the manipulator 117 (FIG. 1) of the robotic system 110 so that it can be automatically placed in and removed from the heating chamber 151.

[0040] FIG. 3 is a detailed perspective view illustrating a side view of the compliant thermally conductive insert 157 of FIG. 2 in additional detail. A frame 158 encapsulates and supports at least one layer of creped graphite material 156, and optionally multiple layers of creped graphite material. The layer of creped graphite material can have a thickness ranging from as little as 10 microns to up to 2,000 microns between the top surface 152 and the bottom surface 154. One or both of the bottom surface 154 and the top surface 152 can include an additional layer of flexible and thermally conductive, but not significantly compliant, supporting material to provide either a protective outer surface or a surface for bonding to a sheet of creped graphite material (or other suitable compliant and highly thermally conductive material). The frame can include single or multiple sheets on one or more of the heat block facing surface, plate facing surface, or interior of the device. The insert frame thickness 152 can similarly be varied, for example, from 10 microns to 2,000 microns, matching the approximate thickness of the creped graphite material 156, or can be significantly thicker to provide a gripping surface for automated handling. The frame 158 can be characterized as an interface device for moving a compliant thermally conductive insert into and out of a thermal cycler, and designed to facilitate movement of the frame by an automated system such as those made for moving SLAS / ANSI standard microplates. The frame 158 can also be molded to removably or permanently connect to a multi-well reaction vessel 119, or suitable microplate, and both the plate and the frame to be movable as a combined assembly.

[0041] The compliant thermally conductive insert 157 can be deformed to accommodate various imperfections or non-flat topographies in the thermal cycler's multi-well reaction vessel 119 or heating element block 155. Some such use cases are described with reference to Figures 4A-4E below, which illustrate various deformation profiles of the compliant thermally conductive insert as shown in Figures 2 and 3.

[0042] For example, FIG. 4A illustrates a first use case 400a in which a multiwell reaction vessel 419a is enclosed and compressed within a heating cavity 451 of a thermal cycler 440. Like the thermal cycler 140 illustrated above, the thermal cycler 440 includes an insulating body 445 having a lid 459 attached at a hinge 449 that can be lowered to apply pressure on the multiwell reaction vessel 419a when the vessel is inserted into the heating cavity 451. A compliant thermally conductive insert 457 is positioned sandwiched between the multiwell reaction vessel 419a and a heating block 455 and associated heating element 453. In the first use case 400a, the multiwell reaction vessel 419a adopts a convex curved configuration in response to thermal cycling. Following conventional approaches, the crimping force by the lid 459 is insufficient to flatten the multiwell reaction vessel 419a in contact with the heating block 455 sufficient to maintain efficient heat transfer. However, the compliant thermally conductive material 456a within the compliant thermally conductive insert 457 can reversibly deform to contact all or substantially all of the bottom surface of the multi-well reaction vessel 419a while maintaining contact with the heating block 455 and thus the heating element 453. According to some embodiments, an additional compliant thermally conductive insert can be placed above the multi-well reaction vessel to accommodate flexure of the reaction vessel while maximizing lateral heat conduction.

[0043] Similarly, FIG. 4B illustrates a second use case 400b in which a multiwell reaction vessel 419b is enclosed and compressed within a heating cavity 451 of a thermal cycler 440. In the second use case 400b, the multiwell reaction vessel 419b adopts a concave configuration in response to thermal cycling. Under conventional approaches, the crimping force by the lid 459 is insufficient to flatten the multiwell reaction vessel 419b in contact with the heating block 455 sufficient to maintain efficient heat transfer. However, the compliant thermally conductive material 456b within the compliant thermally conductive insert 457 can reversibly deform to contact all or substantially all of the bottom surface of the multiwell reaction vessel 419b while maintaining contact with the heating block 455 and thus the heating element 453.

[0044] 4C illustrates a third use case 400c in which a compliant thermally conductive insert 457 accommodates a deformation or misalignment of a heat block 455. For example, in the third use case 400c, the flat bottom surface 419c of the multi-well reaction vessel is no longer parallel to the top surface of the heat block 455. The compliant thermally conductive material 456c within the compliant thermally conductive insert 457 can reversibly deform into a wedge-like shape to contact all or substantially all of the bottom surface of the multi-well reaction vessel 419c while maintaining contact with the heat block 455 and thus the heating element 453.

[0045] 4D illustrates a fourth use case 400d in which a multi-well reaction vessel 419d is enclosed and compressed within a heating cavity 451 of a thermal cycler 440 above a heating block 455d with a surface feature 454d, which may represent, for example, a surface damage or defect, or otherwise a raised topography. A compliant thermally conductive insert 457 is positioned sandwiched between the multi-well reaction vessel 419d and the heating block 455d and associated heating element 453. A compliant thermally conductive material 456d within the compliant thermally conductive insert 457 can reversibly deform to fill the space around the surface feature 454d to minimize air pockets and contact all, or substantially all, of the bottom surface 419d of the multi-well reaction vessel while maintaining contact with the heating block 455d and, therefore, the heating element 453.

[0046] 4E illustrates a fifth use case 400e in which a multi-well reaction vessel 419e enclosed and compressed within a heating cavity 451 of a thermal cycler 440 has a surface feature 418e on its bottom surface that reflects, for example, a surface damage or defect or otherwise a raised topography. A compliant thermally conductive insert 457 is positioned sandwiched between the multi-well reaction vessel 419e and a heating block 455e and associated heating element 453. The compliant thermally conductive material 456e within the compliant thermally conductive insert 457 can reversibly deform to fill the space around the surface feature 418e to minimize air pockets and contact all or substantially all of the bottom surface 419e of the multi-well reaction vessel while maintaining contact with the heating block 455e and thus the heating element 453.

[0047] Data regarding the effective ramp rates for several intervening material options was collected to determine whether the compliant thermally conductive insert was an improvement over conventional materials and is shown in Figure 5. Figure 5 is a graphical diagram illustrating the comparative ramp rates 500 of sample-containing multi-well reaction vessels in a thermal cycler with compliant thermally conductive inserts and alternative materials.

[0048] The comparative slope rate 500 illustrated in Figure 5 was obtained by measuring temperature over time in several cells of a 384-well, flat-bottom acoustic microplate containing an aqueous solution, with each well containing 10 microliters of fluid. Note that these plates have a thicker bottom (nominal 980 microns) than a typical PCR plate (approximately 600 microns thick for the control) and a smaller surface area for heat transfer due to the flat bottom versus the traditional conical shape. Thus, a lower slope rate was expected for the acoustic microplate data compared to the control PCR plate data.

[0049] To obtain temperature ramp data, thermocouples were inserted into the fluid samples in wells E7, L7, E18, and L18 of a 384-well plate. An additional thermocouple was used to monitor the heat transfer block temperature. This additional thermocouple was placed between two layers of aluminum foil, positioned directly on top of the heating block, and pressed firmly against the bottom of the thermal adapter medium. Different thermal adapter media were tested for their performance in well temperature ramp rates during thermal cycling as reflected in Figure 5 and shown in Table 1 below. [Table 1]

[0050] As illustrated in Table 1 above and FIG. 5, the ramp rates achieved between the planar heating element block and the flat-bottomed acoustically enabled plate were much lower than the control ramp rates achieved using a PCR plate. However, the average ramp rates using creped graphite material, i.e., a reversibly compressible thermally conductive material, were significantly higher for the flat-bottomed plate than any other material selected. Graphite sheets (Graphite 1, Graphite 2) were more thermally conductive but not compliant, while silicone rubbers (Silicone Rubber 1, Silicone Rubber 2) were highly compliant but not very thermally conductive. As shown, the combination of reversible deformation (compliance) and thermal conductivity provided a significant improvement in ramp rate.

[0051] Examples of automated and semi-automated processes for sample handling and thermal cycling, for example for use in conjunction with automated PCR, are described below with reference to Figures 6-8. Processes 600, 700, and 800 (or any other process or variant described herein, and / or combinations thereof) may be automated and performed mechanically under the control of one or more computer systems configured with executable instructions, implemented as code (e.g., executable instructions, one or more computer programs, or one or more applications) collectively executed on one or more processors, by hardware or a combination thereof. The code may be stored on a computer-readable storage medium, for example, in the form of a computer program comprising a plurality of instructions executable by one or more processors. The computer-readable storage medium may be non-transient. In some embodiments, aspects of processes 600, 700, and 800 may be performed manually. Although specific process steps are described in each process, each process step of processes 600, 700, and 800 may be performed in any suitable order or sequentially with steps of different processes, unless specifically contradicted. For example, a step of process 700 or process 800 may follow a step of process 600, or vice versa.

[0052] FIG. 6 is a process flow diagram illustrating a first example of a process 600 for sample handling and thermal cycling of samples contained in a multi-well reaction vessel using a compliant thermally conductive insert. In process 600, a first multi-well reaction vessel is inserted into a thermal cycler at 601. The first multi-well reaction vessel can then be enclosed within the thermal cycler, causing the vessel to compress a thermally conductive insert positioned on its heating element according to a first compressed profile at 603. The thermal cycler can then be cycled according to a predefined temperature program to sequentially raise and lower the temperature of the multi-well reaction vessel by applying heat flux from the heating element through the insert at 605. Once completed, the first multi-well reaction vessel can be removed from the thermal cycler, allowing the thermally conductive insert to return to a non-compressed state from the first compressed profile at 607.

[0053] A second multi-well reaction vessel may then be inserted into the thermal cycler, causing the vessel to compress a thermally conductive insert positioned on a heating element according to a second compressed profile at 609, where the second compressed profile is a different geometry than the first compressed profile. For example, the profile may reflect a different surface topography of the bottom surface of the multi-well reaction vessel at each run. The thermal cycler may be cycled to sequentially raise and lower the temperature of the multi-well reaction vessel by applying heat flux from the heating element through the insert at 611, and then the second multi-well reaction vessel may be removed from the thermal cycler, allowing the thermally conductive insert to return to an uncompressed state from the second compressed profile at 613. Importantly, the first and second compressed profiles, although different, do not significantly alter the uncompressed state of the compliant thermally conductive insert. Additionally, the compliant thermally conductive insert compresses in both movements to provide a uniform heat flux across the gap between the heating element and the multi-well reaction vessel.

[0054] 7 is a process flow diagram illustrating a second example of a process for sample handling and thermal cycling of samples contained in a multiwell reaction vessel using a compliant thermally conductive insert. In process 700, either a user or an automated robotic manipulator may insert the multiwell reaction vessel into a heating chamber of a thermal cycler by placing the multiwell reaction vessel on a compliant thermally conductive insert on a heating element of the thermal cycler at 701. The multiwell reaction vessel can then be enclosed in the heating chamber at 703. Securing the thermal cycler cover can include compressing a bottom surface of the multiwell reaction vessel into the compliant thermally conductive insert at 705 to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert.

[0055] Once the multi-well reaction vessel is enclosed within the heating chamber, the thermal cycler can be thermally cycled according to a thermal program by applying a controlled heat flux to the multi-well reaction vessel by a heating element through a compliant thermally conductive insert at 707. According to some embodiments, the thermal cycler can repeatedly increase and decrease the temperature. While some thermal cyclers may simply allow heat to dissipate to cool the multi-well reaction vessel between heating cycles, other thermal cyclers may include a cooling mechanism (e.g., a cold working fluid, refrigeration, or the like). Once the thermal program is completed, the multi-well reaction vessel can be removed from the heating chamber.

[0056] According to some embodiments, after completion of the thermal program, the multi-well reaction vessel can be transported at 709 to an analyzer equipped with an acoustic ejector for sample transport. The acoustic ejector can be configured to acoustically transport droplets of sample contained within the multi-well reaction vessel into the analyzer by an acoustic ejection process whereby focused acoustic energy is transmitted through a bottom surface of the multi-well reaction vessel at 711 to transport droplets of sample into an inlet of the analyzer.

[0057] FIG. 8 is a process flow diagram illustrating a third example of a process for sample handling and thermal cycling of samples contained in a multiwell reaction vessel using a compliant thermally conductive insert. In process 800, a droplet containing a sample from a source well can be acoustically transported into the multiwell reaction vessel at 801, for example, via an acoustic sample handling assembly. The system can also acoustically interrogate sample-containing wells in the multiwell reaction vessel at 803 by transmitting an interrogation tone burst from an acoustic emitter through a bottom surface of the multiwell reaction vessel. Sample interrogation can be used to determine various sample attributes, for example, sample depth, acoustic impedance, viscosity, and other attributes. The multiwell reaction vessel can be inserted into the heating chamber of a thermal cycler at 805, either manually or automatically, by placing the multiwell reaction vessel on the compliant thermally conductive insert on the heating element of the thermal cycler. Encapsulating the multiwell reaction vessel within the thermal cycler compresses the bottom surface of the multiwell reaction vessel into the compliant thermally conductive insert at 807 to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert. The increase in contact area is relative to a hypothetical contact area that may be achieved, for example, between the bottom surface of the multiwell reaction vessel and a flat thermal plate or a non-compliant thermally conductive insert, in which voids or air pockets would tend to form between the adjacent surfaces. This increased contact area is maintained during thermal cycling of the multiwell reaction vessel within the heating chamber at 809 via elastic deformation of the compliant thermally conductive insert in response to thermal deformation of the multiwell reaction vessel or heating element. For example, if the multiwell reaction vessel is deflected either upwards or downwards due to heating or cooling, the compliant thermally conductive insert can elastically deform to conform to its bottom surface as long as it is compressed without permanently flowing or plastically deforming. Removal of the multi-well reaction vessel from the heating chamber allows the compliant thermally conductive insert to return to its uncompressed state at 811.

[0058] The various computational methods discussed above may be implemented in conjunction with or using a computer or other processor having hardware, software, and / or firmware. The various method steps may be implemented by modules, which may comprise any of a wide variety of digital and / or analog data processing hardware and / or software arranged to implement the method steps described herein. The modules optionally comprise data processing hardware adapted to implement one or more of these steps by having appropriate machine programming code associated therewith, with modules for two or more steps (or portions of two or more steps) being integrated into a single processor board or separated on different processor boards into any of a wide variety of integrated and / or distributed processing architectures. These methods and systems will often employ tangible media embodying machine-readable code with instructions for implementing the method steps described above. Suitable tangible media may comprise memory (including volatile and / or non-volatile memory), storage media (such as magnetic recording on a floppy disk, hard disk, tape, or equivalent, optical memory such as a CD, CD-R / W, CD-ROM, DVD, or equivalent, or any other digital or analog storage medium), or equivalent.

[0059] The items set forth herein are presented as examples and for the sole purpose of illustrative discussion of preferred embodiments of the present invention, in order to provide what is believed to be the most useful and readily understood explanation of the principles and conceptual aspects of various embodiments of the present invention. In this regard, no attempt is made to show structural details of the present invention in greater detail than is necessary for a basic understanding of the present invention, a description given with the aid of drawings, and / or examples that will be apparent to those skilled in the art of how some forms of the present invention may be embodied in practice.

[0060] The following definitions and explanations are meant and intended to take precedence in any future structures unless clearly and obviously amended in the examples below, or when application of the meaning would render the structure meaningless or essentially meaningless. If the structure of a term would render it meaningless or essentially meaningless, the definition should be taken from a dictionary known to those skilled in the art, such as Webster's Dictionary, Third Edition, or the Oxford Dictionary of Biochemistry and Molecular Biology (Ed. Anthony Smith, Oxford University Press, Oxford, 2004).

[0061] Unless the context clearly requires otherwise, throughout the description and claims, the words "comprise," "comprising," and the like, should be construed in an inclusive sense, i.e., "including, but not limited to," as opposed to an exclusive or inclusive sense. Words using the singular or plural also include the plural and singular, respectively. In addition, the words "herein," "above," and "below," and words of similar import, when used in this application, shall refer to this application as a whole and not to any particular portions of this application.

[0062] The description of the embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise form disclosed. Specific embodiments and examples of the present disclosure are described herein for illustrative purposes, but various equivalent modifications are possible within the scope of the present disclosure, as those skilled in the relevant art will recognize.

[0063] All references, including patent applications (including patents, patent applications, and patent publications), scientific journals, books, articles, technical references, and other publications and materials discussed in this application are incorporated by reference in their entirety and for all purposes.

[0064] Aspects of the present disclosure can be modified, if necessary, to employ the systems, functions, and concepts of the above references and applications to provide still further embodiments of the present disclosure. These and other changes can be made to the present disclosure in light of the detailed description.

[0065] Specific elements of any of the foregoing embodiments can be combined with or substituted for elements in other embodiments. Additionally, although benefits associated with certain embodiments of the present disclosure are described in the context of those embodiments, other embodiments may also exhibit such benefits, and not all embodiments necessarily need to exhibit such benefits, to fall within the scope of the present disclosure.

[0066] Although the above provides a full and complete disclosure of exemplary embodiments of the present invention, various modifications, alternative constructions, and equivalents may be employed as desired. As a result, although the embodiments have been described in some detail by way of example and for clarity of understanding, various modifications, changes, and adaptations will be apparent to those skilled in the art. Therefore, the above description and illustrations should not be construed as limiting the present invention, which can be defined by the appended claims.

[0067] Other variations are within the spirit of the present disclosure. Thus, while the disclosed techniques are susceptible to various modifications and alternative constructions, certain illustrated embodiments thereof are shown in the drawings and described above in detail. It should be understood, however, that there is no intention to limit the disclosure to the specific forms disclosed, and on the contrary, the intention is to cover all modifications, alternative constructions, and equivalents falling within the spirit and scope of the present disclosure, as defined in the appended claims.

[0068] Use of the terms "a" and "an" as well as "the" and similar references in the context of describing the disclosed embodiments (particularly in the context of the claims that follow) should be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly encompassed by context. The terms "comprising," "having," "including," and "containing" should be construed as open-ended terms (i.e., meaning "including, but not limited to"), unless otherwise stated. The term "connected" should be construed as contained within, attached to, or joined together, either partially or wholly, even if there is something intervening. Recitation of ranges of values ​​herein is merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into this specification as if each separate value were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly encompassed by context. Any and all examples provided herein, or the use of exemplary language (e.g., "such as"), are intended merely to more fully illustrate embodiments of the disclosure and not to impose limitations on the scope of the disclosure unless otherwise claimed. No language in this specification should be construed as indicating any non-claimed element as essential to the practice of the disclosure.

[0069] Disjunctive language such as the phrase "at least one of X, Y, or Z," unless specifically stated otherwise, is intended to be understood in the context of its general use to present items, terms, and the like, and may be either X, Y, or Z, or any combination thereof (e.g., X, Y, and / or Z). Thus, such disjunctive language is generally not intended, and should not imply, that an embodiment requires that at least one of X, at least one of Y, or at least one of Z, each be present.

[0070] Preferred embodiments of the present disclosure, including the best mode known to the inventors of carrying out the disclosure, are described herein. Variations of these preferred embodiments may become apparent to those skilled in the art upon perusal of the foregoing description. The inventors anticipate that those skilled in the art will adopt such variations as necessary, and the inventors intend for the present disclosure to be practiced otherwise than as specifically described herein. Accordingly, the present disclosure includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the present disclosure, unless otherwise indicated herein or otherwise clearly contradicted by context.

[0071] All references cited in this specification, including publications, patent applications, and patents, are herein incorporated by reference to the same extent as if each reference was individually and specifically indicated to be incorporated by reference and was set forth in its entirety herein.

[0072] In the following, further examples are described to facilitate understanding of the present invention.

[0073] Example A 1. A system comprising: a robotic sample handler configured to hold and move multi-well reaction vessels; a thermal cycler comprising a heating chamber containing a heating element, a compliant thermally conductive insert comprising an elastically deformable creped graphite sheet positioned adjacent to the heating element, and a closing mechanism, wherein the heating chamber is shaped to receive a multi-well reaction vessel, and the closing mechanism is configured to urge the multi-well reaction vessel towards the compliant thermally conductive insert and the heating element; a controller operatively connected to the robotic sample handler and the thermal cycler; Equipped with The controller comprises at least one processor and a memory containing executable instructions, which when executed by the at least one processor: causing a robotic sample handler to insert a multi-well reaction vessel into a thermal cycler; causing a closure mechanism to enclose the multi-well reaction vessel within the heating chamber; compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert with a closure mechanism; thermally cycling the multi-well reaction vessel within the heating chamber with a heating element by applying a controlled heat flux from the heating element to the multi-well reaction vessel through a compliant thermally conductive insert; The system includes:

[0074] Example B. compressing the bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert causes a reversible deformation of the creped graphite sheet according to a first compression profile; compressing the second bottom surface of the second multi-well reaction vessel into the compliant thermally conductive insert causes reversible deformation of the creped graphite sheet according to a second compression profile different from the first compression profile; The system of any preceding embodiment, wherein the compliant thermally conductive insert returns to an uncompressed state from the first compressed profile, and from the second compressed profile, without permanently deforming.

[0075] Example C a source vessel containing a reagent or sample; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and a source vessel with a well of a multi-well reaction vessel; Further equipped with The executable instructions, when executed by at least one processor, causing an actuator to selectively align the transducer and source vessel with one or more of the wells of a multi-well reaction vessel; causing an acoustic ejector to eject one or more droplets from a source vessel into a well of a multi-well reaction vessel by applying focused acoustic radiation to a sample contained in the source vessel; The system of any one of the preceding embodiments, further configured to:

[0076] Example D. a multi-well receiving vessel; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and the multi-well reaction vessel with wells of a multi-well receiving vessel; Further equipped with The executable instructions, when executed by at least one processor, causing the actuator to selectively align the transducer and the multi-well reaction vessel with one or more of the wells of the multi-well receiving vessel; causing the acoustic ejector to eject one or more droplets from the multi-well reaction vessel into wells of the multi-well receiving vessel by applying focused acoustic radiation to the sample contained within the multi-well reaction vessel; The system of any one of the preceding embodiments, further configured to:

[0077] Example E an analyzer having a sample inlet; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and the multi-well reaction vessel with a sample inlet of an analyzer; Further equipped with The executable instructions, when executed by at least one processor, selectively aligning the transducer and the multi-well reaction vessel with a sample inlet of the analyzer; causing the acoustic ejector to eject one or more droplets from the multi-well reaction vessel to the sample inlet by applying focused acoustic radiation to the sample contained within the multi-well reaction vessel; The system of any one of the preceding embodiments, further configured to:

[0078] Example F. A method comprising: inserting the multi-well reaction vessel into a heating chamber of a thermal cycler by placing the multi-well reaction vessel on a compliant, thermally conductive insert on a heating element of the thermal cycler, the compliant, thermally conductive insert comprising an elastically deformable creped graphite sheet; Enclosing a multi-well reaction vessel within a heating chamber; compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert; thermally cycling the multi-well reaction vessel within the heating chamber by applying a controlled heat flux to the multi-well reaction vessel by a heating element through a compliant thermally conductive insert; A method comprising:

[0079] Example G The method of any preceding embodiment, wherein the compliant thermally conductive insert has an uncompressed thickness in the range of 250 microns to 2,000 microns.

[0080] Example H. The method of any one of the previous embodiments, wherein the compliant thermally conductive insert has an in-plane thermal conductivity of at least 200 W / mK, preferably at least 700 W / mK.

[0081] Example I The method of any one of the preceding embodiments, wherein the compliant thermally conductive insert has a through-plane thermal conductivity that increases non-linearly with compressive stress, the through-plane thermal conductivity ranging from 1-5 W / mK at 100 kPa compressive stress to 10-30 W / mK at 700 kPa compressive stress.

[0082] Example J. The method of any one of the previous embodiments, wherein the compliant thermally conductive insert reversibly compresses to less than 60% of its original thickness when subjected to a compressive stress of 700 kPa.

[0083] Example K The multi-well reaction vessel comprises a microplate comprising an array of wells having at least one flat bottom surface configured to allow acoustic interrogation of samples contained in the array of wells through the flat bottom surface, and the method further comprises: emitting an interrogation tone burst from an acoustic emitter through the flat bottom surface; detecting an acoustic echo caused by the interrogation tone burst; determining parameters of the sample from the detected acoustic echoes; The method of any one of the preceding Examples, comprising:

[0084] Example L. The method of any one of the preceding Examples, wherein thermally cycling the multi-well reaction vessel in the heating chamber comprises sequentially heating and cooling the sample contained in the multi-well reaction vessel according to a PCR thermal cycling program at a heating or cooling rate of at least 1°C / sec, preferably at least 1.5°C / sec, preferably at least 2°C / sec.

[0085] Example M. aligning one or more wells of a multi-well reaction vessel with a source well and an acoustic ejector positioned to acoustically eject fluid droplets from the source well; ejecting one or more droplets from the source wells into wells of the multi-well reaction vessel by applying focused acoustic radiation from an acoustic ejector to a sample contained in the source well; The method of any one of the preceding Examples, further comprising:

[0086] Example N. aligning one or more wells of the multiwell reaction vessel with an acoustic ejector positioned to acoustically eject fluid droplets from the multiwell reaction vessel and with a multiwell receiving vessel; ejecting one or more droplets from the wells of the multi-well reaction vessel into the wells of the multi-well receiving vessel by applying focused acoustic radiation from an acoustic ejector to the sample contained in the wells of the multi-well reaction vessel; The method of any one of the preceding Examples, further comprising:

[0087] Example O. aligning wells of the multi-well reaction vessel with an acoustic ejector positioned to acoustically eject fluid droplets from the multi-well reaction vessel and with a sample inlet of an analytical device; ejecting one or more droplets from a well of the multi-well reaction vessel into a sample inlet by applying focused acoustic radiation from an acoustic ejector to a sample contained in the well; The method of any one of the preceding Examples, further comprising:

[0088] Example P. 1. A thermal cycler assembly comprising: A heating chamber; a heating element contained in a heating chamber; a closure mechanism configured to enclose the heating chamber and press onto the multi-well reaction vessel when the multi-well reaction vessel is received in the heating chamber; a compliant thermally conductive insert positioned within the heating chamber in contact with the heating element, the compliant thermally conductive insert comprising an elastically deformable creped graphite sheet; A thermal cycler assembly comprising:

[0089] Example Q. A thermal cycler assembly as described in any preceding embodiment, wherein the compliant thermally conductive insert comprises a plurality of layered, elastically deformable creped graphite sheets.

[0090] Example R. The thermal cycler assembly of any one of the previous embodiments, wherein the compliant thermally conductive insert is connected to the elastically deformable creped graphite sheet that is removably insertable into the heating chamber and comprises an interface frame shaped to align the heating element and the multi-well reaction vessel with the elastically deformable creped graphite sheet when the multi-well reaction vessel is inserted into the heating chamber.

[0091] Example S. 2. The thermal cycler assembly of any one of the previous embodiments, wherein the compliant thermally conductive insert has an uncompressed thickness in the range of 250 microns to 2,000 microns and an in-plane thermal conductivity of at least 200 W / mK, preferably at least 700 W / mK.

[0092] Example T 2. The thermal cycler assembly of any one of the preceding embodiments, wherein the compliant thermally conductive insert has a through-plane thermal conductivity that increases nonlinearly with compressive stress, the through-plane thermal conductivity ranging from 1-5 W / mK at 100 kPa compressive stress to 10-30 W / mK at 700 kPa compressive stress, and the compliant thermally conductive insert is reversibly compressible to less than 60% of its original thickness in response to a 700 kPa compressive stress.

[0093] Example U. 1. A method comprising: inserting a first multi-well reaction vessel into a heating chamber of a thermal cycler by placing the multi-well reaction vessel on a compliant, thermally conductive insert on a heating element of the thermal cycler, the compliant, thermally conductive insert comprising an elastically deformable creped graphite sheet; compressing the first bottom surface into the compliant thermally conductive insert such that pressure between the first bottom surface of the first multi-well reaction vessel and the heating element causes reversible deformation of the creped graphite sheet according to a first compression profile; inserting a second multiwell reaction vessel into a heating chamber of a thermal cycler by placing the second multiwell reaction vessel on a compliant thermally conductive insert; compressing the second bottom surface into the compliant thermally conductive insert such that pressure between the second bottom surface of the second multi-well reaction vessel and the heating element causes reversible deformation of the creped graphite sheet according to a second compression profile different from the first compression profile, the compliant thermally conductive insert returning to an uncompressed state from the first compressed profile and from the second compressed profile without permanent deformation; A method comprising:

[0094] Example V The method of any preceding embodiment, further comprising thermally cycling the multi-well reaction vessel within the heating chamber by applying a controlled heat flux to the multi-well reaction vessel with a heating element through a compliant thermally conductive insert.

[0095] Example W. aligning wells of a first or second multiwell reaction vessel with an acoustic emitter positioned to apply focused acoustic radiation from the acoustic emitter to samples contained in the wells of the multiwell reaction vessel; emitting an interrogation tone burst from an acoustic emitter through a flat bottom surface of the first or second multi-well reaction vessel; detecting an acoustic echo caused by the interrogation tone burst; determining a parameter of the sample in the well from the detected acoustic echoes; The method of any one of the preceding Examples, further comprising:

[0096] Example X. The method of any one of the preceding examples, further comprising applying a heat flux from the heating element through the compliant thermally conductive insert to the first or second multi-well reaction vessel sufficient to cause a temperature change of the sample in the first or second multi-well reaction vessel of at least 1° C. / sec, preferably at least 1.5° C. / sec, preferably at least 2° C. / sec.

[0097] Example Y. aligning wells of a first or second multiwell reaction vessel with an acoustic ejector positioned to apply focused acoustic radiation from the acoustic ejector to samples contained in the wells of the multiwell reaction vessel; ejecting droplets from the wells by emitting an ejection tone burst from an acoustic ejector through a flat bottom surface of the first or second multi-well reaction vessel; The method of any one of the preceding Examples, further comprising:

Claims

1. 1. A system comprising: a robotic sample handler configured to hold and move multi-well reaction vessels; a thermal cycler comprising: a heating chamber containing a heating element; a compliant, thermally conductive insert comprising an elastically deformable, creped graphite sheet positioned adjacent to the heating element; and a closing mechanism, the heating chamber shaped to receive the multi-well reaction vessel, the closing mechanism configured to press the multi-well reaction vessel towards the compliant, thermally conductive insert and the heating element, the compliant, thermally conductive insert including a frame for enclosing and supporting an edge of the creped graphite sheet, the frame removably insertable into the heating chamber, the frame sized and shaped to align the heating element and the multi-well reaction vessel with the elastically deformable, creped graphite sheet when the multi-well reaction vessel is inserted into the heating chamber; a controller operatively connected to the robotic sample handler and the thermal cycler; Equipped with The controller comprises at least one processor and a memory containing executable instructions that, when executed by the at least one processor, causing the robotic sample handler to insert the multi-well reaction vessel into the thermal cycler; causing the closure mechanism to enclose the multi-well reaction vessel within the heating chamber; compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert with the closure mechanism; thermally cycling said multi-well reaction vessel within said heating chamber with said heating element by applying a controlled heat flux from said heating element to said multi-well reaction vessel through said compliant thermally conductive insert; and configuring the controller to:

2. compressing the bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert causes a reversible deformation of the creped graphite sheet according to a first compression profile; compressing the second bottom surface of the second multi-well reaction vessel into the compliant thermally conductive insert causes reversible deformation of the creped graphite sheet according to a second compression profile different from the first compression profile; 10. The system of claim 1, wherein the compliant thermally conductive insert returns to an uncompressed state from the first compressed profile and from the second compressed profile without permanently deforming.

3. a source vessel containing a reagent or sample; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and a source vessel with wells of the multi-well reaction vessel; Further equipped with The executable instructions, when executed by the at least one processor, causing the actuator to selectively align the transducer and source vessel with one of the wells of the multi-well reaction vessel; causing the acoustic ejector to eject one or more droplets from the source vessel into the well by applying the focused acoustic radiation to a sample contained within the source vessel; The system of claim 1 , further configured to:

4. a multi-well receiving vessel; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and the multi-well reaction vessel with wells of the multi-well receiving vessel; Further equipped with The executable instructions, when executed by the at least one processor, causing the actuator to selectively align the transducer and multi-well reaction vessel with one or more of the wells of the multi-well receiving vessel; causing the acoustic ejector to eject one or more droplets from the multiwell reaction vessel into the wells of the multiwell receiving vessel by applying the focused acoustic radiation to a sample contained within the multiwell reaction vessel; The system of claim 1 , further configured to:

5. an analyzer having a sample inlet; an acoustic ejector comprising a transducer configured to emit focused acoustic radiation and an actuator configured to align the acoustic ejector and the multi-well reaction vessel with the sample inlet of the analyzer; Further equipped with The executable instructions, when executed by the at least one processor, causing the actuator to selectively align the transducer and multi-well reaction vessel with the sample inlet of the analyzer; causing the acoustic ejector to eject one or more droplets from the multi-well reaction vessel into the sample inlet by applying the focused acoustic radiation to a sample contained within the multi-well reaction vessel; The system of claim 1 , further configured to:

6. 1. A method comprising: inserting a multiwell reaction vessel into a heating chamber of a thermal cycler by placing the multiwell reaction vessel on a compliant, thermally conductive insert on a heating element of the thermal cycler, the compliant, thermally conductive insert comprising an elastically deformable creped graphite sheet, the compliant, thermally conductive insert including a frame for enclosing and supporting an edge of the creped graphite sheet, the frame being removably insertable into the heating chamber, the frame being sized and shaped to align the heating element and the multiwell reaction vessel with the elastically deformable creped graphite sheet when the multiwell reaction vessel is inserted into the heating chamber; enclosing the multi-well reaction vessel within the heating chamber; compressing a bottom surface of the multi-well reaction vessel into the compliant thermally conductive insert to increase the thermal contact area between the bottom surface and the compliant thermally conductive insert; thermally cycling the multi-well reaction vessel within the heating chamber by applying a controlled heat flux to the multi-well reaction vessel by the heating element through the compliant thermally conductive insert; A method comprising:

7. The method of claim 6, wherein the compliant thermally conductive insert has an uncompressed thickness in the range of 250 microns to 2,000 microns.

8. The method of claim 6, wherein the compliant thermally conductive insert has an in-plane thermal conductivity of at least 200 W / mK.

9. 7. The method of claim 6, wherein the compliant thermally conductive insert has a through-plane thermal conductivity that increases non-linearly with compressive stress, the through-plane thermal conductivity ranging from 1-5 W / m-K at 100 kPa compressive stress to 10-30 W / m-K at 700 kPa compressive stress.

10. 7. The method of claim 6, wherein the compliant thermally conductive insert reversibly compresses to less than 60% of its original thickness when subjected to a compressive stress of 700 kPa.

11. The multi-well reaction vessel comprises a microplate comprising an array of wells having at least one flat bottom surface configured to allow acoustic interrogation of samples contained in the array of wells through the flat bottom surface, and the method further comprises: emitting an interrogation tone burst from an acoustic emitter through said flat bottom surface; detecting an acoustic echo caused by said interrogation tone burst; determining parameters of the sample from the detected acoustic echoes; The method according to any one of claims 6 to 10, comprising:

12. The method of any one of claims 6-10, wherein thermally cycling the multi-well reaction vessel within the heating chamber comprises sequentially heating and cooling the sample contained within the multi-well reaction vessel according to a PCR thermal cycling program at a heating or cooling rate of at least 1°C / sec.

13. aligning one or more wells of the multi-well reaction vessel with a source well and an acoustic ejector positioned to acoustically eject fluid droplets from the source well; ejecting one or more droplets from the source wells into the wells of the multi-well reaction vessel by applying focused acoustic radiation from the acoustic ejector to a sample contained in the source well; The method of any one of claims 6-10, further comprising:

14. aligning one or more wells of the multiwell reaction vessel with an acoustic ejector positioned to acoustically eject fluid droplets from the multiwell reaction vessel and with a multiwell receiving vessel; ejecting one or more droplets from the wells of the multiwell reaction vessel into the wells of the multiwell receiving vessel by applying focused acoustic radiation from the acoustic ejector to samples contained in the wells of the multiwell reaction vessel; The method of any one of claims 6-10, further comprising:

15. aligning wells of the multi-well reaction vessel with an acoustic ejector positioned to acoustically eject fluid droplets from the multi-well reaction vessel and with a sample inlet of an analytical device; ejecting one or more droplets from the wells of the multi-well reaction vessel into the sample inlet by applying focused acoustic radiation from the acoustic ejector to the sample contained in the wells; The method of any one of claims 6-10, further comprising:

16. 1. A thermal cycler assembly comprising: A heating chamber; a heating element contained in the heating chamber; and a closure mechanism configured to enclose the heating chamber and press onto the multi-well reaction vessel when the multi-well reaction vessel is received in the heating chamber; a compliant thermally conductive insert positioned within the heat chamber in contact with the heating element, the compliant thermally conductive insert comprising an elastically deformable creped graphite sheet, the compliant thermally conductive insert including a frame for enclosing and supporting an edge of the creped graphite sheet, the frame being removably insertable into the heat chamber, the frame being sized and shaped to match the heating element and the multiwell reaction vessel and the elastically deformable creped graphite sheet when the multiwell reaction vessel is inserted into the heat chamber; A thermal cycler assembly comprising:

17. 17. The thermal cycler assembly of claim 16, wherein the compliant thermally conductive insert comprises a layered, elastically deformable creped graphite sheet.

18. 17. The thermal cycler assembly of claim 16, wherein the compliant thermally conductive insert has an uncompressed thickness in the range of 250 microns to 2,000 microns and an in-plane thermal conductivity of at least 200 W / mK.

19. 17. The thermal cycler assembly of claim 16, wherein the compliant thermally conductive insert has a through-plane thermal conductivity that increases non-linearly with compressive stress, the through-plane thermal conductivity ranging from 1-5 W / m-K at 100 kPa compressive stress to 10-30 W / m-K at 700 kPa compressive stress, and the compliant thermally conductive insert is reversibly compressible to less than 60% of its original thickness in response to a 700 kPa compressive stress.

20. 1. A method comprising: inserting a first multiwell reaction vessel into a heating chamber of a thermal cycler by placing the first multiwell reaction vessel on a compliant thermally conductive insert on a heating element of the thermal cycler, the compliant thermally conductive insert comprising an elastically deformable creped graphite sheet, the compliant thermally conductive insert including a frame for enclosing and supporting an edge of the creped graphite sheet, the frame being removably insertable into the heating chamber, the frame being sized and shaped to align the heating element and the first multiwell reaction vessel and the elastically deformable creped graphite sheet when the first multiwell reaction vessel is inserted into the heating chamber; compressing a first bottom surface of the first multi-well reaction vessel into the compliant thermally conductive insert such that pressure between the first bottom surface and the heating element causes reversible deformation of the creped graphite sheet according to a first compression profile; inserting a second multiwell reaction vessel into the heating chamber of the thermal cycler by placing the second multiwell reaction vessel on the compliant thermally conductive insert; compressing the second bottom surface into the compliant thermally conductive insert such that pressure between the second bottom surface of the second multi-well reaction vessel and the heating element causes a reversible deformation of the creped graphite sheet according to a second compression profile different from the first compression profile, the compliant thermally conductive insert returning to an uncompressed state from the first compressed profile and from the second compressed profile without permanent deformation; A method comprising:

21. 21. The method of claim 20, further comprising thermally cycling the multi-well reaction vessel within the heating chamber by applying a controlled heat flux to the multi-well reaction vessel by the heating element through the compliant thermally conductive insert.

22. aligning wells of the first or second multiwell reaction vessel with an acoustic emitter positioned to apply focused acoustic radiation from the acoustic emitter to samples contained in the wells of the multiwell reaction vessel; emitting an interrogation tone burst from said acoustic emitter through a flat bottom surface of said first or second multi-well reaction vessel; detecting an acoustic echo caused by said interrogation tone burst; determining a parameter of the sample in the well from the detected acoustic echoes; The method of any one of claims 20-21, further comprising:

23. 22. The method of any one of claims 20-21, further comprising applying a heat flux from said heating element through said compliant thermally conductive insert to said first or said second multi-well reaction vessels sufficient to produce a temperature change of a sample in said first or said second multi-well reaction vessels of at least 1°C / sec.

24. aligning wells of the first or second multi-well reaction vessel with an acoustic ejector positioned to apply focused acoustic radiation from the acoustic ejector to samples contained in the wells of the multi-well reaction vessel; ejecting droplets from the wells by emitting an ejection tone burst from the acoustic ejector through a flat bottom surface of the first or second multi-well reaction vessel; The method of any one of claims 20-21, further comprising:

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