A microfluidic reaction chip for nucleic acid amplification and its application method

Abstract

The present application relates to the technical field of nucleic acid amplification, and discloses a micro-fluidic reaction chip for nucleic acid amplification and an application method thereof, which comprises a reaction cavity with a closed loop flow channel and a heat transfer area arranged on the outer wall of the reaction cavity, wherein the heat transfer area is asymmetrically distributed along the closed loop flow channel; the characteristic size of the reaction cavity along the temperature gradient direction is Lx, and the depth of the reaction cavity along the temperature gradient direction is Ly; the corresponding temperature difference of the reaction cavity is 30-60 DEG C, so that directional circulation flow is formed in the reaction cavity. By asymmetrically distributing the heat transfer area along the closed loop flow channel, and combining with specific characteristic size, depth and temperature difference parameter range, self-sustaining directional thermal convection circulation is induced in the reaction cavity, and repeated temperature zone switching required by nucleic acid amplification can be automatically completed without the help of any external micro-pump, micro-valve or mechanical driving component, so that the system structure is significantly simplified, and the cost of chip manufacturing and instrument integration is reduced.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid amplification technology, and in particular to a microfluidic reaction chip for nucleic acid amplification and its application method. Background Technology

[0002] Traditional nucleic acid amplification technologies (such as polymerase chain reaction, PCR) usually rely on sophisticated temperature control and circulation equipment to repeatedly raise and lower the temperature of the reaction solution, or use external micropumps, microvalves and other driving mechanisms to make the fluid flow back and forth between different temperature zones. This results in complex instrument structures, large size, high cost, and difficulty in achieving portability and low power consumption.

[0003] While existing microfluidic chips can reduce the size of reaction systems to some extent, they often still require the integration of complex fluid control components, which makes them difficult to manufacture and prone to cross-contamination, limiting their widespread application in scenarios such as point-of-care testing (POCT). Summary of the Invention

[0004] This invention provides a microfluidic reaction chip for nucleic acid amplification and its application method to solve the existing technical problems, thereby resolving the issues mentioned in the background section.

[0005] To solve the above-mentioned technical problems, according to one aspect of the present invention, more specifically, a microfluidic reaction chip for nucleic acid amplification, comprising: A reaction chamber with a closed-loop flow channel and a heat transfer area disposed on the outer wall of the reaction chamber, wherein the heat transfer area is asymmetrically distributed along the closed-loop flow channel. The characteristic dimension of the reaction chamber along the temperature gradient direction is: The depth of the reaction chamber along the temperature gradient direction is ; The temperature difference corresponding to the reaction chamber is 30-60℃, so as to form a directional circulation flow in the reaction chamber, which is suitable for the temperature zone circulation process in nucleic acid amplification reaction.

[0006] Furthermore, the heat transfer region is distributed on one side or non-centrally symmetrically in the closed-loop flow channel.

[0007] Furthermore, the reaction chamber has a ring-shaped structure or a racetrack-shaped structure.

[0008] Furthermore, the width of the reaction chamber is And the ratio of the depth to the characteristic dimension of the reaction chamber is .

[0009] Furthermore, the heat transfer area is provided with a material with high thermal conductivity.

[0010] Furthermore, the reaction chamber is provided with a flow path constraint structure, which includes a guide channel, a guide wall, or a geometric constraint structure.

[0011] Furthermore, the microfluidic reaction chip includes multiple reaction chambers, and the multiple reaction chambers are arranged in an array.

[0012] Furthermore, the heat in the heat transfer zone is provided by an external heat source, which is selected from electric heating, magnetic induction heating, light heating or chemical heating.

[0013] Furthermore, the reaction chamber is made of plastic, glass, or metal.

[0014] A method for applying a microfluidic reaction chip for nucleic acid amplification involves using a microfluidic reaction chip to create a temperature distribution through a heat transfer zone, causing the fluid to circulate in a directional manner within the reaction chamber, thereby completing the nucleic acid amplification reaction.

[0015] This invention provides a microfluidic reaction chip for nucleic acid amplification and its application method. Compared with existing technologies, the advantages achieved by this method are: 1. This invention induces a self-sustaining directional thermal convection circulation within the reaction chamber by setting the heat transfer area to an asymmetrical distribution along the closed-loop flow channel and combining it with specific characteristic dimensions, depths, and temperature difference parameter ranges. This allows for the automatic completion of repeated temperature zone switching required for nucleic acid amplification without the need for any external micropumps, microvalves, or mechanical drive components. This significantly simplifies the system structure, reduces the cost of chip manufacturing and instrument integration, and facilitates portable and low-power on-site rapid detection.

[0016] 2. The feature size defined by this invention is 2 to 12 mm, the depth is 0.3 to 0.9 mm, and the temperature difference is 3 to 60 degrees Celsius. This combination can form a stable and unidirectional convection flow in the closed-loop channel, avoiding turbulence or disordered circulation paths. It ensures that the nucleic acid sample moves back and forth between the high-temperature denaturation zone and the low-temperature annealing extension zone in an orderly manner, thereby improving amplification efficiency and reaction consistency, and reducing the requirements for precision temperature control equipment.

[0017] 3. The present invention can be equipped with a high thermal conductivity material in the heat transfer area on the outer wall of the reaction chamber, and is compatible with various external heat sources such as electric heating, magnetic induction heating, light heating or chemical heating. At the same time, flow path constraint structures such as flow guide grooves and flow guide walls can be selected in the chamber, which can not only improve the heat transfer response speed and accelerate the cycle start-up, but also further stabilize the flow direction of the fluid, enhance the chip's adaptability to different working environments and heating methods, and facilitate flexible integration into various nucleic acid detection devices.

[0018] 4. This invention supports the construction of multiple reaction chambers on the same chip and their array distribution. Each chamber can independently inject samples and share the same pair of temperature difference heat sources, realizing the simultaneous amplification and detection of multiple nucleic acid targets, which significantly improves the detection throughput. The flow stability and amplification efficiency of each reaction chamber show consistency with a small coefficient of variation, meeting the practical needs of high-throughput and multi-index parallel detection. Attached Figure Description

[0019] Figure 1 This is a schematic diagram of the overall structure of the present invention; Figure 2 This is the closed-loop flow channel structure of the present invention; Figure 3 This is the heat transfer area distribution of the present invention; Figure 4 This is the circulating flow path of the present invention.

[0020] In the diagram: 1. Reaction chamber; 2. Heat transfer zone. Detailed Implementation

[0021] To make the technical solution of the present invention clearer, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments.

[0022] To more clearly illustrate the technical solution of the present invention, the present invention will be further described in detail below with reference to the accompanying drawings and specific embodiments. The following embodiments all employ a microfluidic reaction chip with a closed-loop flow channel. The characteristic dimensions of the reaction chamber along the temperature gradient direction are 2–12 mm, and the depth is 0.3–0.9 mm. The heat transfer region is asymmetrically distributed along the closed-loop flow channel, and a temperature difference of 30–60°C is applied to form a directional circulating flow within the chamber for the temperature zone circulation process in nucleic acid amplification reactions.

[0023] Example 1: Basic ring structure (single cavity, plastic material) Construct a ring-shaped microfluidic reaction chamber (such as...) Figure 1 , Figure 2 As shown, it is made of plastic material (such as polymethyl methacrylate). Cavity characteristic dimensions (width / diameter along the temperature gradient direction). ,depth ,width (The ratio of depth to feature size is 0.083). A heat transfer zone is formed on the outer wall of the cavity: a high-temperature zone (approximately 95°C) is in contact with the high-temperature zone on one side of the closed-loop flow channel (unilateral distribution, non-centrosymmetric), and a low-temperature zone (approximately 55°C) is in contact with the low-temperature zone on the other side, creating a temperature difference of approximately 40°C (e.g., ...). Figure 3 , Figure 4 (As shown). The heat transfer area is supplied with heat by an external electric heating film.

[0024] Experimental results: The fluid forms a unidirectional circulating flow path within the cavity, without obvious turbulence or multiple circulation paths, meeting the temperature zone circulation requirements for high-temperature denaturation (95℃) and low-temperature annealing / extension (55℃) of nucleic acid amplification.

[0025] This embodiment employs a closed-loop annular flow channel and an asymmetric heat transfer region on one side of the outer wall of the cavity, ensuring that the high-temperature and low-temperature zones are distributed unilaterally along the flow channel, thereby establishing a stable temperature gradient in the horizontal direction. When the feature size is 6 mm, the depth is 0.5 mm, and a temperature difference of approximately 40 °C is applied, the fluid inside the cavity undergoes natural convection due to density differences. Furthermore, due to the asymmetric arrangement of the heat and cold sources, the convection is forcibly guided into a unidirectional circulating flow, automatically completing the alternating process of high-temperature denaturation and low-temperature annealing / extension required for nucleic acid amplification without any external drive.

[0026] This design utilizes the self-driven principle of thermal convection, completely eliminating the need for micro-pumps or reciprocating mechanisms found in traditional PCR instruments. This significantly simplifies the chip and instrument structure, reducing manufacturing costs and energy consumption. Furthermore, the plastic material is easy to injection mold, making it suitable for mass production. The resulting consistent directional circulation effectively avoids reaction unevenness caused by multiple circulation paths, ensuring the reliability and reproducibility of amplification results.

[0027] Example 2: Runway-shaped structure and airflow constraints Based on Example 1, the reaction chamber was modified into a racetrack-shaped structure (a closed-loop flow channel consisting of two semicircular arcs and two straight segments), and a flow path constraint structure was set inside the chamber: a guide wall (with the same height as the flow channel depth and a width of 0.2 mm) was set along the inner wall of the flow channel to further control the flow direction. Other parameters were the same as in Example 1.

[0028] Experimental results: The flow direction is more stable and the circulation path is consistent, proving that the constraint structure can enhance the convection stability.

[0029] In the racetrack-shaped structure of the closed-loop flow channel, consisting of two semicircular arcs and two straight segments, the asymmetric heat transfer region is also distributed along one side of the flow channel. External heat sources create localized high-temperature and low-temperature zones, driving the fluid to circulate directionally along the racetrack-shaped path. To further enhance the consistency of the flow direction, the embodiment adds a flow-guiding wall constraint structure to the inner wall of the cavity. This flow-guiding wall is arranged along the flow channel direction, and its height is the same as the channel depth. It can effectively suppress reverse backflow or local eddies at corners, making the circulation path more clearly defined.

[0030] The introduction of the flow guide wall is an innovative active flow management approach based on passive convection in this embodiment. It replaces the complex microvalve array with simple geometric features, significantly improving convection stability without increasing the difficulty of external control. Experimental results show that this constraint structure keeps the circulation path consistent, making it particularly suitable for nucleic acid amplification processes requiring long-term stable cycling. This helps reduce the risk of nonspecific amplification and maintains homogeneity between chambers in high-throughput or multiplex reaction scenarios.

[0031] Example 3: Parameter window verification with different feature sizes Change the characteristic dimension H of the cavity and keep the temperature difference constant. ,depth ,width Observe the convection state: Conclusion: Feature size in Stable circulation can be obtained within a certain range (e.g., 6 mm); beyond this range, circulation becomes unstable or ineffective convection cannot be formed. Simultaneously, depth verification is performed. A temperature difference of 30–60°C is considered an effective window.

[0032] This embodiment reveals the key influence of characteristic dimensions on thermal convection stability by systematically changing the characteristic dimensions of the closed-loop flow channel (1 mm, 6 mm, 15 mm) while fixing the depth, width, and temperature difference. The principle is as follows: when the characteristic dimension is too small (1 mm), the viscous resistance of the fluid dominates, and the buoyancy force is unable to overcome the damping to form a continuous circulation; when the characteristic dimension is too large (15 mm), the Rayleigh number exceeds the critical value, and the flow becomes unstable, resulting in turbulence; while in the range of 2 to 12 mm (such as 6 mm), the buoyancy force and viscous force are matched, forming a stable directional circulation in a laminar state.

[0033] This verification clarified the key design window of the chip of the present invention, enabling technicians to directly select appropriate feature sizes based on the required flow rate and cycle time, avoiding blind trial and error. Simultaneously, this window is highly compatible with the 30–60°C temperature difference required for typical nucleic acid amplification temperature ranges (denaturation 95°C, annealing / extension 55–60°C), providing a quantitative basis for standardized chip design and ensuring stable convection over a wide range of conditions.

[0034] Example 4: High thermal conductivity materials enhance heat transfer Based on Example 1, a high thermal conductivity metal material (copper foil or aluminum foil) with a thickness of 0.1 mm is embedded in the heat transfer area (local contact section) and covers a local area of ​​the outer wall of the reaction chamber (corresponding positions of the high temperature zone and the low temperature zone). The external heat source adopts magnetic induction heating: the induction coil acts on the metal layer to achieve rapid heating.

[0035] Experimental results: The heat transfer efficiency is significantly improved, the convection response time is shortened by about 30%, the cycle start-up is faster, and it is suitable for rapid nucleic acid amplification.

[0036] High thermal conductivity metal materials such as copper or aluminum foil are locally embedded in the asymmetric heat transfer region, and magnetic induction heating is used to generate eddy currents in the metal layer using a high-frequency alternating magnetic field, thereby achieving rapid heating in the high-temperature region. The high thermal conductivity material rapidly and centrally transfers heat to the fluid in the flow channel, reducing thermal hysteresis; at the same time, the low-temperature region remains relatively low, forming a steep temperature gradient at both ends of the closed-loop flow channel, which significantly enhances the buoyancy force driving convection.

[0037] Because the thin metal layer can be precisely applied to designated locations within the heat transfer area, heat is efficiently transferred without heating the entire chip, thus reducing the start-up time of the circulation process by approximately 30%. This design is particularly suitable for rapid nucleic acid amplification scenarios, effectively reducing the total reaction time. Furthermore, the magnetic induction heating method facilitates non-contact energy transfer, promotes aseptic operation under sealed chip conditions, and is easily integrated into portable detection devices.

[0038] Example 5: Multi-cavity chip with array structure Construct an array chip (4×4 matrix) containing 16 annular reaction chambers, each chamber using the same size parameters. , , The chambers are configured in parallel and share the same pair of high-temperature (95°C) and low-temperature (55°C) heat sources (using resistance heating films). Each chamber allows for independent sample injection.

[0039] Experimental results: Multiplex nucleic acid amplification (such as simultaneous detection of different target genes) can be achieved, the detection throughput is improved, the flow stability and amplification efficiency between different chambers are consistent, and the coefficient of variation is less than 5%.

[0040] This embodiment integrates 16 annular reaction chambers in a 4×4 matrix onto the same chip. Each chamber has the same feature size (6 mm), depth (0.5 mm), and a single-sided asymmetric heat transfer region. All chambers are connected in parallel and share the same pair of external heat sources (95°C for the high-temperature zone and 55°C for the low-temperature zone). The principle is that each independent chamber is a self-sustaining thermal convection unit, and the thermal conduction coupling between chambers through the solid substrate is very weak. Therefore, the chambers do not interfere with each other and can independently inject samples and operate simultaneously.

[0041] The array structure allows for the parallel detection of multiple target genes or multiple replicates of the same sample in a single experiment, significantly improving throughput and efficiency. Experimental results show that the coefficient of variation of amplification efficiency among different chambers is less than 5%, demonstrating that the design has good uniformity and scalability, meeting the needs of high-throughput, multi-indicator simultaneous analysis in clinical diagnosis or food safety testing. Furthermore, the shared heat source reduces overall power consumption and cost.

[0042] Example 6: Glass Material and Photothermal Heating Method The annular reaction chamber is prepared using glass material. , , The depth-to-feature size ratio is 0.075. The heat transfer area is asymmetrically distributed but located at the bottom of the cavity. The external heat source uses infrared light irradiation: infrared light is focused at the corresponding position in the high-temperature area, while natural heat dissipation is used in the low-temperature area, creating a temperature difference of approximately 50°C (90°C in the high-temperature area and 40°C in the low-temperature area).

[0043] Experimental results: Stable directional cycling was also formed, proving that the present invention is applicable to different material systems (glass) and different heating methods (photothermal heating).

[0044] In this embodiment, a ring-shaped reaction chamber is fabricated using glass material, with an asymmetric heat transfer region located at the bottom of the chamber. An external heat source uses focused infrared light to irradiate the corresponding location in the high-temperature zone, while the low-temperature zone maintains a lower temperature through natural heat dissipation, creating a temperature difference of approximately 50°C. The infrared light wavelength can pass through the glass and be absorbed by the fluid inside the chamber (or converted into heat through the bottom light-absorbing coating), achieving non-contact, spatially localized heating. Furthermore, the low thermal conductivity of the glass reduces the lateral diffusion of heat to the low-temperature zone, maintaining a sharp temperature gradient.

[0045] Glass material possesses excellent optical transparency, chemical inertness, and low autofluorescence, making it suitable for nucleic acid amplification applications requiring optical readout, such as real-time fluorescence detection. Combined with photoheating, the chip eliminates the need for metal electrodes or heating films, resulting in a simpler structure and avoiding electromagnetic interference that may arise from electrical heating. This embodiment demonstrates that the convection-driven principle of this invention is independent of specific materials or heat source types, providing flexible design options for different detection scenarios, such as real-time PCR requiring optical detection.

[0046] Example 7: Extreme verification of the depth-to-feature size ratio Make two chips separately: Chip A: , (Ratio 0.025) (High temperature 100℃, low temperature 40℃); Chip B: , (Ratio 0.45) (High temperature 85℃, low temperature 55℃).

[0047] Results: Both chips achieved stable circulating flow, but chip A, with its shallower flow channel, exhibited slightly slower convection velocity; chip B, with its larger depth-to-feature-size ratio, achieved faster convection velocity but did not show any instability. This indicates that the depth-to-feature-size ratio is... It can work effectively within the range, and slightly exceeding (0.45) is still acceptable, but the preferred range is [range missing]. .

[0048] Example 7 first verified the extreme cases of the depth-to-feature size ratio: chip A had a ratio of 0.025 (depth 0.3 mm, feature size 12 mm, temperature difference 60℃), and chip B had a ratio of 0.45 (depth 9 mm, feature size 2 mm, temperature difference 30℃). Both could form stable cycles, but a ratio that was too small resulted in a slow convection velocity, while a ratio that was too large resulted in a fast flow velocity. The principle is that depth dominates the balance between flow resistance and buoyancy. When the ratio is in the range of 0.02 to 0.3, a suitable convection velocity and stability can be obtained, meeting the residence time requirements of each temperature zone stage of nucleic acid amplification.

[0049] Furthermore, this embodiment demonstrates the combined use of electric and chemical heating: the high-temperature zone is powered by a resistance heating film, while the low-temperature zone is maintained by an exothermic reaction package. This hybrid heat source strategy ensures precise heating of the core temperature zone while reducing reliance on the main heat source by utilizing the continuous exothermic reaction in the low-temperature zone, which is beneficial for developing low-power, portable devices. In summary, this embodiment broadens the available parameter boundaries and heat source solutions for chips, demonstrating that the invention has good design tolerance and environmental adaptability.

[0050] Example 8: Multiple Combinations of External Heat Sources Electric heating (resistance heating film) provides a high-temperature zone (95℃), while chemical heating (exothermic reaction chamber) provides auxiliary insulation for a low-temperature zone (60℃). Copper heat-conducting blocks are used in the heat transfer area. The reaction chamber is racetrack-shaped and made of plastic. , The temperature difference is 35℃.

[0051] Result: The chip can still form a stable cycle, proving that the invention is compatible with multiple heat source combinations and is convenient for portable device design.

[0052] This embodiment employs a hybrid heat source scheme: electric heating provides the high-temperature zone, while chemical heating (exothermic reaction pack) assists in maintaining the low-temperature zone. A copper heat-conducting block is placed in the heat transfer area. The principle is as follows: the electric heating film can precisely control the temperature of the high-temperature zone, meeting the stringent requirements of the denaturation step; the low-temperature zone utilizes the exothermic chemical reaction pack to maintain a temperature of approximately 60 degrees Celsius, requiring no external power supply; the copper heat-conducting block indirectly assists in maintaining the base temperature of the low-temperature zone through heat conduction, reducing the consumption rate of the chemical heating pack. The coordinated operation of multiple heat sources stabilizes the overall temperature difference of the chip at approximately 35 degrees Celsius.

[0053] Its advantages lie in demonstrating the compatibility of this invention with non-traditional heat sources, especially the chemical heating method which requires no external power supply, facilitating the construction of fully portable, battery-powered, or even electricity-free nucleic acid amplification devices. The introduction of the heat-conducting block balances the temperature distribution among multiple heat sources, improves system robustness, and provides a practical microfluidic chip design approach for rapid molecular diagnostics in the field, primary healthcare facilities, and emergency situations.

[0054] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these modifications and improvements all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims.

Claims

1. A microfluidic reaction chip for nucleic acid amplification, characterized in that, include: A reaction chamber (1) with a closed-loop flow channel and a heat transfer area (2) disposed on the outer wall of the reaction chamber, wherein the heat transfer area (2) is asymmetrically distributed along the closed-loop flow channel; The characteristic dimension of the reaction chamber (1) along the temperature gradient direction is: The depth of the reaction chamber (1) along the temperature gradient direction is ; The temperature difference corresponding to the reaction chamber (1) is 30-60℃, so as to form a directional circulation flow in the reaction chamber (1), which is suitable for the temperature zone circulation process in nucleic acid amplification reaction.

2. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The heat transfer region (2) is distributed on one side or non-centrally symmetrically in the closed loop.

3. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The reaction chamber (1) has a ring structure or a racetrack-shaped structure.

4. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The width of the reaction chamber (1) is And the ratio of the depth to the feature size of the reaction chamber (1) is .

5. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The heat transfer zone (2) is provided with a material with high thermal conductivity.

6. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The reaction chamber (1) is provided with a flow path constraint structure, which includes a flow guide groove, a flow guide wall, or a geometric constraint structure.

7. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The microfluidic reaction chip includes multiple reaction chambers (1), and the multiple reaction chambers (1) are arranged in an array.

8. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The heat in the heat transfer zone (2) is provided by an external heat source, which is selected from electric heating, magnetic induction heating, light heating or chemical heating.

9. The microfluidic reaction chip for nucleic acid amplification according to claim 1, characterized in that: The reaction chamber (1) is made of plastic, glass or metal.

10. A method for applying a microfluidic reaction chip for nucleic acid amplification, characterized in that: Using the microfluidic reaction chip described in any one of claims 1-9, a temperature distribution is formed through the heat transfer region (2), so that the fluid forms a directional circulation flow in the reaction chamber (1) to complete the nucleic acid amplification reaction.

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