A rapid temperature-changing PCR system in a small space

Abstract

This invention discloses a micro-space rapid temperature-changing PCR system, comprising: a PCR chip with a PCR reaction chamber internally connected to a flow channel, a shape memory metal spring and a rubber stopper connected to the shape memory metal spring within the flow channel; and a cooling module including a phase change tank, a flow channel, and a working fluid collection box, wherein the phase change tank is located at the bottom of the PCR chip and is connected to the working fluid collection box via the flow channel. By sealing the reaction chamber with a shape memory metal spring within the flow channel, evaporation of the liquid during high-temperature reactions is prevented; infrared heating technology is employed, allowing the heat source to be directly absorbed by the reaction solution, eliminating thermal resistance between the heat source and the reaction solution; and jet phase change cooling technology is used to cool the PCR reaction solution, achieving rapid cooling.

Description

Technical Field

[0001] This invention relates to the field of microfluidic PCR reaction technology, and in particular to a rapid temperature-changing PCR system in a small space. Background Technology

[0002] Microfluidic PCR technology has developed rapidly in the past decade. Its advantages, such as small size, high integration, and portability, have led to its widespread application in early diagnosis of various diseases, pathogen detection, and prenatal diagnosis. PCR, short for Polymerase Chain Reaction, is a rapid in vitro DNA amplification technique. Under the catalysis of biological enzymes, DNA is broken down into single strands at high temperatures (95℃-98℃). At intermediate temperatures (60℃), the single strands bind to primers, and new double-stranded DNA is synthesized under the action of polymerase. These two steps are repeated more than 40 times.

[0003] Currently, various PCR systems are available on the market, but many products suffer from the following problems: 1. Existing products typically place samples and reaction reagents into centrifuge tubes, which are then placed inside a copper block. The copper block's specific heat capacity and thermal conductivity result in thermal inertia during heating and cooling, leading to a slow temperature response. 2. Most commercially available PCR instruments use semiconductor cooling chips for heating or cooling. By attaching the metal block to the side or bottom, the temperature of the test tube is altered, which can easily cause uneven temperature distribution inside the test tube, resulting in low amplification efficiency. 3. Semiconductor heat dissipation structures generally use metal heat sinks with a cooling fan to dissipate heat from the metal heat sink. This structure has poor heat dissipation and is bulky, making it difficult to integrate. 4. During the high-temperature PCR reaction process, the reaction solution is prone to detaching from the reaction chamber in chip-based PCR systems. Summary of the Invention

[0004] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0005] To solve the above-mentioned technical problems, the present invention provides the following technical solution: A micro-space rapid temperature-changing PCR system includes a PCR chip, which has a PCR reaction chamber inside, the PCR reaction chamber is connected to a flow channel, a memory metal spring and a rubber stopper connected to the memory metal spring are disposed in the flow channel; a cooling module includes a phase change tank, a flow channel and a working fluid collection box, the phase change tank is disposed at the bottom of the PCR chip, and the phase change tank is connected to the working fluid collection box through the flow channel.

[0006] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the PCR chip includes an upper cover plate, a middle layer, and a lower cover plate, and the PCR reaction chamber is disposed in the middle layer.

[0007] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the chip cover plate is provided with a liquid injection port and an exhaust port communicating with the flow channel.

[0008] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the PCR reaction chamber is cylindrical, the side of the cylinder has a frosted surface, and the bottom bonding layer is bonded with a thin copper sheet.

[0009] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, a thin layer of flexible thermally conductive paper and polyethylene high-density foam are embedded between the phase change tank and the PCR chip.

[0010] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the thin-layer flexible thermally conductive paper is a thermally conductive silicone pad or thermally conductive graphite paper.

[0011] In a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the shape memory metal spring is made of nickel-titanium alloy.

[0012] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, a vacuum pump and a solenoid valve are further provided on the flow pipeline.

[0013] In a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the flow channel is connected to the phase change tank through a micro-nozzle array.

[0014] As a preferred embodiment of the micro-space rapid temperature-changing PCR system of the present invention, the PCR reaction chamber is heated by infrared radiation, and the fluorescent detection substance is a fluorescent probe.

[0015] The beneficial effects of this invention are as follows: by setting a memory metal spring in the flow channel to seal the reaction chamber, the evaporation of the liquid during high-temperature reaction is prevented; infrared heating technology is used, and the heat source is directly absorbed by the reaction solution, with no thermal resistance between the heat source and the reaction solution; jet phase change cooling technology is used to cool the PCR reaction solution, achieving rapid cooling; a thin layer of flexible thermally conductive paper and high-density polyethylene foam are embedded on the contact surface between the cooling module and the chip, which can reduce the contact thermal resistance between the phase change tank and the chip. Attached Figure Description

[0016] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort. Wherein:

[0017] Figure 1 This is a schematic diagram of the overall system of the present invention.

[0018] Figure 2 This is a front view of the PCR chip of the present invention.

[0019] Figure 3 This is a schematic diagram of the explosion of the PCR reaction chip of the present invention.

[0020] Figure 4 This is a schematic diagram of the cooling module of the present invention.

[0021] Figure 5 This is a schematic diagram of the cooling module structure of the present invention.

[0022] Figure 6 This is a schematic diagram of the memory spring in the contracted state of the PCR reaction chip of the present invention.

[0023] Figure 7 This is a graph showing the relationship between PCR reaction solution temperature and reaction time.

[0024] Figure 8 This is a graph showing the relationship between the injection height and heat transfer coefficient of a micro-nozzle array.

[0025] Figure 9 This is a schematic diagram of the existing water cooling method.

[0026] Figure 10 This is a time-varying graph comparing the heat exchange of jet phase change cooling technology and water cooling technology. Detailed Implementation

[0027] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the accompanying drawings.

[0028] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0029] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0030] Example 1

[0031] Reference Figures 1 to 2 This is the first embodiment of the present invention, which provides a micro-space rapid temperature-changing PCR system, including a PCR chip 100 and a cooling module 200. The PCR chip 100 has a PCR reaction chamber 101 inside, which is connected to a flow channel 102. A memory metal spring 103 and a rubber stopper 104 connected to the memory metal spring 103 are disposed in the flow channel 102. The cooling module 200 includes a phase change tank 201, a flow pipe 202 and a working medium collection box 203. The phase change tank 201 is disposed at the bottom of the PCR chip 100 and is connected to the working medium collection box 203 through the flow pipe 202.

[0032] The shape memory metal spring 103 is used as a seal during the PCR reaction process to prevent the PCR reaction solution from evaporating at high temperatures. One end of the shape memory metal spring 103 is fixed to one side of the chip channel 102, and the other end is connected to the rubber stopper 104. The shape memory metal spring 103 is initially in an expanded state. After being compressed a certain distance, it is installed in the channel 102 of the PCR chip 100. At this time, there is a certain gap between the shape memory metal spring 103 and the channel 102 for the PCR reaction solution to flow. When the PCR reaction solution is injected into the PCR reaction chamber 101, the sealing area is heated by heating technology, and the shape memory metal spring 103 expands to its initial state, pushing the rubber stopper 104 to block the channel 102, thus achieving a sealing effect.

[0033] The cooling module 200 is used to rapidly cool the PCR reaction solution in the PCR reaction chamber 101. By inputting the coolant in the working medium collection box 203 into the phase change tank 201, which is located below the PCR reaction chamber 101, rapid cooling of the PCR chip 100 is achieved.

[0034] In this embodiment, the PCR chip 100 seals the PCR reaction chamber 101 by setting a memory metal spring 103 in the flow channel 102, changing the temperature and thus changing the extension and contraction state of the memory metal spring 103. Compared with existing chip-type chips, the sealing effect is better, preventing the evaporation of liquid during high-temperature reactions.

[0035] Example 2

[0036] Reference Figures 1 to 6This is the second embodiment of the present invention, which differs from the first embodiment in that: the PCR chip 100 includes an upper cover plate 105, a middle layer 106, and a lower cover plate 107, with the PCR reaction chamber 101 disposed in the middle layer 106. The upper cover plate 105 is provided with an injection port 105a and an exhaust port 105b communicating with the flow channel 102. PCR reaction solution is injected into the PCR reaction chamber 101 through the injection port 105a on the upper cover plate 105, and the exhaust port 105b is used to expel gas from the PCR chip 100.

[0037] The PCR chip 100 reaction process includes a heating stage, a isothermal stage, and a cooling stage. The heating stage uses infrared heating technology to rapidly raise the reaction solution in the PCR reaction chamber 101 to the required temperature. The isothermal stage uses low-power infrared to maintain the temperature of the reaction solution. The cooling stage uses jet phase change cooling technology to rapidly cool the temperature of the reaction solution.

[0038] PCR reaction solutions are substances with a high water content. These substances resonate at their inherent vibrational frequencies, thus absorbing infrared heat energy, increasing the internal heat energy and raising the temperature. Infrared heating technology is advantageous because water has an absorption rate of up to 90% for infrared wavelengths, resulting in high energy utilization. Furthermore, infrared heating is a non-contact heating method, directly transferring energy to the reaction solution and avoiding the influence of contact thermal resistance on temperature rise.

[0039] This system uses fluorescent probes as fluorescent materials to monitor the PCR amplification process in real time during the cooling process. The fluorescent probes and infrared heating are integrated into the same optical head, and the two are used alternately, resulting in higher integration and smaller size.

[0040] Furthermore, the PCR chip 100 is made of PC material, which has good light transmittance, allowing both infrared and fluorescence light to pass through smoothly with minimal energy loss. The PCR reaction chamber 101 is cylindrical, approximately 25 ml in size and 1 mm in height. The cylindrical sides have a frosted surface, facilitating infrared or fluorescence irradiation and providing good reflection, thus improving the absorption of infrared or fluorescence light by the reaction solution. The bottom bonding layer is bonded with a thin copper sheet 108, with a thickness of 0.25 mm. On one hand, the thermal conductivity of copper is approximately 400 W / (m·K), while that of PC is approximately 4 W / (m·K), meaning the thermal conductivity of copper is about 100 times that of PC, thus enhancing thermal conductivity. On the other hand, copper has good reflectivity, reflecting both infrared and fluorescence light, thereby improving energy utilization efficiency.

[0041] Furthermore, the shape memory metal spring 103 is made of nickel-titanium alloy. Because its crystal structure differs above and below 40°C, the alloy contracts or expands with temperature changes, causing its shape to change. The shape memory metal spring 103 is initially in an expanded state. After being compressed a certain distance, it is installed in the flow channel 102 of the PCR chip 100. When the PCR reaction solution is injected into the PCR reaction chamber 101, the sealed area is heated using heating technology. The shape memory metal spring 103 expands to its initial state, pushing the rubber stopper 104 to block the flow channel 102, achieving a sealing effect.

[0042] Furthermore, a thin layer of flexible thermally conductive paper 204 and a high-density polyethylene foam 205 are embedded between the phase change tank 201 and the PCR chip 100. The thin layer of flexible thermally conductive paper 204 is a thermally conductive silicone pad or thermally conductive graphite paper. A vacuum pump 206 and a solenoid valve 207 are also provided on the flow channel 202. The flow channel 202 is connected to the phase change tank 201 through a micro-nozzle array 208.

[0043] Furthermore, the vacuum pump 206 provides power to the cooling module 200, allowing the phase change working fluid to continuously circulate from the working fluid collection tank 203 to the phase change tank 201; the solenoid valve 207 is installed in the flow pipe 202, which can stop or start the flow of the working fluid; the micro-nozzle array 208 connects the phase change tank 201 and the flow pipe 202. When the working fluid flowing out of the flow pipe 202 passes through the micro-nozzle array 208, it can be uniformly flowed into the phase change tank 201, and the micro-nozzle array 208 has tiny pores, allowing the working fluid to flow out evenly. To facilitate phase change, the phase change tank 201 provides a phase change space for the working fluid. A thermally conductive graphene layer is laid on the upper side of the phase change tank 201, which is located directly below the PCR reaction chamber 101. The thermally conductive graphene layer can reduce the contact thermal resistance between the chip and the cooling module 200. The contact material between the phase change tank 201 and other areas of the PCR chip 100, except for the PCR reaction chamber 101, is high-density polyethylene foam 205. This material has a thermal conductivity of 0.03 W / (m·K), which can reduce heat loss during the heating or isothermal stages.

[0044] Solenoid valve 207 is initially closed, vacuum pump 206 is activated, and all phase change working fluid is stored in working fluid collection tank 203. Under the operation of vacuum pump 206, phase change tank 201 will form a vacuum state. When PCR chip 100 needs to be cooled, solenoid valve 207 opens, and phase change working fluid will quickly flow through micro-nozzle array 208 to form a jet state and flow into phase change tank 201 for phase change, rapidly absorbing heat, so that PCR chip 100 is quickly cooled down to 60°C. Solenoid valve 207 closes, waiting for the next cooling to reopen. Compared with ordinary liquid cooling, this cooling method has a higher heat transfer coefficient, and the jet phase change cooling technology can greatly save reaction time.

[0045] Example 3

[0046] The experiment was conducted using the PCR system described in Example 2. The procedure for this experiment is as follows:

[0047] After the nucleic acid extraction is completed, the sample and reaction reagents are injected into the PCR reaction chamber 101 through the injection port 105a. The infrared heater then heats the memory metal spring 103, causing it to expand and block the flow channel 102. During this process, temperature calibration and laser head position adjustment are performed. At this time, the vacuum pump 206 in the cooling module 200 is turned on. The temperature during the heating process is collected by the infrared sensor. The specific heating and cooling steps are as follows:

[0048] (1) 30℃ to 95℃ heating stage 1: The infrared heater is turned on, the solution temperature in PCR reaction chamber 101 is ≤90℃, the infrared heating adopts high power output, when the solution temperature in PCR reaction chamber 101 reaches 90℃, the PID control technology starts to intervene, so that the reaction solution temperature gradually reaches 95℃ from 90℃.

[0049] (2) 95℃ constant temperature stage 1: When the temperature of the PCR reaction chamber 101 solution reaches 95℃, the infrared heater power is quickly switched to the constant power corresponding to 95℃ infrared for heating, so that the temperature of the chamber solution is maintained at 95℃ within 5s.

[0050] (3) Cooling stage 1 from 95℃ to 60℃: At this time, the infrared heater is turned off and the fluorescence detection is turned on to monitor the reaction solution in real time. At the same time, the solenoid valve 207 in the cooling module 200 is turned on, and the phase change working medium is rapidly sprayed from the working medium collection box 203 into the phase change tank 201. The micro-nozzle array 208 sprays at a height of 15mm. The phase change is endothermic, and the temperature in the PCR reaction chamber 101 drops rapidly to 60℃ in about 3s.

[0051] (4) 60℃ constant temperature stage 2: When the solution temperature in PCR reaction chamber 101 reaches 60℃, the solenoid valve 207 is closed, and the infrared heater power is quickly switched to the constant power corresponding to 60℃ infrared for heating, so that the solution temperature in the chamber is maintained at 60℃ for 30s.

[0052] (5) 60℃ to 95℃ heating stage 2: The infrared heater is turned on, the solution temperature in PCR reaction chamber 101 is ≤90℃, the infrared heating adopts high power output, when the solution temperature in PCR reaction chamber 101 reaches 90℃, the PID control technology starts to intervene, so that the reaction solution temperature gradually reaches 95℃ from 90℃.

[0053] After repeating steps 2-5 approximately 40 times, the heating and cooling processes cease, and the results are analyzed.

[0054] Example 4

[0055] During the experiment, it was found that the heat transfer coefficient of the jet phase transition process changes with the jet height. In this embodiment, the PCR chip 100 was cooled by using jet heights of 10mm, 12mm, 14mm, 15mm, 16mm, 18mm, and 20mm for the micro-nozzle array 208. The heat transfer coefficient was measured by the temperature drop of the PCR chip 100 per unit time. The results are as follows: Figure 8 As shown, the optimal spray height of the micro-nozzle array 208 is 15mm, at which point the highest heat exchange efficiency can be achieved, enabling rapid cooling of the PCR chip 100. Therefore, the size of the cooling module 200 is designed to be 40×16×15mm.

[0056] Comparative Example 1

[0057] The PCR chip 100 was cooled using a water-cooling method, such as... Figure 9 The PCR water-cooling device shown includes a main body 300 with multiple sample containers 301 arranged at intervals on the main body 300 for holding samples. Heat dissipation pipes are installed inside the main body 300 and arranged around the sample containers 301. The heat dissipation pipes are connected to an inlet pipe 302 and an outlet pipe 303. A heat exchange box 304 is connected to the inlet pipe 302 and the outlet pipe 303 and contains coolant. A comparative experiment was conducted at 30°C and the same flow rate. The experimental steps for the water-cooling method were the same as in Example 3. During the cooling phase, the water pump was turned on, and the coolant in the heat exchange box 304 entered the heat dissipation pipes inside the main body 300 through the inlet pipe 302, thereby removing heat from the PCR tubes in the sample containers 301. Finally, the coolant flowed back to the heat exchange box 304 through the outlet pipe 303. The temperature in the PCR tubes was collected every second by an infrared sensor and compared with Example 3 to obtain the heat exchange results of water cooling and jet phase change cooling. Figure 10 As shown, by Figure 10 It is known that the time required for jet phase change cooling is 3 seconds, while the time required for water cooling is 9 seconds. Therefore, the jet phase change cooling technology can greatly save reaction time.

[0058] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the claims of the present invention.

Claims

1. A rapid temperature-switching PCR system in a small space, characterized in that: include, A PCR chip (100) has a PCR reaction chamber (101) inside, which is connected to a flow channel (102). A memory metal spring (103) and a rubber stopper (104) connected to the memory metal spring (103) are installed in the flow channel (102). One end of the memory metal spring (103) is fixed to one side of the flow channel (102), and the other end is connected to the rubber stopper (104). The initial state of the memory metal spring (103) is in an expanded state. After being compressed a certain distance, it is installed in the flow channel (102) of the PCR chip (100). At this time, there is a certain gap between the memory metal spring (103) and the flow channel (102) for the PCR reaction liquid to flow. When the PCR reaction liquid is injected into the PCR reaction chamber (101), the sealed area is heated by heating technology. The memory metal spring (103) expands to its initial state and pushes the rubber stopper (104) to block the flow channel (102) to achieve a sealing effect. The cooling module (200) includes a phase change tank (201), a flow pipe (202), and a working fluid collection box (203). The phase change tank (201) is located at the bottom of the PCR chip (100), and the phase change tank (201) is connected to the working fluid collection box (203) through the flow pipe (202). A thin layer of flexible thermally conductive paper (204) and high-density polyethylene foam (205) are embedded between the phase change tank (201) and the PCR chip (100). The PCR reaction chamber (101) is cylindrical with a frosted surface on the side, which facilitates infrared or fluorescent irradiation and can reflect light well, thereby improving the absorption of infrared or fluorescent light by the reaction solution. The bottom bonding layer is bonded with a thin copper sheet (108), which can enhance thermal conductivity and reflect infrared and fluorescent light, thereby improving energy utilization efficiency. The flow channel (202) is connected to the phase change tank (201) through the micro-nozzle array (208); the working fluid flowing out of the flow channel (202) passes through the micro-nozzle array (208), so that the working fluid flows into the phase change tank (201) evenly, and the micro-nozzle array (208) has small pores, so that the working fluid can undergo phase change better when it flows out. The PCR reaction chamber (101) is heated by infrared radiation, and the fluorescent detection substance is a fluorescent probe; the fluorescent probe and infrared heating are integrated in the same optical head, and the two are used alternately.

2. The micro-space rapid temperature-changing PCR system as described in claim 1, characterized in that: The PCR chip (100) includes an upper cover plate (105), a middle layer (106) and a lower cover plate (107), and the PCR reaction chamber (101) is disposed in the middle layer (106).

3. The micro-space rapid temperature-changing PCR system as described in claim 2, characterized in that: The chip cover plate (105) is provided with an injection port (105a) and an exhaust port (105b) that are connected to the flow channel (102).

4. The micro-space rapid temperature-changing PCR system as described in claim 1, characterized in that: The thin-layer flexible thermally conductive paper (204) is a thermally conductive silicone pad or a thermally conductive graphite paper.

5. The micro-space rapid temperature-changing PCR system as described in claim 4, characterized in that: The memory metal spring (103) is made of nickel-titanium alloy.

6. The micro-space rapid temperature-changing PCR system as described in claim 1, characterized in that: The flow pipe (202) is also equipped with a vacuum pump (206) and a solenoid valve (207).

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