WO2021183513A1

WO2021183513A1 - Microfluidic temperature control systems

Info

Publication number: WO2021183513A1
Application number: PCT/US2021/021503
Authority: WO
Inventors: Liangliang Qiang
Original assignee: Esbiolab Llc
Priority date: 2020-03-10
Filing date: 2021-03-09
Publication date: 2021-09-16
Also published as: CN114829016A

Abstract

A microfluidic system includes a microfluidic chamber defined in a substrate of the microfluidic system; multiple microfluidic channels, each microfluidic channel fluidically connected to the microfluidic chamber; and multiple temperature control devices. Each temperature control device is disposed in contact with a wall of a corresponding one of the microfluidic channels and configured to heat or cool a fluid in the microfluidic channel. A method for operating a microfluidic system includes heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature by controlling operation of corresponding temperature control devices; controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber; and causing a biological or chemical reaction to occur in the microfluidic chamber or in a reaction chamber adjacent to the microfluidic chamber.

Description

MICROFLUIDIC TEMPERATURE CONTROL SYSTEMS

Claim of Priority

[001] This application claims priority to U.S. Provisional Application Serial No. 62/987,467, filed on March 10, 2020, the contents of which are incorporated here by reference in their entirety.

Background

[002] Temperature control, such as heating and cooling, is a relevant parameter in microfluidic chip design and fabrication. Temperature control is important for various biological and chemical reactions, including enzymatic reactions such as polymerase chain reaction (PCR), recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), quantitative PCR (qPCR), Droplet Digital PCR (ddPCR), deoxyribonucleic acid (DNA) ligation, ribonucleic acid (RNA) reverse transcription, and other reactions. Chemical reactions, e.g. dye labelling, tag cleavage, and disulfide bond reduction, are often conducted at a controlled temperature. Liquid reagents in a microfluidic reaction environment are heated and cooled by direct heating and cooling of the microchip or microcell, for instance, at a target heating or cooling rate using Peltier (thermoelectric cooling) or resistive heaters.

Summary

[003] We describe here microfluidic systems that enable efficient, precise control of the temperature of the fluids in the system. These systems include microfluidic channels fluidically connected to a microfluidic chamber. The fluid in each channel is heated or cooled independently to a target temperature, and the fluid at the target temperature is flows into the microfluidic chamber. In some examples, biological or chemical reactions, such as temperature- sensitive reactions, occur in the microfluidic chamber. In some examples, the microfluidic chamber containing the fluid at the target temperature serves as a temperature regulator for an adjacent reaction chamber in which biological or chemical reactions, such as temperature- sensitive reactions, occur. For instance, these microfluidic systems can be used for polymerase chain reaction (PCR) testing. Because the volume of fluid to be heated or cooled is small relative to the volume of the microfluidic chamber, the heating or cooling is rapid and energy efficient. The systems are compact, portable, and can be operated using battery power, e.g., power from a rechargeable battery. Applications of these compact, portable systems can include on-site or at- home testing for infectious diseases, such as SARS-CoV-2, influenza, HIV, or other infectious diseases.

[004] In an aspect, a microfluidic system includes a microfluidic chamber defined in a substrate of the microfluidic system; multiple microfluidic channels, in which each microfluidic channel is fluidically connected to the microfluidic chamber; and multiple temperature control devices. Each temperature control device is disposed in contact with a wall of a corresponding one of the microfluidic channels and configured to heat or cool a fluid in the microfluidic channel.

[005] Embodiments can include one or any combination of two or more of the following features.

[006] The microfluidic system includes multiple pumps, each pump configured to pump fluid from a corresponding one of the microfluidic channels into the microfluidic chamber.

[007] The microfluidic system includes multiple valves, each valve disposed along a corresponding one of the microfluidic channels.

[008] The microfluidic system includes a temperature control system configured to control operation of the multiple temperature control devices. The temperature control system is configured to implement closed loop feedback temperature control of the multiple temperature control devices. The temperature control system includes multiple temperature sensors each disposed in contact with a corresponding one of the temperature control devices. The temperature control system includes one or more microcontrollers or microprocessors configured to control operation of each of the temperature control devices based on a signal from the corresponding temperature sensor. The temperature control system is configured to control operation of each temperature control device independently from operation of each other temperature control device. The temperature control system is configured to be powered by a rechargeable battery.

[009] At least one of the temperature control devices includes: an active heating or cooling element; and a thermal sink disposed between the active heating or cooling element and the wall of the respective microchannel.

[010] At least one of the temperature control devices includes a one or more of resistive heater, a radiative heater, or a thermoelectric heating or cooling device. [Oil] A top wall of the microfluidic chamber, a bottom wall of the microfluidic chamber, or both are formed of an optically transparent material.

[012] The microfluidic system includes a secondary channel disposed adjacent to a first one of the microfluidic channels, in which the temperature control device that is disposed in contact with the wall of the first microfluidic channel is configured to heat or cool a material in the secondary channel. The secondary channel contains a material having a melting point at a target temperature. The material includes a combination of fatty acids.

[013] The microfluidic system includes a reaction chamber defined in the substrate of the microfluidic system, in which the reaction chamber is fluidically isolated from the microfluidic chamber. The reaction chamber and the microfluidic chamber share a common wall. The reaction chamber includes an elongated channel. The reaction chamber includes a branched channel. The microfluidic chamber overlaps with at least a portion of the reaction chamber.

[014] In an aspect, a polymerase chain reaction (PCR) test system includes a microfluidic system includes a microfluidic chamber defined in a substrate of the microfluidic system; multiple microfluidic channels, in which each microfluidic channel is fluidically connected to the microfluidic chamber; and multiple temperature control devices. Each temperature control device is disposed in contact with a wall of a corresponding one of the microfluidic channels and configured to heat or cool a fluid in the microfluidic channel.

[015] Embodiments of the PCR test system can include one or any combination of two or more of the foregoing features.

[016] In an aspect, a method for operating a microfluidic system includes heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature by controlling operation of corresponding temperature control devices. Each temperature control device is disposed in contact with a wall of the corresponding microfluidic channel, and each microfluidic channel is fluidically connected to a microfluidic chamber defined in a substrate of the microfluidic system. The method includes controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber; and causing a biological or chemical reaction to occur in the microfluidic chamber or in a reaction chamber adjacent to the microfluidic chamber.

[017] Embodiments can include one or any combination of two or more of the following features. [018] Controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber includes controlling operation of multiple pumps, each pump configured to pump fluid from a corresponding one of the microfluidic channels into the microfluidic chamber.

[019] Controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber includes controlling operation of multiple valves, each valve disposed along a corresponding one of the microfluidic channels.

[020] Heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature includes heating or cooling the fluid in each of the multiple microfluidic channels to a common target temperature.

[021] Heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature includes heating or cooling a fluid in a particular one of the microfluidic channels to a first target temperature that differs from the target temperature for at least one other of the microfluidic channels.

[022] Controlling operation of the temperature control devices includes, for each temperature control device, implementing closed loop feedback control of the temperature control device. Controlling operation of the temperature control devices includes, for each temperature control device, controlling operation of the temperature control device based on a signal from a corresponding temperature sensor disposed in contact with the temperature control device.

[023] Controlling operation of the temperature control devices includes controlling operation of each temperature control device independently from operation of each other temperature control device.

[024] Controlling operation of the temperature control devices includes: controlling a first temperature control device to heat the fluid in the corresponding microfluidic channel to a denaturation temperature (e.g., 95 °C); controlling a second temperature control device to heat the fluid in the corresponding microfluidic channel to an extension temperature (e.g., 72 °C); and controlling a third temperature control device to cool the fluid in the corresponding microfluidic channel to an annealing temperature (e.g., 55 °C).

[025] The method includes providing power to the temperature control devices from one or more rechargeable batteries.

[026] Heating or cooling a fluid in each of multiple microfluidic channels includes operating one or more of a resistive heater, a radiative heater, or a thermoelectric heating or cooling device. [027] Independently controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber includes controlling a flow of fluid adjacent to a reaction chamber of the microfluidic system, in which the reaction chamber is fluidically isolated from the microfluidic chamber. The reaction chamber contains a biological sample. The method includes flowing a fluid into or through the reaction chamber. Flowing a fluid into or through the reaction chamber includes flowing the fluid along an elongated, branched channel. Flowing a fluid into or through the reaction chamber includes flowing a reagent into or through the reaction chamber.

[028] Heating or cooling a fluid in a particular one of the microfluidic channels includes inducing a phase change in a material contained in a secondary channel that is disposed adjacent to the particular one of the microfluidic channels.

[029] The fluid includes a reagent.

[030] The microfluidic chamber includes a biological sample.

[031] In an aspect, a method of conducting a PCR test includes heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature by controlling operation of corresponding temperature control devices. Each temperature control device is disposed in contact with a wall of the corresponding microfluidic channel, and each microfluidic channel is fluidically connected to a microfluidic chamber defined in a substrate of the microfluidic system. The method includes controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber; and causing a biological or chemical reaction to occur in the microfluidic chamber or in a reaction chamber adjacent to the microfluidic chamber.

[032] Embodiments of the method of conducting a PCR test can include one or any combination of two or more of the foregoing features.

[033] In an aspect, a method of conducting a PCR test includes controlling a first temperature control device to heat a fluid in a first microfluidic channel to a denaturation temperature (e.g.,

95 °C); flowing the heated fluid from the first microfluidic channel through a microfluidic chamber and into a fifth microfluidic channel; controlling a fifth temperature control device to heat the fluid in the fifth microfluidic channel to a denaturation temperature (e.g., 95 °C); flowing the heated fluid from the fifth microfluidic channel through the microfluidic chamber and into a second microfluidic channel; cooling the fluid in the second microfluidic channel to a temperature that is around or less than an annealing temperature (e.g., 55 °C); flowing the cooled fluid from the second microfluidic channel through the microfluidic chamber and into a third microfluidic channel; controlling a third temperature control device to heat the fluid in the third microfluidic channel to an annealing temperature (e.g., 55 °C); flowing the heated fluid from the third microfluidic channel through the microfluidic chamber and into a fourth microfluidic channel; controlling a fourth temperature control device to heat the fluid in the fourth microfluidic channel to an extension temperature (e.g., 72 °C); and flowing the heated fluid from the fourth microfluidic channel through the microfluidic chamber and into the first microfluidic channel.

[034] In an embodiment, cooling the fluid in the second microfluidic channel includes controlling a second temperature control device to cool the fluid in the second microfluidic channel to a temperature that is around or less than the annealing temperature (e.g., 55 °C).

[035] In an aspect, a method of conducting an experiment includes controlling a first temperature control device to heat a fluid in a first microfluidic channel to a first target temperature; flowing the heated fluid from the first microfluidic channel through a microfluidic chamber and into a second microfluidic channel; controlling a second temperature control device to heat or cool the fluid in the second microfluidic channel to a second target temperature; and flowing the fluid from the second microfluidic channel through the microfluidic chamber, and optionally to the first microfluidic channel or a third microfluidic channel.

[036] Embodiments can include one or any combination of two or more of the following features.

[037] The first target temperature and the second target temperature are the same.

[038] The first target temperature and the second target temperature are different.

[039] The heated fluid from the second microfluidic channel is flowed through the microfluidic chamber, and to the third microfluidic channel. The method includes controlling the third temperature control device to heat or cool the fluid in the third microfluidic channel to a third target temperature; and flowing the heated fluid from the third microfluidic channel through the microfluidic chamber, and into a fourth microfluidic channel. The method includes: controlling a fourth temperature control device to heat or cool the fluid in the fourth microfluidic channel to a fourth target temperature; and flowing the heated fluid from the fourth microfluidic channel through the microfluidic chamber, and into a fifth microfluidic channel. The method further includes: controlling a fifth temperature control device to heat or cool the fluid in the fifth microfluidic channel to a fifth target temperature; and flowing the heated fluid from the fifth microfluidic channel through the microfluidic chamber, and into a sixth microfluidic channel or into the first, the second, the third, the fourth, or the fifth microfluidic channel.

[040] In an aspect, a method of conducting an experiment includes controlling a first temperature control device to heat a fluid in a first microfluidic channel to a first target temperature; flowing the heated fluid from the first microfluidic channel through a microfluidic chamber and into a second microfluidic channel; controlling a second temperature control device to cool the fluid in the second microfluidic channel to a second target temperature (e.g., room temperature); and flowing the fluid from the second microfluidic channel to a third microfluidic channel without passing through the microfluidic chamber; controlling a third temperature control device to heat the fluid in the third microfluidic channel to a third target temperature; and flowing the fluid from the third microfluidic channel through the microfluidic chamber, and optionally to the first microfluidic channel or a fourth microfluidic channel.

[041] In an aspect, a method of sequencing a nucleic acid includes (a) controlling a first temperature control device to heat a reagent buffer in a first microfluidic channel to a first target temperature (e.g., about 60 °C); (b) flowing the heated reagent buffer from the first microfluidic channel to a microfluidic chamber, wherein a labeled nucleotide is added to a DNA molecule; (c) flowing a wash buffer and an imaging buffer from a second microfluidic channel into the microfluidic chamber, wherein the wash buffer and/or the imaging buffer are optionally heated by a second temperature control device to a second target temperature; (d) imaging the microfluidic chamber, thereby detecting the labeled nucleotide; (e) controlling a third temperature control device to heat a cleavage buffer in a third microfluidic channel to a third target temperature (e.g., about 50-60 °C); (f) flowing the cleavage buffer from the third microfluidic channel to the microfluidic chamber, thereby removing the 3’ terminal blocking group of the labeled nucleotide; (g) flowing wash buffer from a second microfluidic channel into the microfluidic chamber and (h) repeating steps a) to g) until the sequence of the nucleic acid is determined.

[042] The systems and method described here can have one or more of the following advantages. Reactions, e.g., PCR tests, can be completed with low reagent volumes. Rapid, energy efficient, and precise heating and cooling of the fluids in the system can be achieved. The systems are compact, low cost, and portable. [043] The details of one or more implementations are set forth in the accompanying drawings and the description below. Other features and advantages will be apparent from the description and drawings, and from the claims.

Brief Description of Drawings

[044] Fig. l is a diagram of a microfluidic system with temperature control devices.

[045] Fig. 2 is a schematic diagram of a portion of the microfluidic system of Fig. 1.

[046] Fig. 3 is a schematic diagram of a microfluidic system with temperature control devices. [047] Fig. 4 is a side view schematic diagram of a microfluidic channel assembly.

[048] Fig. 5 is a cross-sectional schematic diagram of a portion of a microfluidic system.

[049] Fig. 6 is a diagram of a microfluidic system.

[050] Fig. 7 is a flow chart.

Detailed Description

[051] We describe here microfluidic systems that enable efficient, precise control of the temperature of the fluids in the system. These systems include microfluidic channels fluidically connected to a microfluidic chamber. The fluid in each channel is heated or cooled independently to a target temperature, and the fluid at the target temperature is flows into the microfluidic chamber. In some examples, biological or chemical reactions, such as temperature- sensitive reactions, occur in the microfluidic chamber. In some examples, the microfluidic chamber containing the fluid at the target temperature serves as a temperature regulator for an adjacent reaction chamber in which biological or chemical reactions, such as temperature- sensitive reactions, occur. For instance, these microfluidic systems can be used for polymerase chain reaction (PCR) testing. Because the volume of fluid to be heated or cooled is small relative to the volume of the microfluidic chamber, the heating or cooling is rapid and energy efficient. The systems are compact, portable, and can be operated using a portable power source, e.g., power from a rechargeable battery or mobile device. Applications of these compact, portable systems can include on-site or at-home testing for infectious diseases, such as SARS-CoV-2, influenza, or other infectious diseases.

[052] Referring to Fig. 1, a microfluidic system 100 includes a substrate 101 within which a microfluidic chamber 102 is defined. The microfluidic system 100 also includes multiple microfluidic channels 104a, 104b. Each channel 104a, 104b (collectively referred to as channels 104) is fluidically connected to the microfluidic chamber 102. The channels 104 and the microfluidic chamber 102 are configured to receive fluids, such as liquids or gases. For instance, the microfluidic chamber 102 can be a reaction chamber in which biological or chemical microfluidic reactions occur. A pump 105a is positioned to pump fluid from the channel 104a into the microfluidic chamber, and a pump 105b is positioned to pump fluid from the channel 104b into the microfluidic chamber. In some embodiments, each pump is controlled independently by a controller for that pump. In some embodiments, each pump is controlled by a centralized controller that controls all pumps. In one specific example, one pump is connected to the system (e.g., to the channel 105b). The pump can push the fluid from the fluid channel 104b, to the microfluidic chamber 102, and to the channel 104a. It can also push the fluid in the opposite direction, e.g., it can push the fluid from channel 104a to channel 104b, through the microfluidic chamber 102. Different reagent reservoirs can be connected to the channel 104b, optionally with selective valves.

[053] The microfluidic system 100 includes temperature control devices 108a, 108b (collectively referred to as temperature control devices 108 or temperature-controlled blocks (TCB) 108). Each temperature control device 108 is disposed in contact with one of the walls of a corresponding one of the channels 104. Each temperature control device 108 is configured to heat or cool a fluid in the corresponding channel 104. The temperature control devices 108 can be or include resistive heaters, radiative heaters (e.g., using infrared light, visible light, or other types of radiative heating), thermoelectric heating or cooling devices such as Peltier thermoelectric devices, or other suitable heating or cooling devices. In the example of Fig. 1, the temperature control device 108 extends across the entire width of the corresponding channel 104. In some examples, the temperature control device 108 is wider or narrower than the width of the channel 104.

[054] The configuration of the microfluidic system 100 enables fluid in the system to be heated or cooled to a target temperature in the channels 104 prior to entering the microfluidic chamber 102. Because the volume of fluid contained in each of the channels 104 is relatively small compared to the volume of fluid contained in the microfluidic chamber 102, the fluid can be heated or cooled quickly and efficiently. [055] In some embodiments, the substrate 101 of the microfluidic system has a length of about or less than 80, 70, 60, 50, 40, 30, 20, 10 or 5 mm, a width of about or less than 80, 70, 60, 50,

40, 30, 25, 20, 15, 10 or 5 mm, and a height of about or less than 5, 4, 3, 2, 1.5, or 1 mm. In some embodiments, the microfluidic chamber 102 has a length of about or less than 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 mm, a width of about or less than 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 mm, and a height of about or less than 200, 150, 100, 80, 70, 60, 50, 40, 30, 25, 20, 15, or 10 mih. In some embodiments, the microfluidic chamber 102 has a length of about or less than 10 mm, a width of about or less than 1 mm, and a height of about or less than 80 mih. In some embodiments, the microfluidic chamber 102 has a length of about or less than 5 mm, a width of about or less than 0.5 mm, and a height of about or less than 50 mih. In some embodiments, the microfluidic chamber 102 has a volume of about 50 nL~5000 nL, 100 nL~800 nL, 125 nL~800 nL, 100 nL~4000 nL, 100 nL~3000 nL, 100 nL~2000 nL, or 100 nL~1000 nL. In some embodiments, the microfluidic chamber 102 has a volume of about or at least 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nL. In some embodiments, the microfluidic chamber 102 has a volume of about or at less than 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, or 1500 nL.

[056] In some embodiments, each channel 104 has a volume of about 100 nL~60000 nL, 100 nL~50000 nL, 1000 nL~8000 nL, 1250 nL~8000 nL, 1000 nL~40000 nL, 1000 nL~30000 nL, 1000 nL~20000 nL, or 1000 nL~10000 nL. In some embodiments, each channel 104 has a volume of about or at least 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000,

11000, 12000, 13000, 14000, or 15000 nL. In some embodiments, each channel 104 has a volume of about or less than 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 11000, 12000, 13000, 14000, or 15000 nL. In some embodiments, the volume of the channel is about or at least 5, 6, 7, 8, 9, or 10 times larger than the volume of the microfluidic chamber 102. In a specific example of a microfluidic system having a substrate 101 of dimensions 40 mm x 25 mm x 1.5 mm and channels 104 each of dimensions 20 mm x 5 mm x 0.5 mm, the microfluidic chamber 102 has a volume of about 1 pL and each channel 104 has a volume of about 50 pL.

[057] Because the volume of each channel 104 is so much less than the size of the substrate

101 with the microfluidic chamber (in this example, 30 times less), there is less thermal mass in the channels 104 than the thermal mass of substrate 101. Thus, the fluid in the channels 104 can be heated or cooled at a much quicker rate by the corresponding temperature control devices 108 than the fluid in the microfluidic chamber 102 could be heated or cooled by a heating or cooling device for the entire chamber 102. In some embodiments, the temperature control devices 108 are connected to one or more fluid reservoirs (e.g., about or at least 1, 2, 3, 4, 5, 6, 7, 9, or 10 fluid reservoirs, optionally with one or more selector valves), wherein the fluid can be continuously pumped into the temperature control devices 108, heated or cooled to the target temperature as the fluid goes through the temperature control devices, and then into the microfluidic chamber.

[058] In another specific example of a microfluidic system having a substrate 101 of dimensions 10 mm X 20 mm X 1.5 mm and channels 104 each of dimensions 5 mm X 30 mm X 0.3 mm.

[059] In some examples, both temperature control devices 108 are controlled to heat or cool the fluid in the corresponding channels 104 to the same target temperature. By controlling the pumps 105 to provide an alternating, bi-directional flow of fluid at the target temperature, the temperature in the microfluidic chamber 102 can be precisely regulated to remain at the target temperature. For instance, the fluid in the channel 104a is heated or cooled to the target temperature by the temperature control device 108a and then pumped into the microfluidic chamber 102. This pumping of fluid into the microfluidic chamber 102 pushes fluid from the microfluidic chamber 102 into the channel 104b, where that fluid is heated or cooled to the target temperature by the temperature control device 108b. The fluid in the channel 104b is then pumped into the microfluidic chamber 102, pushing fluid from the microfluidic chamber 102 back into the channel 104a to again be heated or cooled to the target temperature. The flow rate that the fluid (e.g., liquid or gas) is pumped into the microfluidic chamber 102 can be adjusted as needed by the system. In some embodiments, the flow rate is about or less than 3000, 2500,

2000, 1500, 1000, 250 or 50 mΐ/min. In some embodiments, the flow rate is about or more than 2500, 2000, 1500, or 1000 mΐ/min. In some embodiments, the flow rate can be adjusted so that the energy efficiency can be maximized. In some embodiments, a temperature sensor can be used to detect the temperature in the microfluidic chamber 102. A controller can be used to adjust the flow rate and/or one or more temperature control devices based on the signal from the temperature sensor.

[060] In some examples, the microfluidic system 100 can be operated to achieve two- temperature-point thermal cycling. In these examples, the temperature control device 108a is controlled to heat or cool the fluid in the channel 104a to a first temperature, and the temperature control device 108b is controlled to heat or cool the fluid in the channel 104b to a second temperature different from the first temperature. By controlling the pumps 105 to provide an alternating, bi-directional flow of fluid at two different temperatures, the temperature in the microfluidic chamber 102 can be cycled between two different temperatures.

[061] For instance, the fluid in the channel 104a is heated or cooled to the first temperature by the temperature control device 108a and then pumped into the microfluidic chamber 102 (in the direction of a first arrow 120a). When the fluid at the first temperature enters the microfluidic chamber 102, the temperature of the fluid in the microfluidic chamber 102 changes toward the first temperature. This pumping of fluid into the microfluidic chamber 102 pushes fluid from the microfluidic chamber 102 into the channel 104b, where that fluid is heated or cooled to the second temperature by the temperature control device 108b. The fluid in the channel 104b is then pumped into the microfluidic chamber 102 (in the direction of a second arrow 120b), changing the temperature of the fluidic in the microfluidic chamber 102 toward the second temperature and pushing fluid from the microfluidic chamber 102 back into the channel 104a to again be heated or cooled to the first temperature.

[062] This alternating, bi-directional flow of fluid enables rapid reheating or recooling of small volumes of fluid, enabling the temperature in the microfluidic chamber 102 to be maintained or cycled with precision and efficiency. Because the flow of fluid in the microfluidic system 100 is bidirectional (e.g., rather than a unidirectional cycling of fluid into and out of the system), the total volume of reagent remains low. For instance, the microfluidic system 100 can be operable with only the volume of fluid sufficient for the volume of the microfluidic chamber 102 and channels 104.

[063] Fig. 2 shows a side view of a portion of the channel 104. The temperature control device 108 is disposed in contact with a top wall 200 of the channel 104. The top wall 200 of the channel 104 is formed of a thermally conductive material such that heat or cooling capacity generated by the temperature control device 108 can heat or cool fluid in the channel 104. For instance, the top wall 200 of the channel 104 can be formed of a plastic, glass, silicon, or other suitable material.

[064] In the example of Fig. 2, the temperature control device 108 includes an active heating or cooling element 202, such as a resistive or radiative heater or thermoelectric heating or cooling device. The temperature control device 108 also includes a thermal sink 204 disposed between the active heating or cooling element 202 and the wall 200 of the channel 104. The thermal sink 204 is formed of a thermally conductive material, such as a metal, with a high heat capacity. The thermal sink 204 is configured to transfer heat or cooling capacity generated by the active heating or cooling element 202 to the fluid in the channel 104. In some examples, the temperature control device includes only an active heating or cooling element without a thermal sink, such that the active heating or cooling element is disposed directly on the wall 200 of channel 104.

[065] The operation of the temperature control device 108 is controlled by a temperature control system 210. The temperature control system 210 can control independently each of the temperature control devices 108 of the microfluidic system, e.g., temperature control devices 108a, 108b. The temperature control system 210 can implement closed loop feedback temperature control of the temperature control device 108 to maintain the stability of the temperature of the temperature control device 108 at a target temperature.

[066] The temperature control system 210 includes a temperature sensor 212, such as a thermocouple, configured to measure a temperature of the temperature control device 108. In the example of Fig. 2, the temperature sensor 212 is disposed on the thermal sink 204. A signal from the temperature sensor 212 that is indicative of the temperature of the thermal sink 204 is provided to a controller 214, such as one or more processors or microcontrollers. Based on the signal from the temperature sensor 212, the controller 214 controls operation of a heater driver 216. The heater driver 216 in turn controls operation of the active heating or cooling element 202 such that a target temperature is achieved at the temperature sensor 212. The heater driver 216 is powered by a power supply 218, such as a battery, e.g., a rechargeable battery, an alternating current, a mobile device-based power supply (e.g., provided via a universal serial bus (USB) connection), or other suitable power supply.

[067] In some embodiments, the temperature control device is designed to cool the fluid. For example, a coolant control system can continuously circulate a chilled liquid coolant through cooling channels around the fluid channel. The coolant control system controls coolant flow around the fluid channel 104 to cool the fluid. In some embodiments, the coolant control system can cool the fluid to the room temperature, 0 °C or 4 °C. In some embodiments, once the fluid is cooled to the room temperature, the fluid is reheated to a target temperature (e.g., in another temperature control device) before it is pumped into the microfluidic chamber 102.

[068] Referring again to Fig. 1, in some examples, a top wall of the microfluidic chamber 102, a bottom wall of the microfluidic chamber 102, or both are formed of an optically transparent material, such as a transparent plastic, silicon dioxide, or another suitable material. This configuration enables a reaction occurring in the chamber 102 to be observed, e.g., through a microscope. Because the temperature control devices 108 are disposed over the channels 104 rather than over the chamber 102, the view of the chamber 102 is not obscured.

[069] In some examples, the microfluidic chamber 102 is a reaction chamber in which biological or chemical reactions occur. The fluid can be or contain reactants, reagents, or both. In some examples, the microfluidic chamber 102 can contain a biological sample, such as DNA, and the fluid can be or contain a reagent capable of interacting with the DNA. For instance, DNA can be immobilized on one or more interior surfaces of the microfluidic chamber 102.

[070] Referring to Fig. 3, a microfluidic system 300 includes multiple microfluidic channels 304a-304e. Each channel 304a-304e is fluidically connected to a microfluidic chamber 302. Each channel 304a-304e has a corresponding temperature control device 308a-308e. The structure and operation of the temperature control devices 308a-308e are, e.g., as described above for the temperature control device 108.

[071] In the microfluidic system 300 of Fig. 3, each channel 304a-304e has a corresponding valve 312a-312e that can be opened or closed to control fluid flow into our out of the channel. In some examples, a pump (not shown) is associated with each channel 302 and positioned to pump fluid from the corresponding channel 304 into the microfluidic chamber 302. In some examples, the flow of fluid into and out of the channels 304 is a passive flow controlled by the opening and closing of the valves 312. In some examples, a multi-way valve can be used in place of two or more of the individual valves. For instance, the valves 312a-312c can be replaced by a single, three-way valve.

[072] The configuration of the microfluidic system 300 enables multi-stage temperature control to be implemented. Each temperature control device 308a-308e can be controlled to heat or cool the fluid in the corresponding channel 304a-304e to a distinct target temperature. The temperature of the fluid in the microfluidic chamber 302 can be controlled by allowing fluid to flow into the microfluidic chamber 302 from the channel 304 that contains fluid at the desired temperature. This configuration is useful, e.g., for enabling multi-stage reactions that involve different temperature setpoints. For instance, the microfluidic system 300 can be used for PCR testing, in which an annealing temperature (e.g., 55 °C), an extension temperature (e.g., 72 °C), and a denaturation temperature (e.g., 95 °C) are used.

[073] In an example of operation of the microfluidic system 300 for PCR testing, the temperature control devices 308a and 308e are controlled to heat fluid in the channels304a, 304e, respectively, to a denaturation temperature (e.g., 95 °C); the temperature control device 308b is controlled to cool fluid in the channel 304b, e.g., to room temperature, or to around or lower than an annealing temperature (e.g., 55 °C); the temperature control device 308c is controlled to heat fluid in the channel 304c to the annealing temperature (e.g., 55 °C); and the temperature control device 308d is controlled to heat fluid in the channel 304d to an extension temperature (e.g., 72 °C). In operation, fluid flow is controlled by the valves 312, alone or in combination with pumps for actively controlled fluid flow. Fluid in the channel 304a is heated to a denaturation temperature (e.g., 95 °C) by the temperature control device 308a and flowed through the microfluidic chamber 302 and into the channel 304e. In the channel 304e, the fluid is reheated to the denaturation temperature (e.g., 95 °C) by the temperature control device 308e. The fluid with the denaturation temperature (e.g., 95 °C) from the channel 304e is then pushed through the microfluidic chamber 302 and into the channel 304b. Within the channel 304b, the fluid is cooled, e.g., to room temperature, or to around or lower than the annealing temperature (e.g., 55 °C), e.g., by the temperature control device 308b. The cooled fluid from the channel 304b is pushed through the microfluidic chamber 302 into the channel 304c, where it is heated by the temperature control device 308c to the annealing temperature (e.g., 55 °C). The fluid with the annealing temperature (e.g., 55 °C) flows from the channel 304c through the microfluidic chamber 302 and into the channel 304d, where it is heated by the temperature control device 308d to the extension temperature (e.g., 72 °C). That heated fluid is flowed through the microfluidic chamber 302 and into the channel 304a. The temperature control device 308a heats the fluid in the channel 304a back up to the denaturation temperature (e.g., 95 °C) to begin another thermal cycle. [074] Fig. 4 is a side view diagram of a microfluidic channel assembly 400 that forms part of a microfluidic system such as the systems 100, 300. The channel assembly 400 includes a microfluidic channel 404 that is fluidically connected to a microfluidic chamber of a microfluidic system, e.g., as described for the systems 100, 300. The channel assembly 400 also includes secondary channels 412a, 412b disposed adjacent to and on opposite sides of the channel 404 (e.g., on the top and bottom of the channel 404). The channel 404 shares its top and bottom walls 414a, 414b with the respective secondary channels 412a, 412b. A temperature control device (not shown) is disposed over at least a portion of the width of the channel assembly 400 and configured to heat or cool a fluid in the channel 404 and one or both of the secondary channels 412a, 412b. In some examples, only a single secondary channel 412 is present.

[075] The secondary channels 412a, 412b contain a material or combination of materials that have a phase transition temperature (e.g., a melting point) at a target temperature. For instance, the target temperature is the temperature at which the fluid in the microfluidic channel 404 is to be heated or cooled. The presence of a relatively massive reservoir of material having a phase transition temperature at the target temperatures provides a high thermal capacitance to the microfluidic channel assembly 400. For instance, this reservoir of material in the secondary channels 412a, 412b can prevent the temperature of the fluid in the microfluidic channel 404 from exceeding the target temperature. The high thermal capacitance of the reservoir of material in the secondary channels 412a, 412b can also act as a thermal buffer, stabilizing fluctuations in the temperature of the fluid in the microfluidic channel 404.

[076] In a specific example, the secondary channels contain a composition including stearic acid (with a melting temperature (T_m) of 69.3 °C) and arachidic acid (T_m=75.5 °C). By mixing these two fatty acids in a particular ratio, a composition having a melting temperature of the extension temperature (e.g., 72 °C) can be achieved. The presence of this composition in the secondary channels 412a, 412b prevents the temperature of the fluid in the microfluidic channel 404 from exceeding the extension temperature (e.g., 72 °C). Other ratios of stearic acid and arachidic acid can be used to achieve other target temperatures. Other compositions, such as different fatty acids, can be used to achieve other target temperatures.

[077] Fig. 5 is a cross sectional side view diagram of a portion of a microfluidic system 500. The microfluidic system 500 includes a substrate 501 within which a microfluidic chamber 502 and a reaction chamber 520 are defined. The microfluidic chamber 502 and the reaction chamber 520 at least partially overlap one another and share a common wall 505. In the example of Fig. 5, the common wall 505 is the top wall of the microfluidic chamber 502 and the bottom wall of the reaction chamber 520, such that the reaction chamber 520 is positioned directly above the microfluidic chamber 502. In some examples, the reaction chamber 520 is positioned directly below the microfluidic chamber 502, or the reaction chamber 520 and the microfluidic chamber 502 are positioned side-by-side and share a side wall.

[078] The microfluidic system 500 includes one or more microfluidic channels 504 that are fluidically connected to the microfluidic chamber 502. Each channel 504 has a corresponding temperature control device 508. The structure and operation of the temperature control devices are, e.g., as described above for the temperature control device 108. The microfluidic system 500 can also include one or more inlet channels, outlet channels, or both (not shown), that are fluidically connected to the reaction chamber 520. The microfluidic chamber 502 and the reaction chamber 520 are fluidically isolated from one another, meaning that the microfluidic chamber 502 is not in fluidic communication with the reaction chamber 520.

[079] In the microfluidic system 500, reactions, such as biological or chemical reactions, occur in the reaction chamber 520. Temperature control of the fluid in the reaction chamber 520 is provided by heating or cooling fluid in the channels 504, and flowing that heated or cooled fluid into the microfluidic chamber 502, e.g., as described above for Fig. 1. Heat transfer between the microfluidic chamber 502 and the reaction chamber 520 regulates the temperature of the fluid in the reaction chamber 520. The common wall 505 can be thin, thermally conductive, or both, to facilitate efficient heat transfer.

[080] In some examples, the reaction chamber 102 can contain a biological sample, such as DNA. For instance, DNA can be immobilized on one or more interior surfaces of the reaction chamber 102. Fluid in the reaction chamber 102 (e.g., stationary in or flowing through the reaction chamber 102) can be or contain a reagent capable of interacting with the DNA.

[081] In the microfluidic system 500, the temperature in the reaction chamber 520 can be controlled while enabling the fluid flow through the reaction chamber 520 to differ from the fluid flow provided for temperature control. In some examples, the reaction chamber 520 can be in a quiescent state (e.g., with little or no flow) while fluid flow is provided between the channels 504 and microfluidic chamber 502. In some examples, the fluid flow through the reaction chamber 520 can be at a different flow rate than the fluid flow between the channels 504 and the microfluidic chamber 502. In some examples, the fluid flow through the reaction chamber 520 can be unidirectional while the fluid flow between the channels 504 and the microfluidic chamber 502 is an alternating, bidirectional flow.

[082] Referring to Fig. 6, a microfluidic system 600 includes a substrate 601 within which a microfluidic chamber 602 is defined. The microfluidic chamber 602 is fluidically connected to one or more microfluidic channels (not shown) at inlets 603a, 603b, with each microfluidic channel having a corresponding temperature control device, e.g., as discussed above for the microfluidic system 100. An elongated reaction channel 620 is also defined in the substrate 601. In the example of Fig. 6, the reaction channel 620 is a branched channel. In some examples, the reaction channel 620 is a single, elongated channel. The microfluidic chamber 602 and the reaction channel 620 at least partially overlap one another. In the example of Fig. 6, the microfluidic chamber 602 is positioned directly above the reaction channel 620. The microfluidic chamber 602 and the reaction channel 620 are fluidically isolated from one another, meaning that the microfluidic chamber 602 is not in fluidic communication with the reaction channel 620.

[083] Fluid samples, e.g., including reactants, are provided into the reaction channel 620 at the inlet 624. The fluid samples flow through the reaction channel 620 and exit the reaction channel 620 at an outlet 622. Reactions occur in the reaction channel 620. In some embodiments, both 622 and 624 are sealed before reactions (e.g., qPCR). In some embodiments, reagents are added through inlet 624 at one or more of the steps in each reaction cycle. Temperature control in the reaction channel 620 is provided by flowing heated or cooled fluid into the microfluidic chamber 602, e.g., as described above for Fig. 1. Heat transfer between the microfluidic chamber 602 and the reaction channel 620 regulates the temperature of the fluid in the reaction channel 620. In some embodiments, the system or device can run experiments for two or more samples, e.g., 2,

3, 4, 5, 6, 7, 8, 9, or 10 samples, simultaneously.

[084] The fluid flow through the microfluidic chamber 602 can be controlled to provide a temperature gradient within the microfluidic chamber 602. For a branched reaction channel 620, this temperature gradient exposes each branch of the reaction channel 620 to a different temperature. This configuration can be useful in the context of qPCR applications, experimental testing, or other contexts.

[085] In some examples, the microfluidic systems described here form part of a compact, portable PCR testing system. The systems can be powered by a portable power source, such as a battery (e.g., a rechargeable battery) or mobile device. The systems are compact, lightweight, and inexpensive. Applications of these compact, portable systems can include on-site or at-home testing for infectious diseases, such as SARS-CoV-2, influenza, or other infectious diseases. [086] Fig. 7 shows an example process for operating a microfluidic system, such as the microfluidic system 100 of Fig. 1. The operation of each of multiple temperature control devices is controlled by a temperature control system (700). In some examples, the temperature control system implements closed loop feedback control of the temperature control devices, e.g., based on a signal from a sensor, such as a temperature sensor. Operation of each temperature control device causes fluid in a corresponding microfluidic channel to be heated or cooled to a target temperature (702). In some examples, the fluid in each channel is heated or cooled to the same target temperature. In some examples, the fluid in at least one channel is heated or cooled to a target temperature that differs from the target temperature for at least one other channel. In a specific example, the temperature control devices are controlled to heat or cool fluid in the corresponding channels to the denaturation temperature (e.g., 95 °C), the extension temperature (e.g., 72 °C), and the annealing temperature (e.g., 55 °C), respectively. In one aspect, the temperature control system (700) automatically controls heater driver 216 and/or pumps 105 based on a pre-set program for desired reactions, e.g., PCR or QPCR.

[087] Furthermore, because the microfluidic chamber 102 can be heated or cooled rapidly, this significantly reduces the time for running the experiments (e.g., PCR or quantitative PCR). In some embodiments, the peak ramp rate (temperature change rate) at the microfluidic chamber can be at least or about 3, 4, 5, 6, 7, 8, 9, or 10 °C/s. In some embodiments, the temperature increase rate (e.g., peak or average) can be at least or about 3, 4, 5, 6, 7, 8, 9, or 10 °C/s. In some embodiments, the temperature decrease rate (e.g., peak or average) can be at least or about 3, 4, 5, 6, 7, 8, 9, or 10 °C/s. In some embodiments, the system as described herein can significantly reduce the time by about or at least 10%, 20%, 30%, or 40%, e.g., as compared to a standard experimental protocol without using the system as described herein. This is advantageous as it provides an even faster test for numerous biomarkers, including e.g., biomarkers for various diseases, such as SARS-CoV-2, influenza, HIV, or other diseases. In some embodiments, the methods and the systems described herein only take about or less than 3, 5, 7, 8, 9, 10, or 15 minutes to complete QPCR (e.g., for a duration for 30, 35, 40, or 45 cycles of QPCR). In some embodiments, the methods and the systems described herein only take about or less than 8 minutes to complete QPCR (e.g., for a duration for 30 cycles).

[088] Fluid flow from each of the microfluidic channels into a microfluidic chamber is controlled, e.g., by operation of one or more pumps, valves, or both (704). A biological or chemical reaction occurs in the microfluidic chamber or in a distinct reaction chamber whose temperature is regulated by heat transfer from the fluid in the microfluidic chamber (706).

[089] In one aspect, also provided are systems, devices, and/or methods for temperature control for various reactions (e.g., PCR, QPCR, RPA, LAMP, ddPCR, DNA ligation, reverse transcription and sequencing by synthesis). The method involves the steps of: controlling a first temperature control device to heat a fluid in a first microfluidic channel to a first target temperature; flowing the heated fluid from the first microfluidic channel through a microfluidic chamber and into a second microfluidic channel; controlling a second temperature control device to heat/cool the fluid in the second microfluidic channel to a second target temperature; flowing the fluid with the second target temperature from the second microfluidic channel through the microfluidic chamber, thereby changing the temperature of the microfluidic chamber to the second target temperature. This process can be repeated multiple times until the experiment is completed. One or more additional temperature control devices can be used for different target temperatures.

[090] In one aspect, the systems, devices, and/or methods are designed for PCR (e.g., qPCR). A key aspect of PCR is the concept of thermocycling: alternating steps of melting a nucleic acid template, annealing primers to the resulting single strands, and extending those primers to make new copies of double stranded nucleic acid. In thermocycling, a PCR reaction mixture can be repeatedly cycled from high temperatures for melting the DNA, to lower temperatures for primer annealing and extension.

[091] In a typical PCR reaction, the reaction mixture is desirably transitioned and maintained accurately at various temperatures for prescribed time periods with temperature cycling frequently repeated many times. Generally, it is desirable to change the sample temperature to the next temperature in the cycle rapidly for several reasons. First, the chemical reaction may have an optimum temperature for each of its stages. Thus, less time spent at non-optimal temperatures may improve the result product. Another reason is that a minimum time for holding the reaction mixture at each incubation temperature may be desired after each incubation temperature is reached. These minimum incubation times may establish “floor” or minimum time it takes to complete a cycle. Any time transitioning between sample incubation temperatures is time which is added to this minimum cycle time. Since multiple cycles are involved, this additional time lengthens the total time needed to complete the amplification.

[092] Typically, PCR consists of a series of about 20 to about 40 repeated temperature changes, called thermal cycles. In some embodiments, there are about or at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 cycles. Each cycle commonly consisting of two or three discrete temperature steps. In some embodiments, the cycling is often preceded by a single temperature step at a very high temperature (e.g., >90 °C), and followed by one hold at the end for final product extension or brief storage. The temperatures used and the length of time they are applied in each cycle depend on a variety of parameters, including the enzyme used for DNA synthesis, the concentration of bivalent ions and dNTPs in the reaction, and the melting temperature (Tm) of the primers. The individual steps usually include the following:

[093] Denaturation step: this step is the first step in the cycle. The denaturation temperature can cause melting or denaturation of the double-stranded DNA template by breaking the hydrogen bonds between complementary bases, yielding two single-stranded DNA molecules. In some embodiments, the denaturation temperature is about 90-100 °C, or 94-98 °C. In some embodiments, the denaturation temperature is about or at least 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99 °C. During this cycle, the fluid is heated in the temperature control device to the denaturation temperature, and is then pumped in the microfluidic chamber, and optionally into a second temperature control device.

[094] Annealing step: in the next step, the reaction temperature is lowered to the annealing temperature. The annealing temperature allows annealing of the primers to each of the single- stranded DNA templates. Two different primers are typically included in the reaction mixture: one for each of the two single-stranded complements containing the target region. The primers are single-stranded sequences themselves, but are much shorter than the length of the target region, complementing only very short sequences at the 3 ' end of each strand. It is critical to determine a proper temperature for the annealing step because efficiency and specificity are strongly affected by the annealing temperature. This temperature must be low enough to allow for hybridization of the primer to the strand, but high enough for the hybridization to be specific, i.e., the primer should bind only to a perfectly complementary part of the strand, and nowhere else. If the temperature is too low, the primer may bind imperfectly. In some embodiments, the annealing temperature is about 50-65 °C or 50-60 °C. In some embodiments, the annealing temperature is about or at least 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, or 65 °C. In some embodiments, during this step, the fluid (e.g., the fluid from a fluid reservoir or the fluid that has been cooled to the room temperature) is heated in the temperature control device to the annealing temperature, and is then pumped to the microfluidic chamber, and optionally then into a second temperature control device. In some embodiments, the fluid is cooled in a temperature control device to the annealing temperature, and then is pumped into the microfluidic chamber. [095] Extension step: the temperature at this step depends on the DNA polymerase used. In this step, the DNA polymerase synthesizes a new DNA strand complementary to the DNA template strand by adding free dNTPs from the reaction mixture that is complementary to the template in the 5 ' -to-3 ' direction, condensing the 5 ' -phosphate group of the dNTPs with the 3 ' - hydroxy group at the end of the nascent (elongating) DNA strand. The optimum activity temperature for the thermostable DNA polymerase of Taq polymerase is approximately 70-80 °C. In some embodiments, the extension temperature is about or at least 70, 71, 72, 73, 74, 75,

76, 77, 78, 79, or 80 °C. In some embodiments, during this step, the fluid is heated in the temperature control device to the extension temperature, and is then pumped to the microfluidic chamber, and optionally then into another temperature control device.

[096] The processes of denaturation, annealing and elongation constitute a single cycle. Multiple cycles are required to amplify the DNA target to millions of copies. Fluids are heated or cooled in the temperature control device, and are pumped back and forth through the microfluidic chamber, thereby rapidly changing the temperature in the microfluidic chamber during each cycle. In some embodiments, about or at least 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 cycles are involved.

[097] In some embodiments, the fluid comprises reagents for the reactions. The reagents are selected for different reactions, e.g., PCR, QPCR, RPA, LAMP, ddPCR, DNA ligation, reverse transcription reactions. In some embodiments, the fluid comprises 200 mM deoxynucleotide mix, 0.1-0.5 pM forward primer, 0.1-0.5 pM reverse primer, 0.05 units/pL, 0.05 units/pL Taq DNA polymerase, and/or 0.1-0.5 mM MgCh. Because the volume of fluid is small, the system also effectively reduces reagents that are required for the reactions by about or at least 10%, 20%, 30%, or 40%. [098] In some embodiments, when two or more different temperatures are required for different cycles of reactions, a pair of temperature control devices can be used. For example, temperature control device 308a and temperature control device 308e can form a pair for a first target temperature. Temperature control device 308b and temperature control device 308d can form a pair for a second target temperature. The fluid flows from the temperature control device of the first pair into the microfluidic chamber, and then flows into the other temperature control device of the first pair. This is particularly energy efficient if a high temperature (e.g., a denaturation temperature) needs to be maintained. When the temperature needs to be adjusted to the second temperature, the fluid can flow from the temperature control device of the second pair into the microfluidic chamber, and then flows into the other temperature control device of the second pair. In these embodiments, the fluid in the first pair of temperature control devices are not pumped into the second pair of temperature control devices. As the two temperature control devices of the same pair are set for the same temperature and the fluid is only exchanged in the same pair, only a small amount of energy is required to heat or cool the fluid, thereby further improving energy efficiency. In addition, the traditional device needs to heat or cool the entire system (e.g., substrate 101), which has more mass than the fluid in the microfluidic chamber 102. [099] In some embodiments, the system is a portable system. In some embodiments, the weight of the entire system is about or less than 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 kg. In some embodiments, the system is a PCR chip.

[0100] In one aspect, the systems, devices, and/or methods are designed for sequencing (e.g., sequencing by synthesis). The sequencing technology works in three basic steps: amplifying, sequencing, and analyzing. The process begins with purified DNA. The DNA is fragmented and adapters are added that contain segments that act as reference points during amplification, sequencing, and analysis. The modified DNA is loaded onto the substrate (e.g., glass) that forms the surface of the microfluidic chamber 102. The glass can contain nanowells that space out fragments and help with overcrowding. Each nanowell contains oligonucleotides that provide an anchoring point for the adaptors to attach. Once the fragments have attached, a phase called cluster generation begins. This step makes about a thousand copies of each fragment of DNA and is done by bridge amplification PCR. Next, the reagent buffer (including e.g., primers and modified nucleotides) are added to the microfluidic chamber 102. These nucleotides have a reversible 3' fluorescent blocker so the DNA polymerase can only add one nucleotide at a time onto the DNA fragment. After each round of synthesis, a camera takes a picture of the microfluidic chamber 102. A computer determines what base was added by the wavelength of the fluorescent tag and records it for every spot on the microfluidic chamber 102. After each round, non-incorporated molecules are washed away. A chemical deblocking step is then used to remove the 3’ fluorescent terminal blocking group. The process continues until the full DNA molecule is sequenced.

[0101] In an example of operation of the microfluidic system 300 for sequencing by synthesis, the temperature control devices 308a, 308b and 308c are controlled to adjust fluids in the channels 304a, 304b, 304c, respectively, to target temperatures. The channels 304a, 304b, 304c are connected to different reagent reservoirs. The DNA is immobilized in microfluidic chamber 302 (e.g., on the glass surface of microfluidic chamber 302). To incorporate fluorescence labeled nucleotides into the DNA stand, the reagent fluid is heated to about 60 °C in the 304a channel and is pumped into the microfluidic chamber 302. The heated fluid only needs to heat up the glass surface within a few microns where the DNA clusters are attached. It does not need to heat up the entire glass substrate. Then the wash buffer is sent through 304b at room temperature and then the image buffer is followed through 304b. The image of clusters will be taken in microfluidic chamber 302. Then the cleavage buffer is pumped through channel 304c at about 50 °C ~ 60 °C and followed by wash buffer through channel 304b. These steps forms one cycle of sequencing. This cycle can be repeated multiples times until the sequence of the DNA is determined. In some embodiments, the microfluidic chamber 302 is formed by two pieces of glasses. As both sides of the microfluidic chamber 302 are not covered by heating plates, images can be taken from both sides and more DNA molecules can be sequenced simultaneously.

[0102] Particular embodiments of the subject matter have been described. Other embodiments are within the scope of the following claims.

Claims

What is claimed is:

1. A microfluidic system comprising: a microfluidic chamber defined in a substrate of the microfluidic system; multiple microfluidic channels, in which each microfluidic channel is fluidically connected to the microfluidic chamber; and multiple temperature control devices, in which each temperature control device is disposed in contact with a wall of a corresponding one of the microfluidic channels and configured to heat or cool a fluid in the microfluidic channel.

2. The microfluidic system of any of the preceding claims, comprising multiple pumps, each pump configured to pump fluid from a corresponding one of the microfluidic channels into the microfluidic chamber.

3. The microfluidic system of any of the preceding claims, comprising multiple valves, each valve disposed along a corresponding one of the microfluidic channels.

4. The microfluidic system of any of the preceding claims, comprising a temperature control system configured to control operation of the multiple temperature control devices.

5. The microfluidic system of claim 4, in which the temperature control system is configured to implement closed loop feedback temperature control of the multiple temperature control devices.

6. The microfluidic system of claim 4 or 5, in which the temperature control system comprises multiple temperature sensors each disposed in contact with a corresponding one of the temperature control devices.

7. The microfluidic system of claim 6, in which the temperature control system comprises one or more microcontrollers or microprocessors configured to control operation of each of the temperature control devices based on a signal from the corresponding temperature sensor.

8. The microfluidic system of any of claims 4 to 7, in which the temperature control system is configured to control operation of each temperature control device independently from operation of each other temperature control device.

9. The microfluidic system of any of claims 4 to 8, in which the temperature control system is configured to be powered by a rechargeable battery.

10. The microfluidic system of any of the preceding claims, in which at least one of the temperature control devices comprises: an active heating or cooling element; and a thermal sink disposed between the active heating or cooling element and the wall of the respective microchannel.

11. The microfluidic system of any of the preceding claims, in which at least one of the temperature control devices comprises a one or more of resistive heater, a radiative heater, or a thermoelectric heating or cooling device.

12. The microfluidic system of any of the preceding claims, in which a top wall of the microfluidic chamber, a bottom wall of the microfluidic chamber, or both are formed of an optically transparent material.

13. The microfluidic system of any of the preceding claims, comprising a secondary channel disposed adjacent to a first one of the microfluidic channels, in which the temperature control device that is disposed in contact with the wall of the first microfluidic channel is configured to heat or cool a material in the secondary channel.

14. The microfluidic system of claim 13, in which the secondary channel contains a material having a melting point at a target temperature.

15. The microfluidic system of claim 14, in which the material comprises a combination of fatty acids.

16. The microfluidic system of any of the preceding claims, comprising a reaction chamber defined in the substrate of the microfluidic system, in which the reaction chamber is fluidically isolated from the microfluidic chamber.

17. The microfluidic system of claim 16, in which the reaction chamber and the microfluidic chamber share a common wall.

18. The microfluidic system of claims 16 or 17, in which the reaction chamber comprises an elongated channel.

19. The microfluidic system of claim 18, in which the reaction chamber comprises a branched channel.

20. The microfluidic system of any of claims 16 to 19, in which the microfluidic chamber overlaps with at least a portion of the reaction chamber.

21. A polymerase chain reaction (PCR) test system comprising the microfluidic system of any of the preceding claims.

22. A method for operating a microfluidic system, the method comprising: heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature by controlling operation of corresponding temperature control devices, in which each temperature control device is disposed in contact with a wall of the corresponding microfluidic channel, and in which each microfluidic channel is fluidically connected to a microfluidic chamber defined in a substrate of the microfluidic system; controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber; and causing a biological or chemical reaction to occur in the microfluidic chamber or in a reaction chamber adjacent to the microfluidic chamber.

23. The method of claim 22, in which controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber comprises controlling operation of multiple pumps, each pump configured to pump fluid from a corresponding one of the microfluidic channels into the microfluidic chamber.

24. The method of any of claims 22 or 23, in which controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber comprises controlling operation of multiple valves, each valve disposed along a corresponding one of the microfluidic channels.

25. The method of any of claims 22 to 24, in which heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature comprises heating or cooling the fluid in each of the multiple microfluidic channels to a common target temperature.

26. The method of any of claims 22 to 25, in which heating or cooling a fluid in each of multiple microfluidic channels to a corresponding target temperature comprises heating or cooling a fluid in a particular one of the microfluidic channels to a first target temperature that differs from the target temperature for at least one other of the microfluidic channels.

27. The method of any of claims 22 to 26, in which controlling operation of the temperature control devices comprises, for each temperature control device, implementing closed loop feedback control of the temperature control device.

28. The method of claim 27, in which controlling operation of the temperature control devices comprises, for each temperature control device, controlling operation of the temperature control device based on a signal from a corresponding temperature sensor disposed in contact with the temperature control device.

29. The method of any of claims 22 to 28, in which controlling operation of the temperature control devices comprises controlling operation of each temperature control device independently from operation of each other temperature control device.

30. The method of any of claims 22 to 29, in which controlling operation of the temperature control devices comprises: controlling a first temperature control device to heat the fluid in the corresponding microfluidic channel to a denaturation temperature (e.g., 95 °C); controlling a second temperature control device to heat the fluid in the corresponding microfluidic channel to an extension temperature (e.g., 72 °C); and controlling a third temperature control device to cool the fluid in the corresponding microfluidic channel to an annealing temperature (e.g., 55 °C).

31. The method of any of claims 22 to 30, comprising providing power to the temperature control devices from one or more rechargeable batteries.

32. The method of any of claims 22 to 31, in which heating or cooling a fluid in each of multiple microfluidic channels comprises operating one or more of a resistive heater, a radiative heater, or a thermoelectric heating or cooling device.

33. The method of any of claims 22 to 32, in which independently controlling a flow of fluid from each of the microfluidic channels into the microfluidic chamber comprises controlling a flow of fluid adjacent to a reaction chamber of the microfluidic system, in which the reaction chamber is fluidically isolated from the microfluidic chamber.

34. The method of claim 33, in which the reaction chamber contains a biological sample.

35. The method of claim 33 or 34, comprising flowing a fluid into or through the reaction chamber.

36. The method of claim 35, in which flowing a fluid into or through the reaction chamber comprises flowing the fluid along an elongated, branched channel.

37. The method of claim 35 or 36, in which flowing a fluid into or through the reaction chamber comprises flowing a reagent into or through the reaction chamber.

38. The method of any of claims 22 to 37, in which heating or cooling a fluid in a particular one of the microfluidic channels comprises inducing a phase change in a material contained in a secondary channel that is disposed adjacent to the particular one of the microfluidic channels.

39. The method of any of claims 22 to 38, in which the fluid comprises a reagent.

40. The method of any of claims 22 to 39, in which the microfluidic chamber comprises a biological sample.

41. A method of conducting a PCR test comprising the method of any of claims 22 to 40.

42. A method of conducting a PCR test, the method comprising: controlling a first temperature control device to heat a fluid in a first microfluidic channel to a denaturation temperature (e.g., 95 °C); flowing the heated fluid from the first microfluidic channel through a microfluidic chamber and into a fifth microfluidic channel; controlling a fifth temperature control device to heat the fluid in the fifth microfluidic channel to a denaturation temperature (e.g., 95 °C); flowing the heated fluid from the fifth microfluidic channel through the microfluidic chamber and into a second microfluidic channel; cooling the fluid in the second microfluidic channel to a temperature that is around or less than an annealing temperature (e.g., 55 °C); flowing the cooled fluid from the second microfluidic channel through the microfluidic chamber and into a third microfluidic channel; controlling a third temperature control device to heat the fluid in the third microfluidic channel to an annealing temperature (e.g., 55 °C); flowing the heated fluid from the third microfluidic channel through the microfluidic chamber and into a fourth microfluidic channel; controlling a fourth temperature control device to heat the fluid in the fourth microfluidic channel to an extension temperature (e.g., 72 °C); and flowing the heated fluid from the fourth microfluidic channel through the microfluidic chamber and into the first microfluidic channel.

43. The method of claim 42, in which cooling the fluid in the second microfluidic channel comprises controlling a second temperature control device to cool the fluid in the second microfluidic channel to a temperature that is around or less than the annealing temperature (e.g., 55 °C).

44. A method of conducting an experiment, the method comprising:

(a) controlling a first temperature control device to heat a fluid in a first microfluidic channel to a first target temperature;

(b) flowing the heated fluid from the first microfluidic channel through a microfluidic chamber and into a second microfluidic channel;

(c) controlling a second temperature control device to heat or cool the fluid in the second microfluidic channel to a second target temperature; and

(d) flowing the fluid from the second microfluidic channel through the microfluidic chamber, and optionally to the first microfluidic channel or a third microfluidic channel.

45. The method of claim 44, wherein the first target temperature and the second target temperature are the same.

46. The method of claim 44, wherein the first target temperature and the second target temperature are different.

47. The method of any of claims 44 to 46, wherein the heated fluid from the second microfluidic channel is flowed through the microfluidic chamber, and to the third microfluidic channel, wherein the method further comprises: controlling the third temperature control device to heat or cool the fluid in the third microfluidic channel to a third target temperature; and flowing the heated fluid from the third microfluidic channel through the microfluidic chamber, and into a fourth microfluidic channel.

48. The method of claim 47, wherein the method further comprises: controlling a fourth temperature control device to heat or cool the fluid in the fourth microfluidic channel to a fourth target temperature; and flowing the heated fluid from the fourth microfluidic channel through the microfluidic chamber, and into a fifth microfluidic channel.

49. The method of claim 48, wherein the method further comprises: controlling a fifth temperature control device to heat or cool the fluid in the fifth microfluidic channel to a fifth target temperature; and flowing the heated fluid from the fifth microfluidic channel through the microfluidic chamber, and into a sixth microfluidic channel or into the first, the second, the third, the fourth, or the fifth microfluidic channel.

50. A method of conducting an experiment, the method comprising:

(c) controlling a second temperature control device to cool the fluid in the second microfluidic channel to a second target temperature (e.g., room temperature); and

(d) flowing the fluid from the second microfluidic channel to a third microfluidic channel without passing through the microfluidic chamber; (e) controlling a third temperature control device to heat the fluid in the third microfluidic channel to a third target temperature; and

(f) flowing the fluid from the third microfluidic channel through the microfluidic chamber, and optionally to the first microfluidic channel or a fourth microfluidic channel.

51. A method of sequencing a nucleic acid, the method comprising: a) controlling a first temperature control device to heat a reagent buffer in a first microfluidic channel to a first target temperature (e.g., about 60 °C); b) flowing the heated reagent buffer from the first microfluidic channel to a microfluidic chamber, wherein a labeled nucleotide is added to a DNA molecule; c) flowing a wash buffer and an imaging buffer from a second microfluidic channel into the microfluidic chamber, wherein the wash buffer and/or the imaging buffer are optionally heated by a second temperature control device to a second target temperature; d) imaging the microfluidic chamber, thereby detecting the labeled nucleotide; e) controlling a third temperature control device to heat a cleavage buffer in a third microfluidic channel to a third target temperature (e.g., about 50-60 °C); f) flowing the cleavage buffer from the third microfluidic channel to the microfluidic chamber, thereby removing the 3’ terminal blocking group of the labeled nucleotide; g) flowing wash buffer from the second microfluidic channel into the microfluidic chamber; and h) repeating steps a) to g) until the sequence of the nucleic acid is determined.