HK1236465A1 - Floating thermal contact enabled pcr - Google Patents

Description

Floating thermal contact for PCR implementation

Cross Reference to Related Applications

This application claims priority and benefit from U.S. provisional application No. 62/018,893 entitled "FLOATING THERMAL CONTACT for PCR," filed 30/6 2014, the disclosure of which is incorporated herein by reference.

Technical Field

The present invention belongs to the field of molecular biology and instrument technology.

Background

By physically moving the reaction space through separate thermal zones of a microfluidic device using channels of appropriate length and geometry, the temperature of a Polymerase Chain Reaction (PCR) device can be cycled for DNA replication in the reaction space. Typical PCR systems have fluid in a relatively large chamber and circulate the temperature around the chamber. Other systems use flexible tubing to circulate the fluid through the heated zones. One problem with performing PCR in a rigid planar microfluidic device is that it may be difficult to achieve good thermal connections, so the results may be inconsistent and unreliable. To reduce cost and manufacturing complexity, it is desirable for the thermal management system to be an external device that can be attached to the microfluidic component. The number of thermal zones on the microfluidic device, the temperature range of each thermally active region, and the proximity of each zone to each other all limit design choices that can be made with respect to the thermal management device. The following invention uses floating thermal contact to achieve PCR reactions. Such an apparatus is described: the device is used to manage the heating and cooling of multiple zones on an external part or assembly by using isolated, movable and electrically conductive zones, thereby enabling very efficient PCR reactions. Citation or identification of any document in this application is not an admission that such document is available as prior art to the present invention.

Disclosure of Invention

The present invention generally relates to a system for performing PCR in a coupled microfluidic device using floating thermal contact in a thermal management device. The thermal management device includes a plurality of thermal zones attached to a frame, where each zone is an individually actuated isolated subassembly. Actuation of each zone ensures physical contact with a non-uniform, but flat microfluidic chip to achieve efficient conductive heat transfer. The isolation of each zone serves to thermally insulate the thermal zones from each other and minimize undesirable heat transfer between adjacent zones, thereby leaving each zone at an appropriate uniform temperature.

It should be noted that in this disclosure, and particularly in the claims and/or paragraphs, terms such as "comprising," "including," and the like may have the meaning attributed to it in U.S. patent law; for example, they may mean "including," "containing," "including," or the like; and terms such as "consisting essentially of" and "consisting essentially of have the meaning ascribed to them in united states patent law, for example, they allow elements not expressly listed but exclude elements that may be found in the prior art or that affect essential or novel features of the present invention.

t as used herein, "microfluidic" refers to a device, apparatus, or system that includes at least one fluidic channel having a cross-sectional dimension of less than 1mm and a ratio of length to the largest cross-sectional dimension perpendicular to the channel of at least about 3: 1. As used herein, a "microfluidic channel" is a channel that meets these criteria.

In some embodiments, the present disclosure relates to a thermal management device for performing a PCR reaction in a microfluidic channel, wherein the device comprises: a frame; one or more thermal subassembly coupled to the frame, wherein each thermal subassembly can have a thermal control element, and wherein each thermal subassembly can be coupled to the frame by one or more thermal actuation mechanisms; and one or more thermal diffusers configured to contact the thermal control elements of the one or more thermal zone assemblies. The microfluidic device in which PCR can be performed can be mounted on or mechanically coupled to a thermal management device.

In some embodiments, the thermal actuation mechanism further comprises a thermally-insulated bearing, a fastener, and a spring, wherein the spring is configured to apply a force to drive the thermal control element toward one of the one or more thermal diffusers. In some aspects, the thermally-insulated bearing is a polymer-based bearing. In other aspects, each fastener is a shoulder screw and each thermal actuation mechanism includes at least one polymer-based bearing, wherein the bearing can travel up and down on a shaft of the shoulder screw attached to the frame and can be urged by a spring (in some configurations, upward). In further aspects, each thermal control element can be configured to have an operating temperature from about 22 ℃ to about 95 ℃. In some aspects, each of the thermal actuation mechanisms further comprises a first insulating fiber washer surrounding the shoulder screw, the washer holding the spring in place. In such an aspect, the bearing may include a flange that expands on an end of the bearing that contacts the frame. In other aspects, each thermal zone assembly has a minimum spacing of 1mm between itself and any other thermal zone assembly or frame.

In some embodiments, at least one thermal zone assembly has a resistive heating element to maintain the thermal zone at a temperature above room temperature. In further embodiments, at least one thermal zone assembly has a thermoelectric element for maintaining the thermal zone at a temperature at or below room temperature. In such an aspect, at least one thermal zone assembly maintains the thermal zone at a temperature above room temperature, and at least another thermal zone assembly maintains the thermal zone at a temperature at or below room temperature. In some embodiments, the first thermal zone and the fifth thermal zone are maintained at a temperature at or below room temperature, the second thermal zone, the fourth thermal zone, and the sixth thermal zone are maintained at a temperature above room temperature, and the third thermal zone is maintained at a temperature higher than the temperature of at least the second thermal zone. In some aspects, at least one of the thermal zone assemblies is coupled to a heat sink. In such an aspect, the heat sink may be a finned heat sink, at least one of the thermal area assemblies may be remotely connected to the heat sink via a heat pipe, and alternatively or additionally at least another one of the thermal area assemblies may be directly mounted on the heat sink.

In further embodiments, the flexible heater circuit is constructed and arranged as a thermal control element of two or more thermal zone assemblies of a thermal management device. In some aspects, the first region of the flexible heater circuit may be moved into contact with one of the thermal diffusers by one of the thermal actuation mechanisms. In other aspects, a first region of the flexible heater circuit is in contact with one of the thermal diffusers and at least a second region of the flexible heater circuit is in contact with another of the thermal diffusers. In a further aspect, the first region of the flexible heater circuit and the second region of the flexible heater circuit may flex relative to each other a distance of about 0.75mm to about 1.0 mm.

these and other embodiments are disclosed in and are encompassed by the following detailed description.

Drawings

the following detailed description, which is provided by way of example and is not intended to limit the invention solely to the specific embodiments described, may best be understood in conjunction with the accompanying drawings.

Fig. 1 is a schematic layout of thermal zones for PCR arranged in a thermal management device, labeled with exemplary temperature markers indicating each zone, according to aspects of the present disclosure.

Figure 2A is a schematic cross-sectional view of a thermal zone actuation mechanism for a thermal management device for microfluidic chip PCR, according to aspects of the present disclosure.

Figure 2B is a schematic cross-sectional view of a region of a thermal management device for microfluidic chip PCR having a plurality of thermal zone actuation mechanisms, according to aspects of the present disclosure.

Fig. 3 is a schematic design and exploded view of an exemplary thermal zone assembly with a remotely mounted heat sink, with an exploded view of the heat sink, according to aspects of the present disclosure.

Fig. 4 is a schematic design and exploded view of an exemplary thermal zone assembly with a directly mounted heat sink, with an exploded view of the heat sink, according to aspects of the present disclosure.

Fig. 5 is a schematic design and exploded view of an exemplary resistive multi-hot zone assembly, according to aspects of the present disclosure.

Figure 6 is a flow chart of an exemplary process for thermal control of PCR using a thermal management device, according to aspects of the present disclosure.

Detailed Description

the following presents a simplified summary of some embodiments of the invention in order to provide a basic understanding of the invention. This summary is not an extensive overview of the invention. And is not intended to identify key or critical elements of the invention or to delineate the scope of the invention. Its sole purpose is to present some embodiments of the invention in a simplified form as a prelude to the more detailed description that is presented later.

t to replicate DNA using the Polymerase Chain Reaction (PCR) method, the reaction volume is cycled through a series of different temperatures to start or stop different chemical reactions. Traditionally, this is achieved by using an electrothermal module to simultaneously heat and cool the reaction volume. Such thermodynamic cycling can also be achieved by moving the reaction volume through multiple zones maintained at different temperatures. One common solution is to wrap a flexible coil around the thermal zones, with the flexible coil conforming to each thermal zone. To move very small reaction volumes, one can typically use microfluidic devices with channels of appropriate length and geometry in an integrated planar microfluidic circuit. However, these devices are typically rigid, which makes it difficult to achieve good thermal contact with the thermal zones of the thermal management system. The present disclosure provides structures and methods that ensure that each thermal zone of a thermal management device is in effective contact with a microfluidic device at various operating temperatures.

the microfluidic devices contemplated herein (alternatively referred to as cartridges) may be bodies with a series of reservoirs for storing and receiving different chemical reagents that are supplied to microfluidic "chips" that are bound to the body. In some aspects, the reagent may be provided to the sample fluid as a plurality of droplets. Microfluidic chips may be relatively flat portions of devices that contain multiple micron-scale channels and other plumbing features that facilitate different chemical reactions. In exemplary embodiments of the present disclosure, the results of these reactions may be analyzed to determine the DNA sequence of one or more specific genes. To supply or remove heat for these chemical reactions, the microfluidic device may be inserted into a thermal management system, wherein individual thermal zone components of the thermal management device may be adjusted and moved into an operating position to directly contact various regions of the microfluidic device. Each individual thermal zone assembly may have one or more thermal zone actuation mechanisms to align and move a thermal element, such as a thermal spreading element, into contact with the microfluidic device at a specific location.

the geometry of a microfluidic device (including the size and shape of its thermally active region) presents a number of challenges in designing a thermal management device. It is difficult to manufacture microfluidic components or multi-zone thermal devices that have a high enough flatness to ensure proper contact and conduction between the microfluidic device and the various zones. Deviations in the flatness of microfluidic components may simply be the result of unavoidable mechanical deviations that are still within manufacturing tolerances. The thermal zones themselves are in close proximity to each other, and in some cases it is desirable that they be as close to each other as possible in order to effect a reaction in the sample being transferred between the thermal zones. For example, the temperature of each adjacent zone may have a steep temperature gradient of 40 ℃/mm or more. Generally, during operation, each thermal zone may be at a temperature of 35 ℃ or greater, and may be referred to as a heating thermal control zone. In some cases, some of the temperature zones require heating and warming to a temperature of 35 ℃ or higher, but other thermal control zones can cool to below 25 ℃ (room temperature). For example, cooling each thermal control zone may be operable to preserve perishable reagents held within the microfluidic device that may be damaged upon prolonged exposure to relatively high temperatures.

t-conduction is the most efficient form of heat transfer between a solid and a liquid, and it can only be done by direct physical contact. In order for conduction to occur in this case, there must be contact between the microfluidic device and the thermal management device. As previously indicated, ensuring contact over a large surface area is difficult, especially in multi-element assemblies that interact with plastic or glass-based microfluidic devices. Some methods of spreading heat over a large surface area have used thermally conductive fluids or thermal greases between the microfluidic device and the heat source, but such implementations are complex and require extensive maintenance. In order to adequately and accurately dissipate heat over a sufficient surface area, the thermal zones of the present disclosure are each independently actuated so that they can conform to any local or global unevenness in the microfluidic device.

to actuate each zone individually, it is useful to make each zone a separate subassembly, which allows each zone to be thermally isolated from the other zones. In one embodiment of the invention, each zone has a minimum spacing of 1 millimeter (1mm) between itself and any other zone or frame of the device to which the zones are attached. The present invention provides a plurality of bearings and shaft elements for each zone, which elements may be referred to as thermal zone actuation mechanisms. To further insulate each thermal zone from the frame, the present invention may use thermally insulated bearings (e.g., polymer-based bearings) and non-thermally conductive gaskets having much lower conduction rates than any metal equivalent. This configuration will limit the heat transfer path available for each zone to subsequent zones, thereby increasing the energy efficiency of the thermal management device.

In other words, strong thermal conduction to specific sections of the microfluidic device is maintained by direct contact to the thermal subassemblies. Relatively weak thermal gradients may still exist, at least in part due to, for example: convection in the air gap between each zone; conduction between each zone and the support frame of the thermal management device via a thermally insulating fiber gasket; and/or conduction between each zone and the support frame via polymer bearings and stainless steel shoulder screws coupling the thermal management device subassemblies together.

t various heating elements are used to meet the different temperature requirements of the different thermal zones of the device. Some of the heating elements are mounted directly or remotely to a heat sink (e.g., a finned heat sink) to increase thermal mass and facilitate convective heat transfer from the zones to the environment. Each heating element may be a resistance heater, peltier heater, or the like.

When the microfluidic device is inserted, connected and/or coupled to a thermal management device, spring loading in each thermal zone actuation mechanism urges the heating and/or cooling elements of each thermal zone assembly into direct contact with respective associated thermal zones on the microfluidic device. This ensures good contact and thus good heat conduction into the microfluidic chip.

FIG. 1 is a schematic layout of thermal zones for PCR arranged in an example thermal management device 100, labeled with example temperature markers indicating each thermal zone. Generally, it is assumed that the system is operating in a standard 25 ℃ room temperature or slightly elevated environment. Some of the thermal control zones (e.g., zones set to about 22 ℃ or about 35 ℃) may be heated and/or cooled by thermoelectric elements. Some of the thermal control zones that are elevated to relatively high temperatures (e.g., zones set to about 95 ℃) may be heated by resistive heating elements. In some embodiments, each thermal zone of the thermal management device is associated with a respective thermal control subassembly. In some embodiments, each thermal control zone of the thermal management device is independently and separately controlled.

the thermal management device 100 has a plurality of thermal control zones that can be used for heating and/or cooling. A device comprising a microfluidic chip (elements of which are shown overlying the thermal management device 100) may be inserted into the PCR device above and adjacent to the thermal management device 100, wherein the microfluidic chip may have one or more channels aligned to pass over specific thermal control regions of the thermal management device 100. Thus, the sample to be amplified and/or sequenced is moved through the regions via one or more channels (alternatively referred to as serpentine PCR) rather than a single thermal block with temperature ramping up and down.

Once mounted within the PCR device, the sample on the microfluidic chip is positioned over a first thermal zone 102 (alternatively referred to as a sample cooling zone), where the sample is in a main reservoir 103 on the microfluidic device. The first thermal zone 102 may cool or maintain the temperature of the sample in the primary reservoir 103 at a temperature of about 22 ℃ ± 4 ℃. Additional samples, reagents, buffers, oils, and/or other fluids or materials may also be stored on the microfluidic device. The sample fluid is directed outwardly from the main reservoir 103 through a first channel section 101 (alternatively referred to as an upper serpentine region) of the microfluidic chip. Additional samples, reagents, buffers, oils, and/or other fluids or materials may be added to the sample in the first channel section 101, and the reaction may occur within the first channel section 101. The sample may be in the form of droplets while passing through the microfluidic chip channel. In alternative embodiments, additional samples, reagents, buffers, oils, and/or other fluids or materials may be added to the sample within the primary reservoir 103. In further alternative embodiments, the sample may be injected into an existing droplet on the chip with associated reagents, buffers, oils, etc. In other embodiments, the sample may be subjected to PCR in large droplets (called "slugs") that may be subsequently injected into pre-existing droplets on the microfluidic chip following PCR.

the sample is directed out of the main reservoir 103 and away from the first thermal zone 102 by the first channel section 101, and after various reagents are added and the reaction proceeds (in various zones of the microfluidic chip not shown), the sample enters the second thermal zone 104 (alternatively referred to as the main expansion zone) and the third thermal zone 106 (alternatively referred to as the variable zone). In some embodiments, the sample is directed through the upper serpentine region past the denaturing region for a period of time before passing through the primary extension region. As the first channel section 101 alternately passes through the second thermal zone 104 and the third thermal zone 106, the sample circulates through a thermal gradient between the second thermal zone 104 and the third thermal zone 106, which can be set to facilitate PCR. In some embodiments, the second thermal zone 104 can be set to a temperature of about 65 ℃ ± 2 ℃. In some embodiments, the third thermal zone 106 can be set to a temperature of about 95 ℃ ± 5 ℃. In some particular embodiments, the third thermal zone 106 can be set to a temperature of about 98 ℃ ± 2 ℃. The first channel section 101 can continue to direct the sample toward the fourth thermal zone 108 (alternatively referred to as a secondary expansion zone) and alternately pass through the third thermal zone 106 and the fourth thermal zone 108 such that the sample circulates through a thermal gradient between the third thermal zone 106 and the fourth thermal zone 108, which can be set to further facilitate PCR. In some embodiments, the fourth thermal zone 108 can be set to a temperature of about 55 ℃ ± 6 ℃. In some particular embodiments, the fourth thermal zone 108 can be set to a temperature of about 51 ℃ ± 2 ℃.

After passing through the first channel section 101, the sample can be injected with additional temperature controlled reagent(s) (e.g., enzyme solution) as well as other non-temperature controlled reagents to facilitate the sequencing reaction. The temperature controlled sequencing reagent(s) may be held in a secondary reservoir 105 on the microfluidic device and may be introduced into the sample through an injection channel 107 along with other reagents. Secondary reservoir 105 may be positioned over a fifth thermal zone (alternatively referred to as an enzyme cooling zone) that may cool or maintain sequencing reagents (e.g., enzyme solution) in secondary reservoir 105 at a temperature of about 22 ℃ ± 4 ℃. Once injected with sequencing reagents, the sample continues through the second channel section 109 (alternatively referred to as the lower serpentine region) of the microfluidic chip.

the second channel section 109 of the tficrofluidic chip may pass through a sixth thermal zone 112 (alternatively referred to as a sequencing incubation zone), which may be set to a temperature of about 34 ℃ ± 2 ℃. In some particular embodiments, the sixth thermal zone 112 can be set to a temperature of about 35 ℃ ± 1 ℃. While the sample passes through the second channel section 109 at a temperature at or near the temperature of the sixth thermal zone 112, the sample may undergo a sequencing reaction. The second channel segment 109 can be returned from the sixth thermal zone 112 to the second thermal zone 104 such that the sequencing reaction can be completed at about the temperature of the second thermal zone 104. The amplified and sequenced sample can then travel through the second channel section 109 to the optical detection region 111 where the sample can be observed, measured, and/or characterized. After passing through the optical detection region 111, the sample may be directed to a waste reservoir 113. Either or both of the optical detection region 111 and the waste reservoir 113 may be located on the microfluidic chip. In some embodiments, either or both of the optical detection region 111 and the waste reservoir 113 may be located on the microfluidic device, but not on the microfluidic chip. In alternative embodiments, the optical detection region 111 and the waste reservoir 113 may be located in the PCR device, away from the microfluidic device. In some embodiments, the optical detection region 111 is located on a microfluidic chip and the waste reservoir 113 is located on a microfluidic device, but not on the microfluidic chip.

Each of the various thermal control regions of the thermal control device 100 can be a separate subassembly having thermal control elements, a support frame, guide screws, springs, and thermal diffuser elements configured to contact particular regions of the microfluidic device when the microfluidic device is mounted within the PCR device. Each subassembly may also be coupled to a heat sink and/or a thermal insulator. In various configurations, each of the first thermal zone 102, the second thermal zone 104, the third thermal zone 106, the fourth thermal zone 108, the fifth thermal zone 110, and the sixth thermal zone 112 may be directly coupled to an insulating material. In further configurations, each of the first thermal zone 102, the second thermal zone 104, the third thermal zone 106, the fourth thermal zone 108, the fifth thermal zone 110, and the sixth thermal zone 112 may be directly coupled to a heat sink. In a further alternative configuration, each of the first thermal zone 102, the second thermal zone 104, the third thermal zone 106, the fourth thermal zone 108, the fifth thermal zone 110, and the sixth thermal zone 112 may be indirectly coupled to a heat sink via a thermally conductive element, such as a heat pipe. For each thermal zone disclosed herein that is indirectly coupled to a heat sink, the coupling may be by one heat pipe, two heat pipes, or three or more heat pipes. In some embodiments of the present disclosure, the third thermal zone 106 may be coupled to a thermally insulating material, the first, second, and fourth thermal zones 102, 104, 108 may be directly coupled to a respective heat sink, and the fifth and sixth thermal zones 110, 112 may be indirectly coupled to a respective heat sink.

In some embodiments, the microfluidic chip may further comprise an intermediate viewing region. In some aspects, the main measurement region 115 can be positioned along the first channel segment 101 before the sample actually enters the upper serpentine portion of the channel. The primary observation region 115 may be configured to measure and observe the bulk fluid when reagents for PCR are added to the sample. Similarly, in various other aspects, the secondary observation region 117 can be positioned proximate to the interface of the first channel section 101, the injection channel 107, and the second channel section 109 before the sample actually enters the lower serpentine portion of the channel. Secondary observation region 117 may be configured to measure and observe the bulk fluid when reagents for sequencing are added to the sample. The optical detection region 111 remains at the post-sequencing position to measure and observe the sequenced sample.

Figure 2A is a schematic cross-sectional view of a thermal zone actuation mechanism 200 for a thermal management device for microfluidic chip PCR. In some embodiments, each thermal control zone has three to five (3-5) thermal zone actuation mechanisms 200 arranged as part of the respective subassemblies, spaced apart to balance the total spring force at or near the center of each thermal zone. The frame 201 is a fixed element that provides support for both the microfluidic device and the mounting structure for each thermal zone actuation mechanism 200. For each thermal zone actuation mechanism 200, shoulder screws 202 are attached to the entire thermal frame 201 via threaded holes. A thermal module substrate 203 (a thermal module substrate 203 insulated as shown herein) can extend across at least a portion of the area of the thermal force zone, with a hole in the thermal module substrate 203 into which a polymer bearing 204 is inserted. As shown herein, the thermic module substrate 203 can be a single-part component, but in further embodiments, a larger or multi-part thermic module can be positioned where the thermic module substrate 203 is implemented in fig. 2A and 2B. A hole in the polymer bearing 204 allows the shoulder screw 202 to pass through. In some aspects, the thermal module substrate 203 can be mechanically and/or thermally coupled to a heat source or heating element, such that the thermal module substrate 203 can control the conduction of heat to a heat spreading element coupled to the thermal zone actuation mechanism 200. The bearings 204 allow the thermal module substrate 203 to travel up and down the axis of the shoulder screw 202 in the thermal subassembly. In each thermal zone actuation mechanism 200, either or both of the bearing 204 and thermal module substrate 203 are operatively coupled to a spring 205 and urged upward by the spring 205. Accordingly, since the position of the thermal module substrate 203 along the length of the shoulder screw 202 is variable, wherein the spring force from the one or more springs 205 in the thermal force zone pushes upward on the thermal module substrate 203, and wherein any weight or load (e.g., microfluidic chip) exerts a downward force on the thermal module substrate 203 via the frame 201, the thermal insulator 203 and the bearing 204 may be said to be "floating" relative to the frame 201 because they provide contact to the various loads placed on the frame 201.

In some embodiments, a first insulating fiber washer 206 and a second insulating fiber washer 206' may be positioned around the shoulder screw 202. In particular, a second insulating fiber gasket 206' may be positioned between the thermic module substrate 203 and the frame 201 to thermally insulate the zones from the frame 201 and shoulder screws 202. Similarly, the first insulating fiber washer 206 may provide a larger physical diameter to hold the spring 205 in place to distribute the force from compression and insulate the shoulder screw 202 from the spring 205 because the spring 205 is in direct contact with the thermal module substrate 203. When the microfluidic device is clamped to the entire thermal system, the microfluidic chip is secured to the device frame 201 such that the thermal module substrate 203 is pushed down, compressing the springs 205 in all thermal zones. The spring 205 thus applies upward pressure to the various regions of the microfluidic device. In an alternative embodiment, the bearing 204 may be shaped to have a flange in contact with the frame 201 that is large enough to eliminate the need for a second insulating fiber gasket 206'.

the t-spring 205 solves the problem of imperfect contact that might otherwise result from variations in the construction of the microfluidic chip. The spring 205 applies a force to the bearing 204 to place the thermal module, shown here in simplified form as a single-part thermal module substrate 203 (e.g., a heating element), in thermal contact with the microfluidic chip. Each thermal module may have multiple mechanisms for each zone so that the spring force may be applied evenly across any load in a given zone. In some embodiments, a thermic module may be configured with two, three, four, five, six, or more mechanisms to apply a spring force, as disclosed herein. In some aspects, the compression displacement of any spring 205 may be 0.1mm, 0.2mm, 0.5mm, 1.0mm, 1.5mm, 2.0mm, or any increment or gradient of length within this range. In further aspects, the compression displacement of any spring 205 may be less than 0.1mm or greater than 2.0 mm.

In alternative embodiments of the thermal zone actuation mechanism, structures other than the illustrated coil spring 205 may be used, either independently or in combination, to provide a force to ensure contact between the thermal zone assembly and the microfluidic device. In some aspects, such alternatives may include: an extension spring triggered by a binary or "flip" mechanism; elastomeric parts, such as parts made of rubber; a flexure mechanism; a motor; a pneumatic actuator; a gas spring; a belleville spring; a wave-shaped spring; a disc spring; a torsion spring; balancing weight; a magnet; a memory alloy; or gravity in embodiments where the heat source and heat spreader are positioned above the microfluidic device.

t in many embodiments of the thermal zone actuation mechanism, the bearing 204 may be a polymer bearing. The bearing 204 may alternatively be configured as: a metal bushing, optionally lubricated with a coated oil or polymer; a flexure mechanism; or a ball bearing housing. In a further alternative embodiment, the shoulder screw 202 or a bracket coupled to the shoulder screw 202 may be a plastic component impregnated with plastic or oil, wherein the bracket has a flat bore that acts as a bushing, and this configuration does not include a separate bearing.

Figure 2B is a schematic cross-sectional view of two thermal control zones of a thermal management device for microfluidic chip PCR with multiple thermal zone actuation mechanisms located on each thermal control zone. As shown in fig. 2B, the bearing 204 surrounding the shoulder screw 202 has a flange in contact with the frame 201 that facilitates contact and distribution of forces that may be applied from the frame 201 or from a spring 205 coupled to the bearing 204. Also shown is a thermal diffuser 207 (alternatively referred to as a heat diffuser) over which the microfluidic plate may reside during PCR operations. Below (to the right of) one thermal diffuser 207 is an insulating thermal module substrate 203 that provides temperature control by inhibiting heat transfer away from the thermal diffuser. Below the other thermal diffuser 207 (left side) is a heat sink thermal module base plate 208. Each thermal diffuser 207 is thermally coupled to a heating element 209, wherein the heating element 209 may be a flexible resistive heating element, and may also have two, three or more heating power zones that may be set to different temperatures or the same temperature. In other embodiments, the heating element 209 may also be a plurality of elements.

the heating element 209 may be a single flexible body containing multiple independently controlled heating force zones. Accordingly, the heating element 209 may raise the temperature of the one or more thermal diffusers 207 to a particular temperature or temperature range, and various thermal zone actuation mechanisms may provide an upward force to ensure that each thermal diffuser 207 is in direct contact with a corresponding area on the microfluidic chip, where the contact is substantially uniform and has equal force across the entire area of the thermal diffuser 207. In addition, the frame 201 may facilitate positional control between different thermal diffusers 207 or other thermal control zone components. The thermal diffuser 207 may also include channels to allow wires connected to thermistors to be coupled to the subassembly, where the thermistors may be on either or both of the top and bottom sides of the thermal diffuser 207. In some embodiments, the thermal diffuser 207 may be made of copper, other conductive metals or alloys, or a polymer gap pad material. In further embodiments, the thermal diffuser 207 may be sufficiently flexible such that each thermal zone does not require a separate thermal diffuser 207, rather a single thermal diffuser 207 may extend across two or more thermal zones and deflect accordingly when actuated into a position of contact with the microfluidic device.

t in further alternative embodiments of the thermal zone actuation mechanism for a thermal management device, the orientation of the shoulder screw 202 and the bearing 204 may be interchanged. In such a configuration, the shoulder screw 202 may be referred to as a shaft and the bearing 204 may be referred to as a bushing, where the bushing is mechanically coupled to the frame 201 and the shaft may be mechanically coupled to other support structures such as a bracket.

the thermal control zone in devices requiring high temperatures is often heated using resistive heating, thermoelectric elements (peltier), infrared, heated fluids, or other heating methods. For example, a thermal control zone (e.g., a variable zone) elevated to 95 ℃ may use a resistive heating element mounted on a bracket made of a rigid insulating material such as Garolite reinforced phenolic resin to reduce undesirable heat transfer to the environment. In some embodiments, one or two other thermal control zones raised to a temperature of about 65 ℃ (e.g., the primary hybridization zone) and about 55 ℃ (e.g., the secondary hybridization zone) but located in close proximity to (with a small gap of, for example, 1mm between each zone) the thermal control zone set at 95 ℃ can be mounted on a finned heat sink to increase its thermal mass and heat transfer rate to the environment. These finned heat sinks coupled to thermal control zones set at 65 ℃ and 55 ℃ can help dissipate heat transferred from adjacent 95 ℃ zones, and also allow for easier control of the temperature of each zone, as the heat sinks increase the cooling rate. In other words, having the heat sink on a relatively higher temperature zone makes it faster to cool the heat sink when a lower temperature is requested by a user or a process controller, and also makes it more difficult for the heater controller to exceed the desired temperature.

t thermal zones with intermediate temperatures may need to have different functions. For example, a thermal control zone set at about 35 ℃ may be used to heat its own thermal control zone using a resistance or thermoelectric element. Alternatively, a thermal control zone set at about 35 ℃ may be used to act as a heat sink to remove/transfer heat to an adjacent, relatively hotter thermal control zone. Another example is where two thermal control zones are configured close to the reagent reservoir, where the reagent needs to be kept at room temperature or below (e.g., at 22 ℃). Such thermal control zones are cooled by individual thermoelectric elements or other cooling methods.

Alternative heating or cooling elements that can be used in the disclosed thermal control zones include, but are not limited to: a silicone resistance heater; a ceramic resistance heater; a stirling engine; liquid nitrogen, dry ice or other phase change material; a liquid cooling or liquid heating circuit; and infrared illumination such as "heat lamps". Further applications for cooling the hot zone include: a fan or other air source blowing directly on the surface of the thermal control element; cooling the liquid; using a chassis connected to the thermal control element as a cooling sink; and/or piping having a cooling gas in contact with the heated region to provide a heat exchange conduit (e.g., as in a refrigerator).

Fig. 3 is a design schematic of an exemplary thermal zone assembly 300 with a remotely mounted heat sink 302. In some embodiments, the thermal zone assembly 300 may be one of the thermal control zone assemblies that is maintained at a lower temperature relative to one or more of the adjacent thermal control zone assemblies. When in contact with the microfluidic device, thermoelectric module 301 (as part of thermal region assembly 300) may conduct heat into a region of the microfluidic device. Thermal zone assembly 300 transfers excess heat or waste heat from thermoelectric module 301 to remote heat sink 302. In various aspects, this configuration and structure may be used in situations where there is otherwise insufficient space to directly attach a heat sink to the thermoelectric module 301. A thermoelectric module 301 and a remote heat sink 302 may also be attached to the bracket 303 (shown as thermal module body 203 in fig. 2A). With this bracket 303, the plurality of heat pipes 305 may be used to conduct waste heat away from the thermoelectric module 301 through a relatively small space and at a higher rate than can be achieved by conduction through a solid material alone. In areas where there is not enough clearance for heat sinks 302 of sufficient size and heat capacity, heat sinks 302 may be attached to the ends of these heat pipes 305 opposite thermoelectric modules 301.

In some aspects, the thermal zone assembly 300 may also include a heat spreader 306 arranged such that the microfluidic device does not directly contact the thermoelectric module 301, but is arranged such that the thermoelectric module 301 (or other thermal control element) may be spread over a larger area than that occupied by the thermoelectric module 301 alone. In other words, heat from the thermal control elements of the thermal zone assembly 300 is conducted to a surface, which in this case has a larger surface area than the surface of the elements themselves, and can heat corresponding regions of the microfluidic device. Also shown in this figure is a polymeric bushing 304 that is part of the actuation mechanism.

t the thermal interface material 307 may be in contact with both the heat sink 302 and the heat pipe 305, and positioned to ensure thermal conduction between the heat sink 302 and the heat pipe 305. In addition, a top side thermistor 308 and a bottom side thermistor 309 can be positioned above and below the thermoelectric module 301, respectively, to monitor the temperature of the thermoelectric module 301 and the corresponding thermal subassembly. One or more of such thermistors may be in communication with a control system or controller to adjust the temperature of thermoelectric module 301 or stop its operation. In some applications, thermal grease may be used on the thermoelectric module 301 to ensure conduction on one or both sides of the thermoelectric module 301.

FIG. 4 is a design schematic of an exemplary thermal zone assembly with a directly mounted heat sink. In some embodiments, the thermal zone assembly 400 may be one of the thermal control zone assemblies that maintains a lower temperature relative to one or more of the adjacent thermal control zone assemblies. When in contact with the microfluidic device, the thermoelectric module 401 (as part of the thermal electronics module 400) may conduct heat into a region of the microfluidic device. The thermal zone assembly 400 transfers excess heat or waste heat from the thermoelectric module 401 to the directly mounted heat sink 402. In various aspects, this configuration and structure may be used with sufficient space to attach the heat sink 402 directly to the thermoelectric module 401.

In some aspects, the thermal area assembly 400 may also include a heat spreader 406 arranged such that the microfluidic device does not directly contact the thermoelectric module 401, but is arranged such that the thermoelectric module 401 (or other thermal control element) may be spread over a smaller area than that occupied by the thermoelectric module 401 alone. In other words, heat transfer from the thermal control elements of the thermal zone assembly 400 is conducted to a surface, which in this case has a smaller surface area than the surface of the elements themselves, and can transfer heat to corresponding regions of the microfluidic device. Also shown in this figure is a polymer bushing 404 that is part of the actuation mechanism.

Further, a top side thermistor 403 and a bottom side thermistor 405 can be positioned above and below the thermoelectric module 401, respectively, to monitor the temperature of the thermoelectric module 401 and corresponding thermal subassembly. One or more of such thermistors may be in communication with a control system or controller to adjust or stop operation of thermoelectric module 401. In some applications, thermal grease may be used on the thermoelectric module 401 to ensure conduction on one or both sides of the thermoelectric module 401.

t fig. 5 is a schematic design diagram of an exemplary resistive multi-hot zone device. The flexible heater circuit 501 (shown in fig. 2B as heating element 209) is positioned between a set of thermal diffusion elements and a corresponding set of thermal actuation mechanisms in a respective thermal zone assembly. As shown, the flexible heater circuit 501 has three connected but structurally distinct zones, each of which may be set to the same or different temperatures. In other embodiments, the flexible heater circuit 501 may be configured to have two connected but structurally distinct zones, and in other embodiments, the flexible heater circuit 501 may be configured to have four or more connected but structurally distinct zones. In alternative embodiments, the flexible heater circuit 501 may be configured to have a single zone. Disposed below the flexible heater circuit 501 are a first heat sink 502 and a second heat sink 503 (shown in fig. 2B as heat sink thermal module substrate 208), and an insulated thermal module body 505 (shown in fig. 2A and 2B as insulated thermal module body 203), where the first heat sink 502 can be directly mounted to the flexible heater circuit 501 and the second heat sink 503 can be remotely coupled to the flexible heater circuit 501 such that each heat sink and insulated thermal module body correspond to a different thermal force zone. The polymer bearings 504 indicate the locations where force can be applied to move the thermal zone actuation mechanisms of the individual thermal zone modules in order to place the first heat spreader element 509, the second heat spreader element 510, and the third heat spreader element 511 in contact with the microfluidic device. Each region of the flexible heater circuit 501 is connected by a relatively small bump of material, allowing each region to move independently when driven by a corresponding thermal zone actuation mechanism. In some aspects, each zone of flexible heater circuit 501 may move or flex a distance of up to about 0.75mm to 1.0mm relative to other zones of flexible heater circuit 501.

t there are positioned between the first 509, second 510 and third 511 heat spreader elements and the flexible heater circuit 501 a first 506, second 507 and third 508 thermistor each located in a different region of the flexible heater circuit 501. These thermistors may monitor the temperature of the flexible heater circuit 501 and the corresponding thermal sub-assembly connected to the flexible heater circuit 501. One or more of such thermistors may be in communication with a control system or controller to adjust the temperature of or stop operation of one or more of the zones of the flexible heater circuit 501. Above the flexible heater circuit 501 are arranged a first heat spreader 509, a second heat spreader 510 and a third heat spreader 511. When the corresponding thermal zone actuation mechanisms are engaged, the corresponding one or more regions of the flexible heater circuit 501 may be moved (via the polymer bearings 504) into direct contact with one or more of the first heat spreader 509, the second heat spreader 510, and the third heat spreader 511. Because the components are rigidly mounted together, each thermal zone assembly moves in unison when actuated, despite the flexible heater circuit 501 spreading around as part of more than one thermal zone assembly (e.g., as shown in FIG. 5, there are three separate thermal zone assemblies identified by the arrangement of elements 509-506-501-505, 510-507-501-503, and 511-508-501-505). The microfluidic device in contact with one or more of the first heat spreader 509, the second heat spreader 510, and the third heat spreader 511 may thereby be heated (or in some aspects cooled) to a temperature determined by the corresponding region of the flexible heater circuit 501.

In some exemplary aspects, the flexible heater circuit 501 may be controlled to set each zone to a different temperature, wherein the thermal zones may be characterized relative to each other as "low temperature", "medium temperature", and "high temperature". For example, the flexible heater circuit 501 may be configured such that: the first heat spreader 509 acts as a low temperature heat spreader, the second heat spreader 510 acts as a medium temperature heat spreader, and the third heat spreader 511 acts as a high temperature heat spreader. Regions of the microfluidic device that rest on or are otherwise coupled to the heat spreader element may thus be raised (or in some aspects lowered) in temperature to reach the temperature of adjacent regions of the flexible heater circuit 501.

In further embodiments, the entire thermal management device or portions thereof may be covered by a thin thermally conductive film to help seal any or all of the physical gaps in the assembly. Such a seal may prevent liquid from leaking into or out of the assembly.

t as indicated above, many of the heating elements are mounted directly to the finned heat sink to increase thermal mass and facilitate convective heat transfer from the zones to the environment. In some configurations, such zones are constrained within the physical space by separate components, and there is no physical space for direct mounting of the heat sink. These zones transfer heat to a remote heat sink, an example of which is shown in fig. 3, using heat pipes.

t as also noted above, by forming separate modules for each zone, the most efficient path for each thermal zone in a physically isolated and thermally insulated thermal management device to ensure thermal conduction between each zone is the attached microfluidic device itself. The microfluidic device may be made of a polymer that acts as a weak heat conductor, ensuring that there is only a small amount of heat transfer between the zones. In this way, the temperature of each zone can be kept very uniform and constrained to each designated thermal zone.

Figure 6 is a flow chart of an exemplary process for thermal control of a thermal management device for PCR. Generally, the controller 600 is operatively coupled to the thermal management device, wherein the controller 600 is configured to relay instructions to and receive sensor data (e.g., temperature) from the thermal management device and its components. In some aspects, the controller 600 may include a user interface to allow an operator to directly or manually control the operation of the thermal management device. The controller 600 may be a non-transitory computer readable medium that is further configured to store and execute programmed operational instructions for the components of the thermal management device. In some embodiments, the controller 600 may be electronically coupled to: heating and/or cooling elements in each thermal control zone, thermal zone actuation mechanisms, and sensors (e.g., thermistors or optical detectors) as part of a thermal management device. In other embodiments, the controller 600 may also be operatively and electronically coupled to the microfluidic device such that the controller 600 may, in part, control the introduction and flow of reagents and/or samples into the channels of the microfluidic device when the microfluidic device is coupled to and in close proximity to the thermal management device.

In an exemplary process, in step 602, a microfluidic cartridge (alternatively referred to as a chip or plate) is loaded into a thermal management system and mechanically positioned or coupled to an adjacent thermal management system. The microfluidic chip may hold the sample in one or more reservoirs, or the sample may be added to the microfluidic chip immediately upon loading into a thermal control system. In step 604, the various thermal control zones of the thermal management system may be raised or lowered to manually set or preset temperatures. In step 606, the sample is moved from the reservoir into the reaction channel region of the microfluidic chip. In step 608, one or more reagents may be added to the sample at the same reaction region or reaction channel of the microfluidic chip, where such reagents may be PCR reagents for amplification of the sample. In step 610, the sample and any added reagents are directed through a reaction channel region in a microfluidic chip. The process of step 608 and step 610 may be performed simultaneously as additional sample and reagents are added to the microfluidic channel. In step 606, the addition and reaction of reagents (e.g., amplification reagents of a PCR) with the sample may be observed and/or measured by a first sensor, such as an optical detector. Further, during step 606, as the sample fluid passes proximate to the thermal control zone of the thermal management system, the temperature of the sample and reagent is controlled, which in some aspects may be controlled such that the sample and reagent fluids may undergo an amplification reaction.

t in step 612, the (amplified) sample is moved further from the reaction channel region and towards the sequencing channel region of the microfluidic chip. In step 614, one or more reagents may be added to the sample at the same sequencing channel region of the microfluidic chip, where such reagents may be enzymes used for sequencing of the sample. In step 616, the sample and any added reagents are directed through a sequencing channel region in the microfluidic chip. The process of steps 614 and 616 may be performed simultaneously as additional sample and reagents are added to the microfluidic channel. In step 612, the addition and reaction of reagents (e.g., sequencing reagents such as enzymes) with the sample may be observed and/or measured by a second sensor such as an optical detector. Further, during step 612, as the sample fluid passes proximate to the thermal control zone of the thermal management system, the temperature of the sample and reagent is controlled, which in some aspects may be controlled such that the sample and reagent fluids may undergo a sequencing reaction.

After the sample fluid has left each thermal control zone (or in other words, once the amplified and sequenced sample fluid has entered the region of the microfluidic channel(s) not immediately adjacent to the thermal control zone) in step 618, the sample can be observed and/or measured by a sensor such as an optical detector, alternatively referred to as a final sensor or sequencing sensor. In step 620, after the observation/measurement, the sample may be directed to a waste reservoir and the microfluidic cartridge may be removed from the thermal control system.

In an alternative embodiment of the present disclosure, a thermal management device that allows for PCR reactions in microfluidic channels may comprise: a frame having a channel and one or more isolated, movable, and electrically conductive thermal subassembly attached to the frame via thermally insulated bearings and fasteners, wherein each thermal subassembly can be heated or cooled to a temperature suitable for a PCR reaction. In some aspects, the fastener may be a shoulder screw. In other aspects, the thermally insulated bearing may be a polymer-based bearing. In further aspects, each of the thermal zone assemblies may include at least one polymer-based bearing that travels up and down on a shaft attached to at least one of the shoulder screws of the frame and may be urged upward by one or more springs. In some such aspects, each of the thermal zone assemblies may include a polymer-based bearing that may be attached to the frame via a shoulder screw and may be urged upward by a spring. In further embodiments, each of the thermal zone assemblies may further comprise a first insulating fiber gasket surrounding the shoulder screw and between the thermal zone assembly and the frame, and having a second insulating fiber gasket holding the spring in place, wherein each of the thermal zone assemblies may be pushed downward to compress the spring to apply upward pressure to contact the microfluidic device when the microfluidic device is clamped to the thermal management device. In some aspects, each individual thermal zone assembly may have a minimum spacing of 1 millimeter (1mm) between itself and any other thermal zone assembly or frame. In other aspects, at least one thermal zone assembly can have a resistive heating element. In other aspects in which the at least one thermal zone assembly has a resistive heating element, the heating element can be used to maintain the thermal zone at a temperature above room temperature. In other aspects, at least one thermal zone assembly can have a thermoelectric element. In a further aspect, at least one thermal zone assembly having thermoelectric elements can be used to maintain the thermal zone at a temperature at or below room temperature. In some aspects, at least one of the thermal zone assemblies can be mounted on a heat sink, wherein in various aspects, the heat sink can be a finned heat sink. In other aspects, at least one of the thermal zone assemblies may be connected to a remote heat sink via at least one heat pipe. In other aspects, at least one of the thermal area assemblies can have a thermoelectric element and include a heat spreader surrounding the thermoelectric element to spread heat from the element to a surface of the heat spreader.

In an alternative embodiment of the present disclosure, a method of performing a PCR reaction in a microfluidic device is disclosed, the method comprising the steps of: providing a thermal management device as described herein, the device comprising a plurality of droplets comprising reagents necessary for a desired PCR reaction; and performing PCR in the device.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims (20)

1. A thermal management device to enable PCR reactions in a microfluidic channel, the device comprising:

a frame;

one or more thermal area assemblies coupled to the frame, each thermal area assembly having a thermal control element, and wherein each thermal area assembly is coupled to the frame by one or more thermal actuation mechanisms; and

one or more thermal diffusers configured to contact each of the thermal control elements of the one or more thermal zone assemblies.

2. The thermal management device of claim 1, wherein the thermal actuation mechanism further comprises a thermally-insulated bearing, a fastener, and a spring, wherein the spring is configured to apply a force to drive the thermal control element toward one of the one or more thermal spreaders.

3. The thermal management device of claim 2, wherein the thermally insulating bearing is a polymer-based bearing.

4. The thermal management device of claim 2, wherein each fastener is a shoulder screw, and wherein each thermal actuation mechanism comprises at least one polymer-based bearing that travels up and down a shaft of at least one shoulder screw attached to the frame and is urged by the spring.

5. The thermal management device of claim 4, wherein each thermal control element is configurable to have an operating temperature from about 22 ℃ to about 95 ℃.

6. The thermal management device of claim 2, wherein each of said thermal actuation mechanisms further comprises a first insulating fiber washer surrounding said shoulder screw, said first insulating fiber washer holding said spring in place.

7. A thermal management device according to claim 1, wherein each thermal zone assembly has a minimum spacing of 1mm between itself and any other thermal zone assembly or the frame.

8. The thermal management device of claim 1, wherein at least one thermal zone assembly has a resistive heating element for maintaining the thermal zone at a temperature above room temperature.

9. The thermal management device of claim 1, wherein at least one thermal zone assembly has a thermoelectric element for maintaining a thermal zone at a temperature at or below room temperature.

10. The thermal management device of claim 1, wherein at least one thermal zone assembly maintains the thermal zone at a temperature above room temperature, and wherein at least another thermal zone assembly maintains the thermal zone at a temperature at or below room temperature.

11. A thermal management device according to claim 10, wherein the first thermal zone and the fifth thermal zone are maintained at a temperature at or below room temperature, wherein the second thermal zone, the fourth thermal zone and the sixth thermal zone are maintained at a temperature above room temperature, and wherein the third thermal zone is maintained at a temperature higher than at least the temperature of the second thermal zone.

12. The thermal management device of claim 1, wherein at least one of the thermal zone assemblies is coupled to a heat sink.

13. The thermal management device of claim 12, wherein the heat sink is a finned heat sink.

14. The thermal management device of claim 12, wherein at least one of the thermal zone assemblies is remotely connected to a heat sink via a heat pipe.

15. The thermal management device of claim 12, wherein at least one of the thermal zone assemblies is mounted directly on a heat sink.

16. The thermal management device of claim 1 wherein a flexible heater circuit is constructed and arranged to the thermal control elements of two or more thermal zone assemblies of the thermal management device.

17. The thermal management device of claim 16, wherein the first region of the flexible heater circuit is movable by one of the thermal actuation mechanisms into contact with one of the thermal diffusers.

18. A thermal management device according to claim 17, wherein the first region of the flexible heater circuit is in contact with one of the thermal diffusers and at least a second region of the flexible heater circuit is in contact with another of the thermal diffusers.

19. The thermal management device of claim 18 wherein said first region of said flexible heater circuit and said second region of said flexible heater circuit are deflectable relative to each other a distance of about 0.75mm to about 1.0 mm.

20. A method for performing a PCR reaction in a microfluidic device, the method comprising the steps of:

(a) providing a thermal management device according to claim 1 comprising a plurality of droplets comprising reagents necessary for a desired PCR reaction, and

(b) performing PCR in the microfluidic device.