Integrated nucleic acid detection method and device based on micro-fluidic chip

Abstract

The invention relates to the technical field of medical diagnosis, and discloses an integrated nucleic acid detection method and device based on a micro-fluidic chip, the integrated nucleic acid detection device comprises a micro-fluidic chip module, a liquid control module, a temperature control module, an optical detection module and a data processing module, and the multiple modules work cooperatively to realize full-process automatic detection of a sample from splitting decomposition to detection; the micro-fluidic chip module comprises a sample treatment area, a nucleic acid extraction and purification area, a nucleic acid amplification area, a nucleic acid detection area and a waste liquid collection area which are connected through a micro-channel, and a valve is arranged at a key node. Through a fluid control mode combining centrifugal force and air pressure driving and valve combination of non-complex micro-pump micro-valves such as the dissolvable membrane valve, the temperature sensitive valve and the capillary force flow limiting valve, full-flow fluid sequential control can be achieved without integrating a plurality of micro-pump micro-valves; the problems of complex structure, high manufacturing difficulty and high cost caused by integration of multiple micro-pumps and micro-valves of the conventional pressure-driven micro-fluidic chip are solved.

Description

Technical Field

[0001] This invention relates to the field of medical diagnostic technology, specifically to an integrated nucleic acid detection method and device based on microfluidic chips. Background Technology

[0002] As a key tool in fields such as biomedical diagnostics, disease screening, pathogen detection, and life science research, nucleic acid testing technology has placed increasingly stringent demands on the efficiency, accuracy, automation, and portability of testing.

[0003] Among them, the integrated nucleic acid detection method and device based on microfluidic chip is dedicated to completing complex steps such as sample lysis, nucleic acid extraction and purification, amplification reaction and signal detection on a single chip, aiming to achieve fully automated, rapid and low-cost on-site diagnostic applications, thereby greatly expanding the application scenarios and accessibility of nucleic acid detection.

[0004] Research revealed that most pressure-driven microfluidic chips currently available typically control fluid flow sequentially by integrating multiple micropumps and microvalves. This not only leads to a complex chip structure, significantly increasing manufacturing difficulty and cost, but also makes the multiple micropumps and microvalves highly susceptible to failure when working together. This results in a substantial decrease in the stability and accuracy of fluid control, especially during sample and reagent mixing, where insufficient fluid control directly affects mixing uniformity, thereby severely impairing the efficiency of nucleic acid extraction and amplification, as well as the accuracy and repeatability of detection results. Furthermore, while centrifugation-driven microfluidic chips have a relatively simple assembly structure, the inconvenience of simultaneously and precisely controlling mechanical valves during centrifugation limits the selection of valves available for fluid control. This further complicates achieving full automation in end-to-end nucleic acid detection applications; for example, the mixing of samples and lysis buffer still requires external manual intervention. Summary of the Invention

[0005] (a) Technical problems to be solved To address the shortcomings of existing technologies, this invention provides an integrated nucleic acid detection method and device based on microfluidic chips, which solves the problem of "low working efficiency" mentioned above.

[0006] (II) Technical Solution To achieve the above objectives, the present invention is implemented through the following technical solution: an integrated nucleic acid detection device based on a microfluidic chip, comprising a microfluidic chip module, a liquid manipulation module, a temperature control module, an optical detection module, and a data processing module, wherein multiple modules work together to achieve fully automated detection of samples from lysis to detection; The microfluidic chip module includes a sample processing area, a nucleic acid extraction and purification area, a nucleic acid amplification area, a nucleic acid detection area, and a waste liquid collection area connected by microchannels, and valves are set at key nodes; The liquid manipulation module is used to drive the fluid flow and mix the reagents within the microfluidic chip module. The temperature control module is used to achieve the temperature cycle required for nucleic acid amplification; The optical detection module is used to excite and collect fluorescence signals in the nucleic acid detection area; The data processing module is used to receive signals collected by the optical detection module and perform data analysis, result interpretation, and human-computer interaction.

[0007] Preferably, the main material of the microfluidic chip module is polymethyl methacrylate, and a predetermined microchannel structure is formed by micromachining technology and laser etching technology. The size of the microfluidic chip module is set to a standardized cartridge form for easy single use.

[0008] Preferably, the valve includes at least one of a soluble membrane valve, a temperature-sensitive valve, a pneumatically driven valve, and a capillary flow restrictor valve.

[0009] Preferably, the liquid control module achieves fluid control by combining centrifugal force and precise air pressure drive, and includes a high-precision rotary motor, a micro air pressure pump and a solenoid valve. The air pressure pump can provide a pressure range of 0.05MPa-0.2MPa with a response time of less than 100ms, and the opening and closing time of the solenoid valve is less than 10ms.

[0010] Preferably, the liquid control module also integrates an ultrasonic generator, the frequency of which can be set to 40kHz to 60kHz and the power is adjustable.

[0011] Preferably, the temperature control module includes an array of multiple semiconductor coolers, a high-precision thermistor, and a proportional-integral-derivative controller. The semiconductor cooler array is attached to the bottom of the amplification chamber of the microfluidic chip module. The temperature control module can achieve a heating rate of 5℃ / s-10℃ / s and a cooling rate of 10℃ / s, switching the amplification chamber temperature between 25℃ and 95℃.

[0012] Preferably, the optical detection module includes a high-brightness light-emitting diode, a filter, an optical fiber, a lens group, and a high-sensitivity photomultiplier tube or complementary metal-oxide-semiconductor sensor. The emission spectrum center of the high-brightness light-emitting diode is located between 480nm and 520nm. The lens group is used to focus the fluorescence signal. The minimum detection area can reach 0.5mm², and the background noise is less than 10 photons / second.

[0013] Preferably, an integrated nucleic acid detection method based on a microfluidic chip is characterized by comprising the following steps: S1, Sample lysis buffer loading and mixing: The sample to be tested is added to the microfluidic chip module and mixed with the lysis buffer to release nucleic acids; S2, Nucleic acid extraction solution loading and nucleic acid capture washing: The nucleic acid-containing mixture is brought into contact with the nucleic acid adsorption medium to capture nucleic acid and remove impurities by washing; S3, Nucleic acid elution loading and elution: The elution buffer is brought into contact with the medium on which nucleic acid is adsorbed, releasing the nucleic acid from the medium and transferring it to the amplification chamber; S4, Amplification reagent loading and nucleic acid amplification reaction: Mix the amplification reagent with nucleic acid and achieve nucleic acid amplification through temperature cycling; S5, Fluorescent detection solution loading and nucleic acid fluorescence detection: Excitation, acquisition and analysis of fluorescence signals of amplified products to obtain detection results; S6, Waste Liquid Collection and Treatment: Collect all waste liquid generated during the testing process and treat it safely.

[0014] Preferably, in step S1, the microfluidic chip module is driven by the liquid control module to perform a preset oscillation or rotation program to fully mix the sample and the lysis buffer. The oscillation or rotation program is to rotate alternately in both directions at a speed of 800-1200 rpm for 20 ms in each direction, and repeat three times. The mixing of the sample and the lysis buffer is carried out at room temperature for 5 min to 15 min.

[0015] Preferably, in step S4, the temperature cycling program is as follows: pre-denaturation at 95°C for 2 minutes; followed by 40 cycles, each cycle including: denaturation at 95°C for 15 ms, annealing at 60°C and extension for 30 ms; and finally extension at 72°C for 5 minutes, with the deviation between the actual temperature and the target temperature controlled within ±0.1°C.

[0016] (III) Beneficial Effects This invention provides an integrated nucleic acid detection method and device based on a microfluidic chip. It has the following beneficial effects: (1) This invention uses a fluid control method that combines centrifugal force and air pressure drive, and combines a valve combination of non-complex micro-pumps and micro-valves such as soluble membrane valves, temperature-sensitive valves, and capillary flow limiting valves. It can achieve sequential control of the fluid throughout the entire process without integrating multiple micro-pumps and micro-valves. This solves the problems of complex structure, high manufacturing difficulty and high cost caused by the integration of multiple micro-pumps and micro-valves in existing pressure-driven microfluidic chips. At the same time, it reduces the risk of failure caused by the collaboration of multiple components and improves the overall reliability and economy of the device.

[0017] (2) Based on centrifugal-driven fluid flow, this invention introduces a micro air pressure pump, a solenoid valve and a micro heating element. The soluble membrane valve can be ruptured by air pressure pulse control and the temperature-sensitive valve can be melted by micro heating control, so as to realize the step-by-step automatic release of pre-loaded reagents. At the same time, the liquid control module can execute the centrifugal rotation and oscillation program simultaneously. The fluid control of the entire process of sample lysis, nucleic acid extraction and purification, amplification and detection can be completed without external manual intervention. This completely solves the problem of the difficulty in realizing full automation of centrifugal-driven chips and meets the application requirements of on-site diagnosis for unmanned intervention. Attached Figure Description

[0018] Figure 1 This is a schematic diagram of the modular architecture of the device of the present invention; Figure 2 This is a schematic diagram of the method flow of the present invention. Detailed Implementation

[0019] 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.

[0020] Please see Figure 1 - Figure 2 This invention provides an integrated nucleic acid detection device based on a microfluidic chip, comprising a microfluidic chip module, a liquid manipulation module, a temperature control module, an optical detection module, and a data processing module. These modules work collaboratively to achieve fully automated, high-precision fluid control, and efficient, contamination-free detection throughout the entire sample lysis and detection process. Specifically: The microfluidic chip module is the core detection unit of this device, comprising a sample processing area, a nucleic acid extraction and purification area, a nucleic acid amplification area, a nucleic acid detection area, and a waste collection area. The microfluidic chip module connects these functional areas via microchannels and incorporates various types of valves at key nodes, such as soluble membrane valves, temperature-sensitive valves, pneumatically driven valves, and capillary flow-limiting valves, to achieve precise liquid pathway control and stepwise reagent release. The main material of the microfluidic chip module is polymethyl methacrylate (PMMA), with predetermined microchannel structures formed using micromachining and laser etching techniques. The microfluidic chip module is sized in a standardized, single-use cartridge format, ensuring single-use and preventing cross-contamination. The sample inlet is located on the outer edge of the microfluidic chip module for initial loading of samples and some pre-loaded reagents. The lysis chamber and extraction and purification chamber are connected via microchannels, with the extraction and purification chamber's internal surface fixed with a nucleic acid adsorption medium. The amplification chamber is designed as a microcavity with excellent thermal conductivity to work closely with the temperature control module for rapid and accurate temperature cycling. The detection chamber is an optically transparent area used to receive the excitation light emitted by the optical detection module and collect the target fluorescence signal. The waste liquid collection chamber is designed to be large enough to completely contain all the waste liquid generated during the detection process, and a one-way valve prevents backflow or leakage of the waste liquid.

[0021] The liquid manipulation module is responsible for driving fluid flow and reagent mixing within the microfluidic chip module, employing a combination of centrifugal force and precise pneumatic pressure actuation to achieve fluid control. The module includes a high-precision rotary motor to provide controllable centrifugal force, driving liquid flow along radial channels within the microfluidic chip module. It also includes a micro-pneumatic pump and solenoid valves to control the pneumatic pressure applied to specific areas of the microfluidic chip module, thereby assisting in liquid transfer, valve opening, or liquid mixing. The pneumatic pump provides a pressure range of 0.05MPa-0.2MPa with a response time of less than 100ms, and the solenoid valves have an opening and closing time of less than 10ms, ensuring instantaneous and precise fluid control. For example, changing the centrifugal speed controls the liquid to cross the threshold of a capillary valve, or pneumatic pressure drives the rupture of a soluble membrane valve or the melting of a temperature-sensitive valve. Furthermore, the liquid manipulation module integrates an ultrasonic generator to provide high-frequency vibrations in specific steps, such as sample lysis or magnetic bead mixing, to enhance reagent mixing efficiency and ensure reaction homogeneity. The ultrasonic frequency can be set from 40kHz to 60kHz, and the power is adjustable to avoid damage to nucleic acid or enzyme activity.

[0022] The temperature control module primarily enables rapid and accurate temperature cycling. Specifically, it consists of an array of multiple semiconductor coolers attached to the bottom of the amplification chamber of the microfluidic chip module. A high-precision thermistor monitors the amplification chamber temperature in real time, and a proportional-integral-derivative (PID) controller provides feedback control. The temperature control module can achieve heating and cooling rates of 5℃ / s-10℃ / s, rapidly switching the amplification chamber temperature between 25℃ and 95℃ to meet the temperature requirements of polymerase chain reaction (PCR) cycles for denaturation, annealing, and extension.

[0023] The optical detection module is used to excite and acquire fluorescence signals in the detection chamber. It includes a high-brightness light-emitting diode (LED) as the excitation source, with an emission spectrum centered between 480nm and 520nm, matching the excitation spectra of commonly used fluorescent dyes. The excitation light, after passing through a filter, is coupled through an optical fiber into the detection chamber of the microfluidic chip module, exciting the fluorescent probes bound to the amplified products. A high-sensitivity photomultiplier tube (PMT) or complementary metal-oxide-semiconductor (CMOS) sensor is positioned above the detection chamber to acquire the fluorescence signal. The acquired fluorescence signal passes through another set of filters before entering the PMT for amplification and conversion into an electrical signal. The optical detection module also includes a lens group for focusing the fluorescence signal, achieving a minimum detection area of ​​0.5mm², thereby improving detection sensitivity. Background noise is designed to be less than 10 photons / second, ensuring reliable detection of low-concentration samples.

[0024] The data processing module is responsible for receiving the raw signals acquired by the optical detection module, performing data analysis, result interpretation, and human-computer interaction. Specifically, it includes a high-performance microprocessor running an embedded operating system, integrating signal filtering algorithms, baseline correction algorithms, threshold setting algorithms, and quantitative analysis algorithms. The data processing module is also equipped with a memory unit for storing preset detection protocols, calibration data, and historical detection results, and has external interfaces such as a universal serial bus and an Ethernet interface for data transmission and software upgrades. The detection results are presented on the display screen in an intuitive graphical or numerical form and can be automatically interpreted as positive, negative, or invalid according to preset standards. Real-time graphs and raw data are also provided for user review.

[0025] Based on the above, the present invention also provides an integrated nucleic acid detection method based on a microfluidic chip, comprising the following steps: S1, Sample lysis buffer loading and mixing, mainly involves lysing the cells or virus particles in the sample to be tested, releasing the internal nucleic acid. The microfluidic chip module has a pre-set sample inlet and a lysis buffer reservoir. The lysis buffer reservoir is pre-filled with chemical lysis buffer, and specifically includes the following steps: Step 1: Add the sample to be tested, such as 100 microliters of swab washing solution or plasma, into the sample chamber of the microfluidic chip module through the sample inlet. The sample chamber is designed to accommodate a microcavity of a specified volume of sample, and a one-way valve is installed at the inlet to prevent backflow, ensuring that the sample can only enter along the predetermined channel.

[0026] Step Two: Activate the liquid manipulation module to drive the sample into the pre-set lysis chamber using centrifugal force. Simultaneously, a pneumatic control mechanism precisely controls the rupture of the soluble membrane valve between the lysis buffer reservoir and the lysis chamber, or a micro-heating element melts the temperature-sensitive valve, thereby mixing the pre-filled lysis buffer with the sample within the lysis chamber. The rupture of the soluble membrane valve is typically achieved by applying a specific pneumatic pulse.

[0027] Step 3: To ensure thorough mixing of the sample and lysis buffer, the liquid manipulation module drives the microfluidic chip module to perform a preset oscillation or rotation program, such as alternating forward and reverse rotation at 800-1200 rpm for 20 ms in each direction, repeated three times to achieve effective convective mixing. Mixing efficiency can also be enhanced by incorporating microstructures within the lysis chamber. During this mixing process, surfactants in the lysis buffer disrupt cell membranes or viral envelopes, and proteases degrade proteins, releasing intact nucleic acid molecules. This process typically lasts 5-15 minutes at room temperature to ensure complete lysis. Mixing efficiency is a key parameter in this step, affecting the yield of subsequent nucleic acid extraction. The effective diffusion coefficient during mixing can be approximated by the following formula: in, It is the effective diffusion coefficient. It is the static diffusion coefficient. It is a mixing factor related to the geometry and fluid properties of the microfluidic chip. Rotational angular velocity, It is the feature blending length. Through optimization and This maximizes mixing efficiency and ensures complete lysis. Finally, the lysed mixture is driven by centrifugal force and flows through a preset microchannel to the nucleic acid extraction and purification zone. During this process, a capillary flow-limiting valve prevents it from flowing prematurely before reaching the predetermined area.

[0028] S2, Nucleic Acid Extraction Buffer Loading and Nucleic Acid Capture Washing, primarily separates nucleic acids from complex cell debris and proteins in the lysis buffer and adsorbs them onto a solid-phase support, while removing impurities through washing. The nucleic acid extraction and purification zone includes one or more chambers, each containing a fixed nucleic acid adsorption medium, such as a silica gel membrane or magnetic beads, and specifically includes the following steps: Step 1: The lysed mixture is centrifuged and enters the nucleic acid extraction and purification chamber through a specific microchannel, where it comes into full contact with the nucleic acid adsorption medium. The centrifugal force is controlled, for example, at 2000 rpm for 2 minutes, to ensure that all liquid passes through the adsorption medium. The nucleic acid adsorption medium specifically binds to nucleic acid molecules, while other impurities such as proteins and lipids do not bind or bind weakly.

[0029] Step 2: After nucleic acid adsorption is complete, activate the liquid control module to dissipate the pre-filled washing solution in an independent reservoir via pneumatic or centrifugal force. The washing solution reservoir is isolated from the extraction and purification chamber by a soluble membrane valve or a temperature-sensitive valve. The washing process is typically repeated 1–3 times, with 100–150 μL of washing solution each time. After each wash, centrifugal force drives the waste liquid through a one-way valve into the waste liquid collection chamber. The purpose of washing is to remove non-specifically bound impurities and residual lysis buffer components to the greatest extent possible, ensuring the purity of subsequent amplification reactions.

[0030] Step 3: After washing, to thoroughly remove residual washing liquid, the liquid control module applies a short high-speed centrifugation step, such as 4000 rpm for 1 minute, to completely remove all liquid from the adsorption medium into the waste liquid collection chamber, followed by brief air drying to ensure no residue of substances inhibiting the amplification reaction remains. Nucleic acid capture efficiency is a key performance indicator in this step, influenced by factors such as the properties of the adsorption medium, nucleic acid concentration, and ionic strength. Finally, the washed and purified nucleic acid adsorption medium is ready for the next elution step.

[0031] S3, Nucleic acid elution loading and elution: This process efficiently releases the purified nucleic acid from the adsorption medium and transfers it to the solution required for the amplification reaction. The elution reservoir contains elution buffer, such as nuclease-free deionized water or low-salt buffer. The specific steps include: Step 1: Activate the liquid control module to open the valve of the eluent reservoir, allowing the pre-filled eluent to be driven by centrifugal force or air pressure into the nucleic acid extraction and purification chamber, where it comes into full contact with the medium adsorbed with nucleic acids. The eluent typically has low ionic strength or high alkalinity, which can disrupt the binding force between nucleic acids and the adsorption medium, promoting the dissociation of nucleic acids from the medium surface.

[0032] Step 2: In order to improve the elution efficiency of nucleic acid, the liquid control module drives the microfluidic chip module to perform a short oscillation or rotation program again, or the eluent is briefly heated to 55℃-65℃ by the micro heating element for 1min to 3min to enhance the dissolution and release of nucleic acid.

[0033] Step 3: After the nucleic acids are fully eluted, the eluent containing the purified nucleic acids is precisely guided into the amplification chamber through a microchannel under centrifugal force. During the guiding process, the microchannel is designed with structures to prevent evaporation and contamination, such as narrow capillary channels or hydrophobic surface treatment areas, to ensure the integrity and purity of the purified nucleic acids. The volume tolerance of the eluent reservoir is controlled within ±2%.

[0034] S4, Amplification reagent loading and nucleic acid amplification reaction, mainly involves the efficient amplification of the trace amounts of nucleic acid obtained from elution to achieve detectable abundance. The amplification reagent reservoir is pre-loaded with amplification reagents, such as polymerase, primers, probes, deoxyribonucleoside triphosphates, and buffer solutions, and specifically includes the following steps: Step 1: Activate the liquid control module and open the valve of the amplification reagent reservoir. This allows the pre-filled amplification reagent to be driven by centrifugal force or air pressure into the amplification chamber, where it mixes with the purified nucleic acid that has been transferred there. Reagent mixing is a prerequisite for successful amplification; therefore, the amplification chamber is also designed with a micro-mixing structure or uses brief fluid oscillations to ensure uniform mixing of reagents and nucleic acids. After mixing, the total volume of the entire reaction system is typically 50 μL.

[0035] Step Two: Activate the temperature control module to precisely heat and cool the amplification chamber according to the preset temperature cycling program. For example, for polymerase chain reaction (PCR), a typical temperature cycle includes: 95°C pre-denaturation for 2 minutes; followed by 40 cycles, each cycle consisting of: 95°C denaturation for 15 ms, 60°C annealing and extension for 30 ms; and finally, 72°C extension for 5 minutes. The temperature control module monitors the temperature inside the amplification chamber in real time through a feedback control system, controlling the deviation between the actual temperature and the target temperature within ±0.1°C, ensuring that the temperature transition rate and residence time of each cycle meet the requirements.

[0036] Step 3: During temperature cycling, the polymerase uses deoxyribonucleoside triphosphates as raw materials, starting with primers, to synthesize nucleic acid sequences according to the template sequence, thereby exponentially amplifying the target nucleic acid sequence. Simultaneously, the fluorescent probe hydrolyzes and releases a fluorescent signal when the amplification products accumulate to a certain level. The fluorescent signal is acquired in real time by the optical detection module during the annealing / extension phase of each cycle to plot the amplification curve. After the amplification reaction is complete, the amplification products proceed to the detection step.

[0037] S5, Fluorescent detection solution loading and nucleic acid fluorescence detection, mainly uses optical detection methods to collect and analyze the fluorescence signal generated by the amplification product, thereby achieving qualitative or quantitative detection of the target nucleic acid. The detection chamber is set as an optically transparent microcavity with low autofluorescence background, and specifically includes the following steps: Step 1: After the amplification reaction is complete, if additional fluorescent detection solution is required for testing, such as in certain specialized fluorescence resonance energy transfer (FRET) detection methods, the liquid manipulation module controls the opening of the valve in its reservoir, allowing the pre-loaded fluorescent detection solution to be driven by centrifugal force or gas pressure into the detection chamber to mix with the amplification products. However, for most real-time fluorescence polymerase chain reactions (RT-PCRs), the fluorescent probe is pre-loaded in the amplification reagent.

[0038] Step 2: Activate the optical detection module. The excitation light source emits excitation light of a specific wavelength, which shines through the bottom or side window of the detection chamber of the microfluidic chip module onto the liquid region containing the amplification product and the fluorescent probe. After being irradiated by the excitation light, the fluorescent probe emits fluorescence of a characteristic wavelength.

[0039] Step 3: The fluorescence signal emitted by the fluorescent probe is acquired by a photomultiplier tube or complementary metal-oxide-semiconductor sensor in the optical detection module. The acquired raw electrical signal undergoes preprocessing in the data processing module, including signal amplification, filtering, and analog-to-digital conversion, to form digitized fluorescence signal data. The data processing module then analyzes the fluorescence signal according to preset thresholds and algorithms.

[0040] Step 3: The data processing module automatically interprets the test results according to the set detection standards. For example, if the fluorescence signal intensity exceeds a specific threshold before reaching the preset number of cycles, it is judged as positive; otherwise, it is negative. Simultaneously, the amplification of the internal control is used to verify the effectiveness of the entire detection process. The test results are clearly displayed on the device's screen, and a test report can be generated.

[0041] S6, Waste Liquid Collection and Treatment, primarily involves the safe and efficient collection and storage of all waste liquids generated during the reaction process to prevent biocontamination and reagent residue. The waste liquid collection chamber is the largest cavity in the microfluidic chip module, located at the distal end of the module, and is equipped with multiple one-way valves connecting to each reaction zone. The specific steps include: Step 1: During the nucleic acid extraction and purification stage, the waste liquid generated in the washing step is automatically driven by centrifugal force or air pressure through a pre-designed microchannel and one-way valve into the waste liquid collection chamber. This one-way valve design ensures that the waste liquid can only enter the waste liquid collection chamber and cannot flow back into the reaction area, thus effectively avoiding cross-contamination.

[0042] Step Two: After the amplification and detection reactions are completed, all residual reaction liquids, including unconsumed reagents, amplification products, and detection solutions, are completely diverted to the waste liquid collection chamber through a preset fluid control program. The liquid control module executes a final centrifugation process, such as 5000 rpm for 2 minutes, to ensure that all liquids in the microchannels and reaction chambers are completely emptied and collected in the waste liquid collection chamber.

[0043] Step 3: The waste liquid collection chamber is designed as a fully sealed structure, with an impermeable sealing membrane at the top to ensure that the waste liquid will not leak after collection, maintaining a complete seal even when the microfluidic chip module is tilted or vibrated. The materials selected for the waste liquid collection chamber have strict requirements for biocompatibility and chemical inertness to ensure that the waste liquid will not react with the container to produce toxic substances or corrode the container.

[0044] Step 4: After all testing processes are completed, the microfluidic chip module containing all waste liquid can be safely removed as a whole and disposed of. This integrated waste liquid management method simplifies the handling process for operators, reduces biosafety risks, and complies with relevant regulations for medical waste disposal.

[0045] 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. An integrated nucleic acid detection device based on a microfluidic chip, characterized in that, It includes a microfluidic chip module, a liquid manipulation module, a temperature control module, an optical detection module, and a data processing module. Multiple modules work together to achieve fully automated detection of samples from lysis to detection. The microfluidic chip module includes a sample processing area, a nucleic acid extraction and purification area, a nucleic acid amplification area, a nucleic acid detection area, and a waste liquid collection area connected by microchannels, and valves are set at key nodes; The liquid manipulation module is used to drive the fluid flow and mix the reagents within the microfluidic chip module. The temperature control module is used to achieve the temperature cycle required for nucleic acid amplification; The optical detection module is used to excite and collect the fluorescence signal in the nucleic acid detection area; The data processing module is used to receive signals collected by the optical detection module and perform data analysis, result interpretation, and human-computer interaction.

2. The integrated nucleic acid detection device based on a microfluidic chip according to claim 1, characterized in that: The main material of the microfluidic chip module is polymethyl methacrylate. A predetermined microchannel structure is formed by micromachining and laser etching technology. The size of the microfluidic chip module is set to a standardized cartridge form for easy single use.

3. The integrated nucleic acid detection device based on a microfluidic chip according to claim 1, characterized in that: The valve includes at least one of a soluble membrane valve, a temperature-sensitive valve, a pneumatically driven valve, and a capillary flow-limiting valve.

4. The integrated nucleic acid detection device based on a microfluidic chip according to claim 1, characterized in that: The liquid control module achieves fluid control by combining centrifugal force and precise air pressure drive. It includes a high-precision rotary motor, a micro air pressure pump and a solenoid valve. The air pressure pump can provide a pressure range of 0.05MPa-0.2MPa with a response time of less than 100ms, and the opening and closing time of the solenoid valve is less than 10ms.

5. The integrated nucleic acid detection device based on a microfluidic chip according to claim 4, characterized in that: The liquid control module also integrates an ultrasonic generator, the frequency of which can be set from 40kHz to 60kHz and the power is adjustable.

6. The integrated nucleic acid detection device based on a microfluidic chip according to claim 1, characterized in that: The temperature control module includes an array of multiple semiconductor coolers, a high-precision thermistor, and a proportional-integral-derivative controller. The semiconductor cooler array is attached to the bottom of the amplification chamber of the microfluidic chip module. The temperature control module can achieve a heating rate of 5℃ / s-10℃ / s and a cooling rate of 10℃ / s, switching the amplification chamber temperature between 25℃ and 95℃.

7. The integrated nucleic acid detection device based on a microfluidic chip according to claim 1, characterized in that: The optical detection module includes a high-brightness light-emitting diode, a filter, an optical fiber, a lens group, and a high-sensitivity photomultiplier tube or complementary metal-oxide-semiconductor sensor. The emission spectrum center of the high-brightness light-emitting diode is located between 480nm and 520nm. The lens group is used to focus the fluorescence signal. The minimum detection area can reach 0.5mm², and the background noise is less than 10 photons / second.

8. An integrated nucleic acid detection method based on a microfluidic chip as described in any one of claims 1-7, characterized in that, Includes the following steps: S1, Sample lysis buffer loading and mixing: The sample to be tested is added to the microfluidic chip module and mixed with the lysis buffer to release nucleic acids; S2, Nucleic acid extraction solution loading and nucleic acid capture washing: The nucleic acid-containing mixture is brought into contact with the nucleic acid adsorption medium to capture nucleic acid and remove impurities by washing; S3, Nucleic acid elution loading and elution: The elution buffer is brought into contact with the medium on which nucleic acid is adsorbed, releasing the nucleic acid from the medium and transferring it to the amplification chamber; S4, Amplification reagent loading and nucleic acid amplification reaction: Mix the amplification reagent with nucleic acid and achieve nucleic acid amplification through temperature cycling; S5, Fluorescent detection solution loading and nucleic acid fluorescence detection: Excitation, acquisition and analysis of fluorescence signals of amplified products to obtain detection results; S6, Waste Liquid Collection and Treatment: Collect all waste liquid generated during the testing process and treat it safely.

9. The integrated nucleic acid detection method based on a microfluidic chip according to claim 8, characterized in that: In step S1, the microfluidic chip module is driven by the liquid manipulation module to perform a preset oscillation or rotation program to fully mix the sample and the lysis buffer. The oscillation or rotation program is to rotate alternately in the forward and reverse directions at a speed of 800-1200 rpm, each direction lasting 20 ms, for three cycles, and the mixing of the sample and the lysis buffer continues for 5 min to 15 min at room temperature.

10. The integrated nucleic acid detection method based on a microfluidic chip according to claim 8, characterized in that: In S4, the temperature cycling program is as follows: pre-denaturation at 95°C for 2 minutes; followed by 40 cycles, each cycle including: denaturation at 95°C for 15 ms, annealing at 60°C and extension for 30 ms; and finally extension at 72°C for 5 minutes, with the deviation between the actual temperature and the target temperature controlled within ±0.1°C.

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