EWOD microfluidic integrated nucleic acid fluorescence PCR detection method and system

Abstract

This invention discloses a microfluidic integrated nucleic acid fluorescence PCR detection method and system based on EWOD, belonging to the field of microfluidic nucleic acid molecular diagnostic technology. This method uses the electrowetting effect on the medium to drive micro-droplets, sequentially completing nucleic acid adsorption and separation by magnetic beads, nucleic acid elution, and in-situ reconstitution of lyophilized reagents to prepare the amplification solution. The amplification solution is driven to move back and forth between a spatially separated 95℃ constant denaturation zone and a 60℃ constant annealing extension zone, completing 40 rounds of PCR thermal cycling, simultaneously acquiring fluorescence signals and analyzing the results. The two temperature zones maintain a constant temperature throughout the process, eliminating the need for overall temperature adjustment of the reaction chamber. The system includes a microfluidic reagent card integrating all functional areas, as well as a matching EWOD drive module, a controllable magnet displacement mechanism, a dual-path independent temperature control module, and a fluorescence detection module. This invention eliminates the need for mechanical pumps and valves and chamber thermal cycling, resulting in fast detection speed, low contamination risk, and low reagent consumption, making it suitable for rapid on-site nucleic acid testing and primary care diagnostic scenarios.

Description

Technical Field

[0001] This invention relates to the field of nucleic acid molecular diagnostic technology, specifically to the EWOD microfluidic integrated nucleic acid fluorescence PCR detection method and system. Background Technology

[0002] Polymerase chain reaction (PCR) is a core technology in molecular diagnostics, widely used in pathogen detection, genotyping, and tumor marker analysis. Real-time quantitative PCR (qPCR) enables qualitative or quantitative detection of nucleic acids by monitoring fluorescence signals in real time during amplification, and is currently the gold standard for clinical molecular diagnostics.

[0003] However, existing qPCR detection equipment and methods have the following main drawbacks: (1) Traditional nucleic acid extraction, purification, amplification and detection are carried out on separate equipment (such as nucleic acid extractor and PCR instrument), which requires manual transfer of samples, addition of reagents and opening of the lid. This is not only cumbersome and requires high professional skills from the operators, but also easily introduces cross-contamination and aerosol contamination, leading to false positive results.

[0004] (2) Some microfluidic PCR chips attempt to integrate extraction and amplification functions, but most rely on external mechanical pumps, syringe pumps, solenoid valves or centrifugal force to drive liquid flow. These structures not only increase the size, cost and control complexity of the instrument, but also make it difficult to achieve precise control of nanoliter / microliter level liquids, and the miniaturization advantages of microfluidic chips cannot be fully utilized.

[0005] (3) Traditional PCR thermal cycling relies on repeated heating and cooling of the entire reaction chamber or metal hot block. The heating rate is usually only 4-6℃ / s and the cooling rate is 3-5℃ / s. It takes 60-90 minutes to complete 40 cycles, which cannot meet the needs of rapid detection. At the same time, frequent large-scale heating and cooling lead to thermal fatigue of the device, affecting the service life of the instrument and the accuracy of temperature control. Summary of the Invention

[0006] The purpose of this invention is to provide an EWOD microfluidic integrated nucleic acid fluorescence PCR detection method and system to solve the problems mentioned in the background art.

[0007] To achieve the above objectives, the present invention provides the following technical solution: The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method includes the following steps: S1. Sample and Reagent Loading: Transfer the nucleic acid lysis buffer to the first loading well of the microfluidic reagent card and transfer the nucleic acid elution buffer to the second loading well; the bottom of the first and second loading wells corresponds to independent EWOD driving electrodes; S2. Nucleic Acid Adsorption and Waste Liquid Separation Using Magnetic Beads: The electrode in the magnetic bead adsorption zone is activated, and the EWOD effect drives the lysis buffer in the first sample well to move to the magnetic bead adsorption zone of the pre-coated nucleic acid extraction magnetic beads. The nucleic acid in the sample is adsorbed by the modified groups on the surface of the magnetic beads. The controllable magnet built into the reagent card is brought close to the magnetic bead adsorption zone to fix the magnetic beads to the electrode surface. The electrode in the waste liquid zone is activated, and the EWOD effect drives the residual lysis waste liquid to move directionally to the waste liquid zone for storage, thus achieving solid-liquid separation. S3, Nucleic Acid Elution: Controlling the built-in controllable magnet away from the magnetic bead adsorption area releases the magnetic bead limitation, connects the second sample loading hole electrode to the magnetic bead adsorption area electrode, and the EWOD drives the eluent to move to the magnetic bead adsorption area. The nucleic acid bound to the surface of the magnetic bead dissociates and enters the eluent, resulting in an eluent containing nucleic acid. S4. Preparation of amplification solution: The EWOD-driven elution solution containing nucleic acid moves to the dry reagent reconstitution zone electrode, dissolving the lyophilized primers, fluorescent probes and PCR buffer powder reagents pre-placed in this zone, forming a fluorescent PCR amplification solution in situ. S5. Dual Isothermal Zone Cyclic Amplification and Real-time Detection: The denaturation zone electrode is switched on, and the PCR amplification solution is moved to the denaturation zone, which maintains a constant temperature of 95°C throughout the process, and held for 10 seconds to allow the DNA double strands to unwind. The annealing extension zone electrode is then switched on, and the amplification solution is moved to the annealing extension zone, which maintains a constant temperature of 60°C throughout the process, and held for 30 seconds to complete primer annealing and extension. After each annealing extension pause, the fluorescence detection module located directly above the annealing extension zone is triggered to collect a fluorescence signal. The amplification solution oscillates between the 95°C denaturation zone and the 60°C annealing extension zone, cycling a total of 40 times. S6. Results Analysis: Integrate the fluorescence signals from 40 acquisitions, plot the fluorescence intensity-cycle number amplification curve, and determine whether the nucleic acid is positive or negative based on the threshold cycle number Ct value.

[0008] As a further aspect of the present invention: in step S2, the surface modification group of the magnetic bead is any one of hydroxyl, carboxyl or oligo dT; the controllable magnet is an electromagnet or permanent magnet combined with a linear drive mechanism, and the dual-position switching of magnetic bead adsorption and release is realized by current switching or mechanical displacement.

[0009] As a further aspect of the present invention: in step S5, the denaturation zone and the annealing extension zone are spatially separated and spaced apart by a distance of 2-5 mm. The two temperature zones maintain their own constant temperature independently throughout the process, and there is no overall heating and cooling process of the instrument cavity. The amplification solution is controlled by the timing of the EWOD electrode to move directionally between the two temperature zones to complete the thermal cycle.

[0010] As a further aspect of the present invention: in step S5, the temperature control accuracy of the denaturation region is ±0.5℃, and the temperature control accuracy of the annealing extension region is ±0.5℃; the duration of each complete PCR cycle is 40s, and the total amplification time for 40 cycles is 26-28 minutes.

[0011] As a further aspect of the present invention: In step S4, the freeze-dried reagent pre-placed in the dry reagent reconstitution zone is prepared by vacuum freeze-drying using trehalose as a protective agent, and the reagent has a shelf life of not less than 6 months at room temperature; after the eluent moves to the dry reagent reconstitution zone, an alternating voltage is applied through the EWOD electrode to drive the droplets to oscillate back and forth for 5-10 seconds to accelerate the dissolution and mixing of the dry powder reagent.

[0012] As a further aspect of the present invention: In step S2, after the lysis buffer moves to the magnetic bead adsorption zone, the droplets are driven to oscillate back and forth in the magnetic bead adsorption zone 3-5 times by EWOD electrode timing control to promote the full binding and adsorption of nucleic acid to the surface of the magnetic beads; after the waste liquid is separated, the EWOD drives the washing liquid to move to the magnetic bead adsorption zone to wash the magnetic beads, fix the magnetic beads again and discharge the washing waste liquid.

[0013] The present invention also provides an EWOD microfluidic integrated nucleic acid fluorescence PCR detection system for implementing the above detection method, including a microfluidic reagent card body, an EWOD driving module, a partitioned independent electrode group, a controllable magnet displacement mechanism, a dual-path independent temperature control module and a fluorescence detection module; The microfluidic reagent card body is composed of a bottom substrate, an intermediate insulating dielectric layer, a top hydrophobic layer and a cover plate. The body is provided with integrated and interconnected functional areas: a first sample dispensing hole, a second sample dispensing hole, a magnetic bead adsorption area, a waste liquid area, a dry reagent reconstitution area, a denaturation temperature area and an annealing extension temperature area. The partitioned independent electrode group is arranged according to functional areas, including at least a first sample feeding port electrode sheet, a second sample feeding port electrode sheet, a magnetic bead adsorption zone electrode sheet, a waste liquid zone electrode sheet, a dry reagent reconstitution zone electrode sheet, a denaturation temperature zone electrode sheet, and an annealing extension zone electrode sheet; each electrode is an independent metal film electrode, which is connected to the control circuit contacts through leads and can be driven by an independent voltage. The EWOD drive module includes a high-voltage power supply, a switch matrix, and a controller, which couples all the electrode plates. By controlling the on / off state and voltage polarity of each electrode in a timing sequence, it enables the droplet to move in a directional manner, stay at a fixed point, reciprocate bidirectionally, and oscillate to mix. The controllable magnet displacement mechanism is installed in the magnetic bead adsorption area to realize the switching between fixing and releasing the magnetic bead. The dual-path independent temperature control module independently controls the temperature of the denaturation zone and the annealing extension zone, maintaining a constant temperature of 95°C and 60°C respectively throughout the process. The fluorescence detection module is fixedly installed directly above the annealing extension zone, with its optical path aligned with the electrode surface of that temperature zone, and is used to collect fluorescence signals at the end of each annealing extension stage.

[0014] As a further aspect of the present invention: the bottom substrate of the microfluidic reagent card body is a glass substrate, a silicon substrate, or a carbon-doped polycarbonate substrate; the independent metal film electrode is an Au electrode, a Pt electrode, or an ITO transparent electrode, with an electrode thickness of 100nm-1μm and an insulation resistance between electrodes greater than 100MΩ; the driving voltage is a DC or low-frequency AC voltage with a voltage amplitude of 30-200V.

[0015] As a further aspect of the present invention: the dual-channel independent temperature control module uses a thin-film platinum resistance thermometer (Pt100) as a temperature sensor, in conjunction with a PID controller and a thin-film heating element, to achieve a temperature fluctuation of ≤0.5℃ between the two temperature zones; a heat insulation groove is provided between the denaturation temperature zone and the annealing extension temperature zone to reduce thermal crosstalk between the temperature zones.

[0016] As a further aspect of the present invention: the fluorescence detection module includes an excitation source and a photodetector; the excitation source is an LED or a laser diode, with an emission wavelength adapted to the excitation peak of the fluorescence probe; the photodetector is a photomultiplier tube (PMT) or a silicon photodiode, equipped with a filter corresponding to the emission wavelength; the microfluidic reagent card cover is provided with a transparent optical window at the corresponding fluorescence detection position.

[0017] The beneficial effects of this invention are: (1) This invention abandons the traditional PCR chamber overall heating and cooling mode and innovatively adopts the dual fixed constant temperature zone spatial thermal cycling technology. The 95℃ denaturation temperature zone and the 60℃ annealing extension temperature zone maintain a constant temperature throughout the process. The thermal cycle is completed by driving nano-level droplets to move rapidly between the two temperature zones through EWOD. The droplet movement time is less than 1s, and each cycle only takes 40s. The total amplification time for 40 cycles is shortened to 26-28 minutes, which is 1-2 times faster than the traditional qPCR detection speed of 60-90 minutes, and the energy consumption is significantly reduced.

[0018] (2) This invention integrates all steps of sample lysis, magnetic bead nucleic acid extraction, washing, elution, dry reagent reconstitution, PCR amplification and real-time fluorescence detection in a single microfluidic reagent card, truly realizing fully enclosed automated detection of "sample in, result out". Users only need to add samples and wait for the results without professional training. Moreover, the fully enclosed operation completely avoids aerosol contamination and cross-contamination.

[0019] (3) The droplets are driven entirely by the electrowetting effect on the EWOD medium. No external mechanical pumps, solenoid valves, syringe pumps or centrifuges are required. The instrument has no moving liquid circuit parts, which greatly reduces the system complexity, cost and failure risk, and improves the reliability and portability of the equipment.

[0020] (4) The reaction system can be reduced to 1-5 μL, which reduces the reagent consumption by more than 80% compared with the traditional 20-50 μL system. Key reagents such as PCR primers, probes, and enzymes are pre-placed in the chip in a lyophilized form, so users do not need to prepare the reaction system themselves. This not only reduces the number of operation steps, but also ensures good reagent stability and can be stored at room temperature for more than 6 months, thus reducing the cost of cold chain transportation and storage.

[0021] (5) By using the timing coordination of controllable magnets and EWOD drive, solid-liquid separation of nucleic acid extraction by magnetic beads is efficiently achieved, resulting in high purity and recovery rate of nucleic acid extraction. The extraction process is fully automated and requires no manual intervention. Attached Figure Description

[0022] Figure 1 This is a schematic diagram of the overall structure of the detection system of the present invention; Figure 2 for Figure 1 A schematic diagram of the chip's top cover structure; Figure 3 for Figure 1 A schematic diagram of the distribution structure of the chip sample loading apertures; Figure 4 for Figure 1 A schematic diagram of the bottom structure of a microfluidic reagent card; Figure 5 for Figure 1 A schematic diagram of the bottom structure of the chip substrate; Figure 6 This is a diagram showing the electrode layout of the microfluidic reagent card of the present invention; Figure 7 For the present invention Figure 6 A schematic diagram of the droplet path; Figure 8 , 9 10 is a schematic diagram of step S2 of the present invention: nucleic acid adsorption by magnetic beads; Figure 11 , 12 13 is a schematic diagram of step S2 of the present invention: waste liquid separation; Figure 14 , 15 16 and 17 are schematic diagrams of step S3 of the present invention: nucleic acid elution; Figure 18 , 19 This is a schematic diagram of step S4 of the present invention: preparation of amplification solution; Figure 20 , 21 22, 23, 24, and 25 are schematic diagrams of step S5 of the present invention: cyclic amplification and real-time detection in dual isothermal zones; Figure 26 , 27Figures 28, 29, 30, 31, 32, and 33 are amplification curves and result interfaces of positive samples for nucleic acid detection of eight target animal-derived components in this invention; Figure 34 This is a diagram illustrating the method steps of the present invention.

[0023] Figures 1 to 5 Markings: 1. 60℃ annealing and extending isothermal plate; 2. 95℃ denaturation isothermal plate; 3. Magnet; 4. Fluorescence detector acquisition window; 5. Positioning pin; 6. Electrode contact interface; 7. Chip top cover; 8. Chip second sample loading port; 9. Chip first sample loading port; 10. Chip fluorescence acquisition window; 11. Chip electrode array; 12. Chip electrode contact; 13. Chip 60℃ annealing and extending isothermal plate contact window; 14. Chip 95℃ denaturation isothermal plate contact window; 15. Chip magnet contact window; 16. Chip positioning hole; 17. Chip substrate.

[0024] Figure 6 , Figure 7 Marking Explanation: Area A1 is the first sample dispensing well, consisting of 2 electrodes. Area A2 is the second sample dispensing well, consisting of 3 electrodes. Area B is the magnetic bead adsorption area, consisting of 4 electrodes. Area C is the waste liquid adsorption area, consisting of 3 electrodes. Area D is the dry reagent fixation area, consisting of 2 electrodes. Area E is the 95℃ denaturation temperature area, consisting of 2 electrodes. Area F is the 60℃ annealing extension temperature area, consisting of 2 electrodes. Detailed Implementation

[0025] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0026] Example 1 Please see Figure 1-34 As shown, the present invention is an EWOD microfluidic integrated nucleic acid fluorescence PCR detection system, which includes two main parts: the main unit of the detector and the disposable microfluidic reagent card.

[0027] The main unit of the detector integrates an EWOD drive module, a dual-channel independent temperature control module, a controllable magnet displacement mechanism, a fluorescence detection module, a main control circuit, a touch screen, and a power supply module. The upper surface of the main unit has a reagent card slot, within which are a positioning pin 5, an electrode contact interface 6, a 60℃ annealing and extending constant temperature plate 1, a 95℃ denaturing constant temperature plate 2, a magnet 3, and a fluorescence detector acquisition window 4.

[0028] The microfluidic reagent card is a single-use consumable, with overall dimensions of 76mm × 26mm × 2mm. It is constructed by bonding a chip substrate 17 and a chip cover plate 7. The chip substrate 17 is a 0.5mm thick glass substrate, on which a Cr / Au electrode array 11 is fabricated by magnetron sputtering. The electrode thickness is 200nm, the electrode width is 1.5mm, and the electrode gap is 50μm. A 1μm thick Si3N4 insulating dielectric layer and a 50nm thick TeflonAF hydrophobic layer are sequentially deposited on the electrode surface. The chip cover plate 7 is made of 0.3mm thick PMMA material and is bonded to the substrate with double-sided adhesive to form a closed droplet manipulation chamber with a height of 100μm.

[0029] The microfluidic reagent card features integrated, interconnected functional areas: a first sample dispensing port 9 (lysis buffer dispensing port), a second sample dispensing port 8 (elution buffer dispensing port), a magnetic bead adsorption area, a waste liquid area, a dry reagent reconstitution area, a 95°C denaturation temperature area, and a 60°C annealing extension temperature area. Each functional area has an independent driving electrode below it, which is connected to the chip electrode contact 12 at the chip end via leads. When the reagent card is inserted into the host slot, the chip electrode contact 12 is pressed against the host's electrode contact interface 6, establishing an electrical connection.

[0030] The bottom surface of the reagent card has a chip 60℃ annealing extension isothermal contact window 13, a chip 95℃ denaturation isothermal contact window 14, and a chip magnet contact window 15, corresponding to the positions of the two isothermal plates and the magnet on the main unit, ensuring heat conduction and magnetic field effect. Chip positioning holes 16 are located at the four corners of the bottom surface of the reagent card, which cooperate with the positioning pins 5 on the main unit for precise positioning. The chip cover plate 7 has a chip fluorescence acquisition window 10 above the 60℃ annealing extension temperature zone, made of transparent optical-grade PMMA material, ensuring efficient transmission of fluorescence signals.

[0031] The dual-channel independent temperature control module uses a thin-film platinum resistance thermometer (Pt100) as the temperature sensor, combined with a PID algorithm and a polyimide thin-film heating element, to independently control the temperature of the modification zone and the annealing extension zone. The system reaches the preset temperature within 3 minutes of startup, with the modification zone stably maintained at 95℃±0.3℃ and the annealing extension zone stably maintained at 60℃±0.3℃. A 3mm gap exists between the two temperature zones, with a 0.5mm wide insulation groove filled with air for insulation, controlling thermal crosstalk between the two zones to within 1℃.

[0032] The controllable magnet displacement mechanism uses a 5mm diameter neodymium iron boron permanent magnet, which is driven by a micro linear servo motor to achieve switching between two positions: in the upper position, the magnet is 0.5mm away from the electrode surface of the magnetic bead adsorption area, and the magnetic field strength is sufficient to capture the magnetic bead; in the lower position, the magnet is 5mm away from the electrode surface, and the magnetic field strength is insufficient to adsorb the magnetic bead, so the magnetic bead can move freely with the droplet.

[0033] The fluorescence detection module uses a 470nm blue LED as the excitation source, which illuminates the droplet in the annealed extension region after passing through an excitation filter (470±20nm) and a focusing lens. The fluorescence signal generated by the droplet is received by a silicon photodiode after passing through a dichroic mirror and an emission filter (520±20nm), thus achieving quantitative detection of fluorescence intensity. The integration time for each detection is 100ms, and the detection sensitivity can reach the nM level of fluorophore concentration.

[0034] The PCR reagents pre-placed in the dry reagent reconstitution zone were prepared using a vacuum freeze-drying process: 2×PCR buffer, dNTPs, Taq DNA polymerase, upstream and downstream primers, TaqMan fluorescent probe, and trehalose protectant were mixed in a specific ratio, and 2 μL was spotted onto the electrode surface of the dry reagent reconstitution zone. After pre-freezing at -80℃ for 2 hours, the mixture was then vacuum freeze-dried for 24 hours to form stable freeze-dried reagent beads. This freeze-dried reagent can be stored at room temperature for more than 6 months without significant decrease in activity.

[0035] 1 μL of carboxyl-modified magnetic beads (1 μm in diameter) with a concentration of 10 mg / mL were pre-coated in the magnetic bead adsorption area and then fixed on the electrode surface by freeze-drying.

[0036] Example 2 The EWOD microfluidic integrated nucleic acid fluorescence PCR detection was performed using the system described in Example 1. The specific steps are as follows: Step S1: Sample and Reagent Loading. Take 200 μL of the sample to be tested, add 200 μL of nucleic acid lysis buffer (containing 4M guanidine hydrochloride, 1% Triton X-100, 10 mM Tris-HCl pH 8.0), mix thoroughly, and then add 5 μL to the first sample well 9; separately add 3 μL of nucleic acid elution buffer (10 mM Tris-HCl pH 8.0, nuclease-free water) to the second sample well 8. After loading the samples, close the sealing plug of the sample well, insert the reagent card into the main unit slot, and close the hatch.

[0037] Step S2: Nucleic acid adsorption and waste liquid separation using magnetic beads. The host starts the detection program, first controlling magnet 3 to move to a lower position (away from the magnetic bead adsorption area), and sequentially applying an 80V AC voltage (1kHz) to the electrodes according to the preset timing sequence: first sample well electrode → magnetic bead adsorption area electrode, driving the lysis buffer droplet to move to the magnetic bead adsorption area at a speed of approximately 10mm / s. After reaching the magnetic bead adsorption area, the droplet is driven to oscillate back and forth in this area 5 times by alternating current through adjacent electrodes, promoting the full binding and adsorption of nucleic acids in the sample with carboxyl groups on the surface of the magnetic beads. The oscillation frequency is 2Hz. Figure 8 , 9 10) After adsorption is complete, magnet 3 is moved to the upper position (closer to the magnetic bead adsorption area), and 2 seconds are waited for the magnetic beads to be completely captured and fixed on the electrode surface by the magnetic field. Then, the electrodes in the magnetic bead adsorption area and the waste liquid area are sequentially switched on, driving the residual pyrolysis waste liquid without magnetic beads to move to the waste liquid area for storage. The waste liquid area is a closed, large-volume chamber that can hold more than 10 μL of waste liquid. Figure 11 , Figure 12 , Figure 13 ) Step S3: Nucleic acid elution. Control magnet 3 to move to the lower position to release the magnetic bead constraint, sequentially connecting the second sample well electrode to the magnetic bead adsorption zone electrode, driving 3 μL of eluent to move to the magnetic bead adsorption zone. The eluent oscillates back and forth 10 times in the magnetic bead adsorption zone, allowing the nucleic acid bound to the magnetic bead surface to fully dissociate and enter the eluent under low-salt conditions, yielding purified nucleic acid eluent. Figure 14 , Figure 15 , Figure 16 , Figure 17 ) Step S4: Amplification buffer preparation. Sequentially connect the magnetic bead adsorption electrode to the dry reagent reconstitution electrode, driving 3 μL of elution buffer containing nucleic acid to move to the dry reagent reconstitution zone. Upon arrival, use electrode timing control to drive the droplet to rapidly oscillate back and forth in this zone for 10 seconds, ensuring complete dissolution of the pre-lyophilized PCR reagent and mixing with the nucleic acid elution buffer, forming approximately 3 μL of fluorescent PCR amplification buffer in situ. Figure 18 , Figure 19 ) Step S5: Dual-isothermal zone cyclic amplification and real-time detection. Following the preset timing sequence, the electrodes are switched on, driving the PCR amplification solution to the 95°C denaturation zone, where it is precisely held for 10 seconds to ensure complete denaturation and unwinding of the DNA double strands. Then, the electrode conduction state is switched, driving the droplet to the 60°C annealing and extension zone rapidly. The droplet's movement time between the two temperature zones is approximately 0.5 seconds. The droplet is precisely held at 60°C for 30 seconds to complete the primer annealing and DNA strand extension reactions. Figure 20 , Figure 21 ) During the last 100ms of the 30s annealing extension period, the fluorescence detection module is triggered to acquire a fluorescence signal intensity. After signal acquisition, the droplet is immediately driven back to the 95℃ denaturation temperature zone to begin the next cycle. This process is repeated for a total of 40 cycles, with a total amplification time of approximately 27 minutes. Figure 22 , Figure 23 , Figure 24 , Figure 25 ) Step S6: Result Analysis. The main control system automatically records fluorescence intensity data for 40 cycles. After background subtraction and smoothing, an amplification curve showing fluorescence intensity changing with the number of cycles is plotted. A threshold line is automatically calculated, and the result is determined based on the cycle number Ct value corresponding to the fluorescence signal exceeding the threshold line: Ct value ≤ 38 is considered positive, Ct value > 40 or no amplification curve is considered negative, and 38 < Ct value ≤ 40 is considered suspicious and requires retesting.

[0038] This embodiment tested samples from eight rare animal species: saiga antelope, sika deer, red deer, red-spotted newt, pangolin, white rhinoceros, musk deer, and black bear. The results showed that the Ct values ​​were all less than 35. Figures 26 to 33 This demonstrates that the system can stably detect nucleic acid samples as low as 100 copies / mL, with a consistency rate of over 98% with traditional qPCR instruments. The detection time has been reduced from the traditional 80 minutes to less than 30 minutes, and the entire process requires no manual intervention or opening of the lid, effectively avoiding the risk of aerosol contamination.

[0039] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method, characterized in that, Includes the following steps: S1. Sample and Reagent Loading: Transfer the nucleic acid lysis buffer to the first loading well of the microfluidic reagent card and transfer the nucleic acid elution buffer to the second loading well; the bottom of the first and second loading wells corresponds to independent EWOD driving electrodes; S2. Nucleic Acid Adsorption and Waste Liquid Separation Using Magnetic Beads: The electrode in the magnetic bead adsorption zone is activated, and the EWOD effect drives the lysis buffer in the first sample well to move to the magnetic bead adsorption zone pre-coated with nucleic acid extraction magnetic beads. Nucleic acids in the sample are adsorbed by the surface-modified groups of the magnetic beads. A controllable magnet built into the reagent card is brought close to the magnetic bead adsorption zone, fixing the magnetic beads to the electrode surface. The electrode in the waste liquid zone is activated, and the EWOD effect drives the residual lysis waste liquid to move directionally to the waste liquid zone for storage, achieving solid-liquid separation. S3, Nucleic Acid Elution: Controlling the built-in controllable magnet away from the magnetic bead adsorption area releases the magnetic bead limitation, connects the second sample loading hole electrode to the magnetic bead adsorption area electrode, and the EWOD drives the eluent to move to the magnetic bead adsorption area. The nucleic acid bound to the surface of the magnetic bead dissociates and enters the eluent, resulting in an eluent containing nucleic acid. S4. Preparation of amplification solution: The EWOD-driven elution solution containing nucleic acid moves to the dry reagent reconstitution zone electrode, dissolving the lyophilized primers, fluorescent probes and PCR buffer powder reagents pre-placed in the dry reagent reconstitution zone, forming a fluorescent PCR amplification solution in situ. S5. Dual Isothermal Zone Cyclic Amplification and Real-time Detection: The denaturation zone electrode is switched on, and the PCR amplification solution is moved to the denaturation zone, which maintains a constant temperature of 95°C throughout the process, and held for 10 seconds to allow the DNA double strands to unwind. The annealing extension zone electrode is then switched on, and the amplification solution is moved to the annealing extension zone, which maintains a constant temperature of 60°C throughout the process, and held for 30 seconds to complete primer annealing and extension. After each annealing extension pause, the fluorescence detection module located directly above the annealing extension zone is triggered to collect a fluorescence signal. The amplification solution oscillates between the 95°C denaturation zone and the 60°C annealing extension zone, cycling a total of 40 times. S6. Results Analysis: Integrate the fluorescence signals from 40 acquisitions, plot the fluorescence intensity-cycle number amplification curve, and determine whether the nucleic acid is positive or negative based on the threshold cycle number Ct value.

2. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method according to claim 1, characterized in that, In step S2, the surface modification groups of the magnetic beads are one or more of hydroxyl, carboxyl, or oligomeric dT; the controllable magnet is an electromagnet or permanent magnet combined with a linear drive mechanism, which realizes the dual-position switching of magnetic bead adsorption and release through current switching or mechanical displacement.

3. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method according to claim 1, characterized in that, In step S5, the denaturation region and the annealing extension region are spatially separated and spaced apart by a distance of 2-5 mm. The two temperature zones maintain their own constant temperatures independently throughout the process. The amplification solution is directionally moved between the two temperature zones by the timing control of the EWOD electrode to complete the thermal cycle.

4. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method according to claim 1, characterized in that, In step S5, the temperature control accuracy of the denaturation region is ±0.5℃, and the temperature control accuracy of the annealing extension region is ±0.5℃; the duration of each complete PCR cycle is 40s, and the total amplification time for 40 cycles is 26-28 minutes.

5. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method according to claim 1, characterized in that, In step S4, the freeze-dried reagent pre-placed in the dry reagent reconstitution zone is prepared by vacuum freeze-drying using trehalose as a protective agent. The reagent is stored stably at room temperature for more than 6 months. After the eluent is moved to the dry reagent reconstitution zone, an alternating voltage is applied through the EWOD electrode to drive the droplets to oscillate back and forth for 5-10 seconds to accelerate the dissolution and mixing of the dry powder reagent.

6. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection method according to claim 1, characterized in that, In step S2, after the lysis buffer moves to the magnetic bead adsorption zone, the droplets are driven to oscillate back and forth in the magnetic bead adsorption zone 3-5 times by EWOD electrode timing control to promote the full binding and adsorption of nucleic acid to the surface of magnetic beads. After the waste liquid is separated, the EWOD drives the washing liquid to move to the magnetic bead adsorption zone to wash the magnetic beads, fix the magnetic beads again and discharge the washing waste liquid.

7. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection system, used to implement the detection method according to any one of claims 1-6, characterized in that, It includes a microfluidic reagent card body, an EWOD driving module, a zoned independent electrode group, a controllable magnet displacement mechanism, a dual-channel independent temperature control module, and a fluorescence detection module; The microfluidic reagent card body is composed of a bottom substrate, an intermediate insulating dielectric layer, a top hydrophobic layer and a cover plate. The body is provided with integrated and interconnected functional areas: a first sample dispensing hole, a second sample dispensing hole, a magnetic bead adsorption area, a waste liquid area, a dry reagent reconstitution area, a denaturation temperature area and an annealing extension temperature area. The partitioned independent electrode group is arranged according to functional areas, including at least a first sample loading port electrode sheet, a second sample loading port electrode sheet, a magnetic bead adsorption zone electrode sheet, a waste liquid zone electrode sheet, a dry reagent reconstitution zone electrode sheet, a denaturation temperature zone electrode sheet, and an annealing extension temperature zone electrode sheet; each electrode is an independent metal film electrode, which is connected to the control circuit contacts through leads and can be driven by an independent voltage. The EWOD drive module includes a high-voltage power supply, a switch matrix, and a controller, which couples all the electrode plates. By controlling the on / off state and voltage polarity of each electrode in a timing sequence, it enables the droplet to move in a directional manner, stay at a fixed point, reciprocate bidirectionally, and oscillate to mix. The controllable magnet displacement mechanism is installed in the magnetic bead adsorption area to realize the switching between fixing and releasing the magnetic bead; The dual-path independent temperature control module independently controls the temperature of the denaturation zone and the annealing extension zone, maintaining a constant temperature of 95°C and 60°C respectively throughout the process. The fluorescence detection module is fixedly installed directly above the annealing extension zone, and the optical path of the fluorescence detection module is aligned with the electrode surface of the annealing extension temperature zone to collect fluorescence signals at the end of each annealing extension stage.

8. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection system according to claim 7, characterized in that, The bottom substrate of the microfluidic reagent card body is a glass substrate, a silicon substrate, or a carbon-doped polycarbonate substrate; the independent metal film electrode is an Au electrode, a Pt electrode, or an ITO transparent electrode with an electrode thickness of 100nm-1μm and an insulation resistance greater than 100MΩ between electrodes; the driving voltage is a DC or low-frequency AC voltage with a voltage amplitude of 30-200V.

9. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection system according to claim 7, characterized in that, The dual-channel independent temperature control module uses a thin-film platinum resistance thermometer (Pt100) as a temperature sensor, along with a PID controller and a thin-film heating element, to achieve a temperature fluctuation of ≤0.5℃ between the two temperature zones. A heat insulation groove is set between the denaturation temperature zone and the annealing extension temperature zone to reduce thermal crosstalk between the temperature zones.

10. The EWOD microfluidic integrated nucleic acid fluorescence PCR detection system according to claim 7, characterized in that, The fluorescence detection module includes an excitation source and a photodetector; the excitation source is an LED or a laser diode, with an emission wavelength adapted to the excitation peak of the fluorescence probe; the photodetector is a photomultiplier tube (PMT) or a silicon photodiode, equipped with a filter corresponding to the emission wavelength; the microfluidic reagent card cover has a transparent optical window at the corresponding fluorescence detection position.

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