A self-driven digital microfluidic chip and its application in digital recombinase polymerase amplification

Abstract

This invention relates to the fields of microfluidic chip technology and molecular diagnostic technology, and discloses a self-driven digital microfluidic chip and its application in digital recombinase polymerase amplification. The chip comprises two layers: an upper PDMS sheet and a lower glass plate. The lower surface of the upper PDMS sheet is etched with a microfluidic channel structure, including a sample loading port, a droplet reactor array, microchannels, and a pump chamber. The upper and lower layers are thermally bonded to form a sealed flow channel. Based on the gas permeability of PDMS material, this invention achieves self-driven sample introduction and digital dispensing by pre-degassing to create negative pressure, eliminating the need for any external equipment. The chip structure of this invention is rationally designed, with a simple preparation process, low cost, and convenient operation. It can automatically complete sample digitization within 5-10 minutes, supports multi-channel parallel detection, is highly compatible with RPA reaction systems, and can achieve absolute quantitative analysis of target nucleic acids. It has advantages such as high detection sensitivity, accurate quantification, and good repeatability, and has broad application prospects in the field of molecular diagnostics.

Description

Technical Field

[0001] This invention relates to the fields of microfluidic chip technology and molecular diagnostic technology, and particularly to a self-driven digital microfluidic chip and its application in digital recombinase polymerase amplification. Background Technology

[0002] Recombinase polymerase amplification (RPA) is a novel isothermal nucleic acid amplification technique that can rapidly amplify target nucleic acid sequences at a constant temperature of 37–42°C. It offers advantages such as rapid reaction speed, no need for thermal cycling equipment, and suitability for rapid on-site detection. However, conventional RPA techniques rely on time thresholds for quantitative analysis, making accurate quantification of target nucleic acids difficult, which limits its application in precision molecular diagnostics to some extent.

[0003] Digital recombinase polymerase amplification (dRPA) technology achieves absolute quantification of target nucleic acid molecules by dispersing the RPA reaction mixture into a large number of independent microreaction units, based on the statistical principle of Poisson distribution. This technology avoids the complex thermal cycling control required in PCR-based methods and overcomes the shortcomings of conventional RPA in accurately quantifying nucleic acids, providing a new technical approach for absolute quantitative analysis of nucleic acids.

[0004] Microfluidic technology provides an ideal platform for realizing dRPA. Currently, digital nucleic acid amplification technologies based on microfluidic chips mainly include droplet-based microfluidic chips, valve-controlled microfluidic chips, positive pressure-driven microfluidic chips, and electrowetting digital microfluidic chips. However, these technologies still have many shortcomings in practical applications: droplet-based microfluidic chips usually require complex pumping equipment and long droplet readout times, and are prone to droplet aggregation; valve-controlled microfluidic chips require complex structural designs and external pumping or mechanical control systems; positive pressure-driven microfluidic chips still rely on external component control and require pre-wetting of the flow channels with an immiscible phase; while electrowetting-based digital microfluidic chips improve automation, they have limitations in achieving high-throughput digitization.

[0005] Polydimethylsiloxane (PDMS) is a polymer material with excellent gas permeability, optical transparency, and biocompatibility, and is widely used in the fabrication of microfluidic chips. Based on the properties of PDMS, researchers have developed various forms of microfluidic chips for nucleic acid amplification reactions. However, existing PDMS-based microfluidic chips, when applied to dRPA technology, still suffer from problems such as complex structure, high fabrication cost, and cumbersome operation, making it difficult to meet the requirements of portability and ease of use for rapid on-site detection.

[0006] Therefore, developing a PDMS-based microfluidic chip with a reasonable structural design, simple fabrication process, and convenient use, and applying it to digital recombinase polymerase amplification technology, is of great significance for promoting the portability, automation, and cost reduction of nucleic acid absolute quantitative detection technology. Summary of the Invention

[0007] To address the aforementioned technical problems, this invention provides a self-driven digital microfluidic chip and its application in digital recombinase polymerase amplification, aiming to simplify chip structure, reduce preparation costs, achieve self-driven sample digitization without external equipment, and provide a high-throughput analysis platform for digital recombinase polymerase amplification technology that is easy to operate, has uniform reaction units, and provides accurate quantification.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows: A self-driven digital microfluidic chip includes a two-layer structure: an upper PDMS sheet and a lower glass sheet. The lower surface of the upper PDMS sheet is etched with a microchannel structure, which includes a sample loading port, a droplet reactor array, a microchannel, and a pump chamber. One end of the microchannel is connected to the sample loading port, and the other end is connected to the pump chamber. The droplet reactor array is distributed on both sides of the microchannel. The upper PDMS sheet and the lower glass sheet are encapsulated by thermal bonding to form a sealed flow channel. The chip achieves self-driven sample injection and digital dispensing by pre-degassing to create a negative pressure.

[0009] In the above scheme, the microchannel between the droplet reactor array and the pump chamber has a tortuous structure to increase flow resistance and prolong the time for the sample to fill the micropores.

[0010] In the above scheme, the chip has an overall length of 75 mm, a width of 25 mm, and a thickness of 7 mm.

[0011] In the above scheme, the chip is provided with three independent detection areas. Each detection area includes a sample loading hole, a main channel, eight branch channels, a droplet reactor array consisting of 400 micropores, and a pump chamber.

[0012] In a further technical solution, the height of the microchannel is 30 μm, the width of the branch channel is 50 μm, and the width of the main channel is 100 μm; the diameter of the micropores in the droplet reactor array is 120 μm, the depth is 120 μm, the spacing between adjacent micropores is 80 μm, the center of the micropore is 125 μm away from the center of the microchannel and is connected to the microchannel by a 50 μm wide branch.

[0013] In the above scheme, the bends and branch points of the microchannel are designed with 1 / 4 rounded corners.

[0014] In the above scheme, the pump chamber is 15.2 mm long and 500 μm wide, and is equipped with multiple support columns with a diameter of 100 μm inside to prevent the chamber from collapsing.

[0015] Application of a self-driven digital microfluidic chip as described above in digital recombinase polymerase amplification.

[0016] The above solution includes the following steps: (1) Place the chip in an environment below 10 kPa for 20-30 minutes to degas it and create a negative pressure; (2) Add the sample solution into the sample well, and then cover it with mineral oil; (3) Transfer the chip to a thermostat amplification device and perform the RPA reaction at 39~42°C for 20~40 minutes; (4) After the reaction is complete, the fluorescence signal of the droplet reactor array is collected using a fluorescence microscope; (5) Calculate the absolute copy number of the target nucleic acid based on the number of positive and negative reaction units and the Poisson distribution.

[0017] In a further technical solution, the amount of sample solution added is 7.5~8.5 μL, Tween 20 with a volume fraction of 0.1% is added to the sample solution, and Span 80 with a volume concentration of 2% is added to the mineral oil as a surfactant.

[0018] Through the above technical solution, the self-driven digital microfluidic chip provided by the present invention and its application in digital recombinase polymerase amplification have the following beneficial effects: 1. Achieve true self-driven sample digitization without the need for external equipment. This invention leverages the permeability of PDMS material, creating a negative pressure driving force through pre-degassing. The chip requires no integrated microvalve, micropump, or electrode structure, nor does it need to be connected to any external injection pump, gas source, or control system. The sample loading process simply involves adding the sample solution and mineral oil sequentially; the chip automatically completes sample aspiration, dispensing, and droplet formation, greatly simplifying the operation and reducing reliance on specialized equipment and operators.

[0019] 2. The chip structure is rationally designed, and the reaction units have good uniformity. The chip employs a two-layer flow channel structure with different heights. The height and diameter of the micropores in the droplet reactor array are equal, ensuring that the formed droplets are nearly spherical, with low surface energy and stable states. Parameters such as the distance between the micropore center and the flow channel center, and the branch width, have been optimized to ensure both smooth filling of the micropores by the sample solution and effective cutting off of the sample flow by the mineral oil, forming closed, independent reaction units. Each detection area contains 3200 highly uniform microreaction units, providing a reliable guarantee for the accurate quantification of dRPA technology.

[0020] 3. High integration, supporting multi-channel parallel detection The chip features three independent detection areas, each containing an independent sample loading port, droplet reactor array, and pump chamber. This allows for the simultaneous detection of three different samples or targets, or as three independent parallel experiments of the same set of detection parameters. This multi-channel parallel design significantly improves detection throughput and experimental efficiency, meeting the needs of various application scenarios.

[0021] 4. The preparation process is simple and the cost is low. The chip is fabricated by thermal bonding of PDMS wafers and glass slides. The process is mature and does not require complex photolithography, etching, or precision machining. PDMS material is low in cost and suitable for mass production. The overall size of the chip is consistent with that of a conventional microscope slide, making it compatible with existing microscopes, temperature control equipment, etc., without the need for additional customized equipment.

[0022] 5. Easy to operate, fast and efficient digitalization process Before use, the chip only needs to be degassed in a low-pressure environment for 20-30 minutes. During sample loading, the sample solution and mineral oil are added sequentially; the entire process requires no complex manual control. Under optimized conditions, the sample solution can complete the filling of all micropores and mineral oil separation within 5-10 minutes, forming a uniform droplet array, significantly shortening sample digitization time and improving detection efficiency.

[0023] 6. Highly compatible, adaptable to RPA reaction systems The chip design is highly compatible with the RPA reaction system, and parameters such as channel height and micropore size fully consider the viscosity and diffusion characteristics of the RPA reagents. By adding surfactants such as Tween 20 to the reaction mixture and Span 80 to the mineral oil, the sample viscosity is effectively reduced, interfacial stability is improved, and the smooth progress of droplet formation and reaction stability are ensured. The chip can operate stably at RPA reaction temperatures of 39–42°C, meeting the amplification time requirements of 20–40 minutes.

[0024] 7. Quantitative accuracy, conforming to the statistical principle of Poisson distribution. In the droplet reactor array formed by the chip, each microwell serves as an independent reaction unit. After the reaction, the endpoint fluorescence signal can be collected using a fluorescence microscope, and the number of positive and negative reaction units can be counted. The absolute copy number concentration of the target nucleic acid molecule can then be calculated based on the Poisson distribution principle. This method does not require the establishment of a standard curve, and the quantitative results are accurate and reliable.

[0025] 8. The structure is expandable and has broad application prospects. The chip structure design of this invention has good scalability, and parameters such as the number of detection areas, the size of the microwell array, and the flow channel size can be adjusted according to actual detection needs to adapt to different detection throughput and sensitivity requirements. In addition to dRPA technology, this chip platform can also be extended to other isothermal nucleic acid amplification technologies, digital PCR technology, single-cell analysis, protein detection, and other fields, and has broad application prospects. Attached Figure Description

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the description of the embodiments or the prior art will be briefly introduced below.

[0027] Figure 1 This is a schematic diagram of a self-driven digital microfluidic chip disclosed in an embodiment of the present invention; Figure 2 This is a schematic diagram of a PDMS sheet as disclosed in an embodiment of the present invention; Figure 3 This is a schematic diagram of the microchannel structure disclosed in the embodiments of the present invention; wherein, (a) is the structure at the sample feeding port; (b) is the droplet reactor array; and (c) is the pump chamber inlet structure. Figure 4 A schematic diagram of the degassing self-driven digital chip principle; Figure 5 The images show the sample loading process, where (a) the sample enters the flow channel, (b) the sample enters the micropore, (c) the sample fills the micropore, (d) the sample is fully filled, (e) the mineral oil sealing droplet, and (f) the digital completion.

[0028] In the figure, 1. PDMS sheet; 2. Glass slide; 3. Sample loading port; 4. Droplet reactor array; 5. Microchannel; 6. Pump chamber; 7. Main channel; 8. Branch channel; 9. Support column. Detailed Implementation

[0029] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention.

[0030] I. Structural Design of Self-Driven Digital Microfluidic Chips This embodiment provides a self-driven digital microfluidic chip for digital recombinase polymerase amplification, such as... Figure 1 and Figure 2 As shown, it includes a two-layer structure: the upper layer is a PDMS sheet 1, and the lower layer is a clean glass sheet 2.

[0031] The lower surface of the upper PDMS sheet 1 is etched with a microchannel structure, which consists of two layers of different heights. The main areas include the sample inlet 3 at the front end, the droplet reactor array 4 forming independent reaction units, the microchannels 5, and the pump chamber 6 at the end. The upper PDMS sheet 1 and the lower glass sheet 2 are encapsulated by thermal bonding to form a sealed flow channel system. The microchannels 5 include main channels and branch channels.

[0032] In this embodiment, the chip has an overall length of 75 mm, a width of 25 mm, and a thickness of 7 mm, which is consistent with the size of a conventional microscope slide, so as to be compatible with the size of various instruments and equipment such as microscopes.

[0033] like Figure 2 As shown, the chip has three identical detection areas. Each detection area includes a sample loading well 3, a main channel 7, and eight branch channels 8 extending from it. Each branch channel 8 is connected in series with a droplet reactor array 4 consisting of 400 micropores and a pump chamber 6 at one end. In practical use, the three detection areas of each chip can detect three different samples or targets respectively, or they can be used as three independent parallel experiments for the same set of detection contents.

[0034] like Figure 3 As shown in (a), the microchannel 5 has a height of 30 μm, the branch channel 8 has a width of 50 μm, and the main channel 7 has a width of 100 μm. The main channel 7 is wider than the branch channel 8 to allow sufficient liquid flow. The bends and branch points of the microchannel are designed with 1 / 4 fillet to reduce flow resistance.

[0035] like Figure 3 As shown in (b), the droplet reactor array 4 is designed with a height of 120 μm, the micropore diameter on both sides of the flow channel is 120 μm, the spacing between adjacent micropores is 80 μm, the center of the micropore is 125 μm away from the center of the microchannel, and it is connected to the microchannel by a 50 μm wide branch. The height of the micropore is designed to be equal to the bottom diameter so that the shape of the droplets formed inside is as close to spherical as possible, thereby achieving a stable state with low surface energy.

[0036] like Figure 3As shown in (c), a special structure is provided at the inlet of pump chamber 6. Pump chamber 6 has an overall length of 15.2 mm and a width of 500 μm to provide a larger gas storage space. To prevent chamber collapse during chip fabrication and degassing, a series of 100 μm diameter support pillars 9 are installed inside pump chamber 6. Simultaneously, to maintain sufficient distance between adjacent pump chambers, the center-to-center spacing between adjacent flow channels is set to 700 μm. The portion of the flow channel between the droplet reactor array 4 and the inlet of pump chamber 6 is designed with a meandering structure to increase the friction of the liquid at the end of the flow channel and on the sidewalls, thereby allowing the RPA reaction solution more time to fill the micropores, ensuring sufficient and uniform micropore filling.

[0037] II. Fabrication Method of Self-Driven Digital Microfluidic Chips This embodiment provides a method for fabricating a self-driven digital microfluidic chip as described above, including the following steps: (1) Substrate cleaning and pretreatment The glass slides used were pre-cleaned in an ultrasonic cleaner with ethanol and ultrapure water for 15 minutes each, and then spun dry in a micro glass slide centrifuge and purged with nitrogen to prevent surface dust and impurities from clogging the channels or affecting the bonding effect. Special adhesive tape was used to clean the dust from the surface of the PDMS sheet channel layer.

[0038] (2) Plasma surface treatment Place the PDMS sheet with its flow channel layer and the glass slide (without markings) face up into the plasma cleaner and evacuate for 5 minutes. Open the gas source valve, introduce oxygen, and adjust the gas flow rate to maintain a grayish-white color inside the instrument chamber. Continue this treatment for 5 minutes. Plasma treatment activates the PDMS and glass surfaces, generating hydrophilic groups that facilitate subsequent bonding.

[0039] (3) Adhesion and thermal bonding The plasma-treated PDMS thin film flow channel layer is quickly bonded to the glass substrate, and gently pressed to remove air bubbles at the interface, ensuring tight contact between the two layers. The bonded chip is then placed in a 110°C constant temperature drying oven for 12 hours for thermal bonding, forming an irreversible seal between the PDMS and the glass.

[0040] (4) Chip storage After thermal bonding, the chip is placed in a clean environment and cooled to room temperature. The prepared chip can be used immediately or vacuum-sealed for storage.

[0041] III. Method for Digital Recombinase Polymerase Amplification Using Self-Driven Digital Microfluidic Chips This embodiment provides a method for digital recombinase polymerase amplification using the chip described above, including the following steps: (1) Chip degassing treatment The prepared chip was sealed with transparent tape over the sample application port 3, and then placed in a vacuum desiccator under low pressure (below 10 kPa) for 20-30 minutes. During this process, the gas in the chip channel, droplet reactor array 4, and pump chamber 6 was gradually discharged through the PDMS material, causing the internal pressure to drop below atmospheric pressure and creating a negative pressure driving force. Sealing the sample application port with transparent tape prevents air from directly entering the channel through the sample application port when the chip is removed from the low-pressure environment, thus avoiding a weakening of the negative pressure effect.

[0042] (2) Preparation of RPA reaction system Prepare the RPA reaction mixture according to the detection requirements of the target nucleic acid. The reaction system contains recombinase, single-stranded binding protein, polymerase, dNTPs, specific primers, and fluorescent probes. To facilitate the sample digitization process, add 0.1% (v / v) of Tween 20 surfactant to the reaction mixture to reduce reagent viscosity and improve interfacial stability. Add the nucleic acid sample to be tested to the reaction mixture and mix thoroughly.

[0043] (3) Sample loading After degassing, remove the chip from the vacuum environment and peel off the transparent tape on sample well 3. Using a micropipette, draw 7.5–8.5 μL of the prepared sample solution, insert the pipette tip into the bottom of sample well 3, and slowly inject the sample solution. Next, using another pipette, draw sufficient mineral oil and add it above the sample solution surface, completely covering the sample well. The mineral oil contains 2% Span 80 (by volume) as a surfactant to improve the interfacial stability between the sample solution and the immiscible phase.

[0044] (4) Sample self-driving and digitization Under the influence of negative pressure, the sample solution automatically enters the microchannel 5 from the sample inlet 3 and flows towards the pump chamber 6 along the distribution channel. As the sample solution flows through the droplet reactor array 4, it gradually fills the micropores under capillary action. Figure 4 and Figure 5 As shown, the sample loading process includes the following stages: sample entering the flow channel, sample entering the micropore, sample filling the micropore, sample filling completed, mineral oil sealing the droplets, and digitization completed. Under optimized conditions, the entire digitization process can be completed within 5-10 minutes, forming a uniform droplet array.

[0045] (5) Isothermal amplification reaction After sample addition, the chip is smoothly transferred to an isothermal amplification device, and the reaction temperature is set to 39-42℃ (preferably 41℃) for RPA reaction. The amplification time is set to 20-40 minutes (preferably 30 minutes) depending on the target nucleic acid concentration and detection sensitivity requirements. During isothermal amplification, the reaction unit in each microwell undergoes nucleic acid amplification independently.

[0046] (6) Fluorescence signal acquisition and data analysis After the reaction, the chip was placed under a fluorescence microscope to collect the endpoint fluorescence signal of the droplet reactor array 4. The number of positive reaction units (with fluorescence signal) and negative reaction units (without fluorescence signal) was counted based on the fluorescence images of the droplet array. According to the Poisson distribution principle, the average copy number of each reaction unit was calculated using the formula λ = -ln(1 - k / N), where k is the number of positive reaction units and N is the total number of reaction units. The absolute copy number concentration of the target nucleic acid molecule can be calculated by dividing the average copy number by the sample volume.

[0047] IV. The Influence of Different Surfactants on Droplet Formation This embodiment investigates the effects of different surfactants on droplet formation in a self-driven digital microfluidic chip.

[0048] Experimental group 1: 0.1% Tween 20 was added to the RPA reaction mixture, and 2% Span 80 was added to the mineral oil.

[0049] Experimental group 2: 0.1% Tween 20 was added only to the RPA reaction mixture, and Span 80 was not added to the mineral oil.

[0050] Experimental group 3: 2% Span 80 was added only to the mineral oil, and Tween 20 was not added to the RPA reaction mixture.

[0051] Experimental Group 4: No surfactants were added to either the RPA reaction mixture or the mineral oil.

[0052] The chip degassing, sample loading, and digitization processes were performed according to the above method, and the droplet formation effect was observed. The results showed that experimental group 1 exhibited the best droplet formation effect, with sufficient micropore filling, a clear oil-water interface, regular droplet shapes, and no aggregation. Experimental groups 2 and 3 showed slightly less effective droplet formation, with some micropores not fully filled or droplet shapes being irregular. Experimental group 4 showed the worst droplet formation effect, with obvious bubbles and droplet aggregation.

[0053] Therefore, the addition of Tween 20 to the RPA reaction mixture and Span 80 to the mineral oil have a synergistic effect, which can significantly improve the digitization effect of the sample solution and ensure the formation quality of the droplet reactor array.

[0054] V. The Influence of Chip Degassing Conditions on Sample Self-Driven Performance This embodiment investigates the effect of different degassing conditions on the self-driving performance of a self-driven digital microfluidic chip sample.

[0055] Different degassing pressures were set: 5 kPa, 10 kPa, 20 kPa, and atmospheric pressure control.

[0056] Set different degassing times: 10 minutes, 20 minutes, 30 minutes, and 60 minutes.

[0057] The chip degassing, sample loading, and digitization processes were performed according to the above method, and the self-driving speed and micropore filling effect of the sample were observed. The results showed that the lower the degassing pressure and the longer the degassing time, the stronger the negative pressure inside the chip, and the faster the sample's self-driving speed. Degassing for 20-30 minutes in an environment below 10 kPa yielded good self-driving performance, and the sample could complete the digitization process within 5-10 minutes. When the degassing pressure was higher than 20 kPa or the degassing time was shorter than 10 minutes, the self-driving force was insufficient, and the sample could not completely fill all the micropores. Although a degassing time exceeding 60 minutes could obtain a stronger negative pressure, the preparation efficiency decreased, and the improvement in droplet formation was limited.

[0058] Taking into account both self-driving effect and preparation efficiency, the preferred degassing condition is to place the product in an environment below 10 kPa for 20-30 minutes.

[0059] VI. Chip Testing Performance Verification This embodiment utilizes a self-driven digital microfluidic chip to detect target nucleic acid standards of different concentrations, verifying the chip's quantitative detection performance.

[0060] The target nucleic acid standard of known concentration was serially diluted 10-fold to obtain a concentration range of 10. 1 ~10 5 A series of samples were prepared in copies / μL. Chip degassing, sample loading, RPA amplification, and fluorescence signal acquisition were performed according to the above method. Three parallel chips were set up for each concentration sample (i.e., simultaneous detection using three detection areas on the chip), and each concentration was detected three times.

[0061] The results showed that the number of positive reaction units increased with increasing sample concentration, consistent with the expected Poisson distribution. In 10... 1 ~10 5 Within the concentration range of copies / μL, the detection results showed a good linear relationship with the expected concentration (R0). 2>0.99). The chip's detection sensitivity reaches 10 copies / μL, and its dynamic range covers four orders of magnitude. The results from the three parallel detection regions show good consistency, with a coefficient of variation of less than 10%, indicating that the chip has good repeatability and stability.

[0062] VII. Practical Sample Testing Applications This embodiment applies a self-driven digital microfluidic chip to the detection of target nucleic acids in actual clinical samples.

[0063] Ten clinical pharyngeal swab samples were collected, and nucleic acids were extracted from each sample. The samples were then subjected to dRPA detection using the method described above, targeting respiratory pathogen nucleic acids. Real-time quantitative PCR (qPCR) was used as a control.

[0064] The results showed that out of 10 samples, the dRPA method detected 6 positive and 4 negative results, which was completely consistent with the qPCR method. The quantitative results of the dRPA method showed a good correlation with the concentration calculated from the Ct values ​​obtained by the qPCR method. Furthermore, the dRPA method does not require the establishment of a standard curve, is simple to operate, and the detection time (including nucleic acid extraction) is controlled within 1.5 hours, significantly shorter than the 3-4 hours of the qPCR method.

[0065] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A self-driven digital microfluidic chip, characterized in that, The chip comprises a two-layer structure: an upper PDMS sheet and a lower glass sheet. The lower surface of the upper PDMS sheet is etched with a microfluidic structure, which includes a sample loading port, a droplet reactor array, a microchannel, and a pump chamber. One end of the microchannel is connected to the sample loading port, and the other end is connected to the pump chamber. Droplet reactor arrays are distributed on both sides of the microchannel. The upper PDMS sheet and the lower glass sheet are thermally bonded to form a sealed flow channel. The chip is pre-degassed to create a negative pressure, enabling self-driven sample injection and digital dispensing.

2. The self-driven digital microfluidic chip according to claim 1, characterized in that, The microchannel between the droplet reactor array and the pump chamber has a meandering structure.

3. The self-driven digital microfluidic chip according to claim 1, characterized in that, The chip has an overall length of 75 mm, a width of 25 mm, and a thickness of 7 mm.

4. The self-driven digital microfluidic chip according to claim 1, characterized in that, The chip has three independent detection areas. Each detection area includes a sample loading well, a main channel, eight branch channels, a droplet reactor array consisting of 400 micropores, and a pump chamber.

5. A self-driven digital microfluidic chip according to claim 4, characterized in that, The height of the microchannel is 30 μm, the width of the branch channel is 50 μm, and the width of the main channel is 100 μm; the diameter of the micropores in the droplet reactor array is 120 μm, the depth is 120 μm, the spacing between adjacent micropores is 80 μm, the center of the micropore is 125 μm away from the center of the microchannel and is connected to the microchannel by a 50 μm wide branch.

6. The self-driven digital microfluidic chip according to claim 1, characterized in that, The bends and branch points of the microchannel are designed with 1 / 4 rounded corners.

7. A self-driven digital microfluidic chip according to claim 1, characterized in that, The pump chamber is 15.2 mm long and 500 μm wide, and has multiple support columns with a diameter of 100 μm inside.

8. The application of a self-driven digital microfluidic chip as described in any one of claims 1-7 in digital recombinase polymerase amplification.

9. The application according to claim 8, characterized in that, Includes the following steps: (1) Place the chip in an environment below 10 kPa for 20-30 minutes to degas it and create a negative pressure; (2) Add the sample solution into the sample well, and then cover it with mineral oil; (3) Transfer the chip to a thermostat amplification device and perform the RPA reaction at 39~42°C for 20~40 minutes; (4) After the reaction is complete, the fluorescence signal of the droplet reactor array is collected using a fluorescence microscope; (5) Calculate the absolute copy number of the target nucleic acid based on the number of positive and negative reaction units and the Poisson distribution.

10. The application according to claim 9, characterized in that, The sample solution was added in an amount of 7.5~8.5 μL, and 0.1% Tween 20 was added to the sample solution by volume. The mineral oil contained 2% Span 80 by volume as a surfactant.

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