Portable nucleic acid extraction, amplification and detection integrated device and detection method thereof

Abstract

The invention discloses a portable nucleic acid extraction, amplification and detection integrated device and a detection method thereof, and belongs to the technical field of nucleic acid detection.The portable nucleic acid extraction, amplification and detection integrated device comprises a rack, an upper cover hinged to the rack, a centrifugal module arranged on the rack, a heating module, a laser module, a fluorescence detection module, a printing module, a code scanning module, a main control chip and an Android all-in-one machine, the centrifugal module drives the micro-fluidic chip to rotate so as to provide a driving force for liquid in the chip, the heating module heats the micro-fluidic chip so as to provide a temperature required in a nucleic acid extraction and amplification process, and the laser module emits laser to release a reagent. And the fluorescence detection module is used for carrying out fluorescence signal detection on an isothermal amplification product in the micro-fluidic chip. According to the present invention, the rapid detection of nucleic acid can be achieved, the reaction process is completely closed, the pollution is low, the detection flux is high, the sample demand is small, the steps are simple, the operation is easy, the portability is good, and the clinical and on-site rapid detection requirements can be met.

Description

Technical Field

[0001] The present invention relates to the technical field of nucleic acid detection, and particularly to a portable integrated device for nucleic acid extraction, amplification and detection based on isothermal amplification. Background Art

[0002] Nucleic acid detection technology is an important tool in modern molecular biology and is widely used in fields such as disease diagnosis, gene analysis, and environmental monitoring. Generally speaking, in order to improve the detection sensitivity, nucleic acid extraction and amplification are required before nucleic acid detection. The traditional nucleic acid extraction and amplification detection process usually requires multiple steps and equipment, with complex operations and time-consuming processes. In order to simplify the operation process and improve the detection efficiency, in recent years, portable integrated devices for nucleic acid extraction, amplification and detection have been developed in the industry, which can complete the extraction, amplification and detection of samples in one system, greatly improving the convenience and flexibility of detection. For example, portable PCR instruments integrate nucleic acid extraction and PCR amplification functions and can quickly detect samples in the field environment. Among them, the portable PCR instrument of ThermoFisher Scientific can complete the nucleic acid extraction and amplification process within a few minutes, and the portable nucleic acid detection device of Bio-Rad can complete the nucleic acid extraction, amplification and fluorescence detection processes within a few minutes. However, the types of samples processed by such instruments are limited, and multiple sample addition operations are required, greatly increasing the overall cost. At the same time, the stability of the device in different environments is poor. In addition, current nucleic acid extraction and detection devices are all open, which is prone to nucleic acid cross-contamination, and there are also safety risks to people when detecting pathogenic bacteria or viruses.

[0003] Isothermal amplification technology is a currently favored amplification technology. Its reaction process always maintains a constant temperature and achieves rapid amplification by adding enzymes with different activities and their respective specific primers. It has the advantages of simple operation, short reaction time, high sensitivity, etc., and is suitable for the field of rapid diagnosis. It is expected to go out of the laboratory, be applied to emergency departments for immediate testing, and even reach every household. Truly, it can greatly promote the application of molecular diagnosis. However, the integrated device for nucleic acid extraction, amplification and detection based on loop-mediated isothermal amplification technology is large in volume and low in portability, and is prone to contamination when manually loading samples or reagents. At the same time, multiple reactions are required when detecting multiple targets, with cumbersome operations and low throughput. Therefore, the shortcomings in terms of high efficiency, result accuracy and operation convenience still need to be solved through technological innovation to meet the broader clinical and on-site rapid detection needs. Summary of the Invention

[0004] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a portable integrated device for nucleic acid extraction, amplification and detection, which can achieve rapid detection of nucleic acids. The reaction process is fully enclosed, with little pollution, high detection throughput, small sample requirements, simple steps, easy to operate, and good portability, and can well meet the needs of clinical and on-site rapid detection.

[0005] To solve the above problems, the technical solution adopted by the present invention is as follows: A portable integrated device for nucleic acid extraction, amplification and detection, which includes a frame for installing other components of the integrated device, and an upper cover is hinged on the frame; a microfluidic chip, which includes a chip body and a reagent storage box arranged in the chip body. The chip body is provided with a sample lysis area, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative dispensing area and a reaction detection area that are connected through microchannels and distributed radially along the chip body. The reaction detection area includes a plurality of reaction chambers; a centrifugal module installed on the frame, which is used to drive the microfluidic chip to rotate to generate centrifugal force as the driving force of the liquid inside the microfluidic chip; a heating module, which is arranged on the upper and lower sides of the microfluidic chip and is used to heat the microfluidic chip to provide the temperature required during nucleic acid extraction and isothermal amplification; a laser module arranged on the upper cover and used to release the reagent pre-placed inside the reagent storage box; a fluorescence detection module, which is used to perform real-time detection of the fluorescence signal of the product after isothermal amplification in the reaction chamber and convert the fluorescence signal into an electrical signal for output; a printing module, which is used to output the detection result; a barcode scanning module, which is used to identify the two-dimensional code information on the chip body or the reagent storage box and output the data information of the two-dimensional code; a main control chip, which is electrically connected to the centrifugal module, the heating module, the laser module and the fluorescence detection module respectively, and the centrifugal module, the heating module, the laser module and the fluorescence detection module are controlled by the main control chip; an Android all-in-one machine, which is sequentially connected to the main control chip, the printing module and the barcode scanning module through digital or analog interfaces.

[0006] As a preferred embodiment of the present invention, the reagent storage box includes a box body and a sealing film covering the box body; a plurality of independent reagent storage cavities are recessed downward from the top of the box body, and a release hole is provided at a position on the side wall of each reagent storage cavity close to the bottom wall, and an envelope for controlling the release of the reagent is provided in the release hole; a convex block extends outward from one side of the box body, and a sample addition port is provided on the convex block; an accommodation cavity adapted to the reagent storage box is provided on the chip body, and the reagent storage box is arranged in the accommodation cavity.

[0007] As a preferred embodiment of the present invention, the nucleic acid extraction area includes a mixing buffer pool and a nucleic acid extraction column; the mixing buffer pool is respectively communicated with the sample lysis pool and the nucleic acid extraction column, and the nucleic acid extraction column is respectively communicated with the nucleic acid dilution area and the waste liquid tank through microchannels; the nucleic acid extraction column includes an extraction cavity in a cylindrical structure and a pressing ring hermetically installed in the extraction cavity, a small hole for liquid to flow out is provided on the bottom wall of the extraction cavity, an extraction membrane is provided on the bottom wall of the extraction cavity, and the pressing ring abuts against the extraction membrane and is used to limit the position of the extraction membrane in the liquid flow direction.

[0008] As a preferred embodiment of the present invention, the centrifugation module includes a centrifugation motor fixed on the frame and a centrifugation turntable drivingly connected to the output shaft of the centrifugation motor, the centrifugation turntable includes at least two symmetrically arranged and hollowed-out card slots, and the microfluidic chip is installed in the card slots of the centrifugation turntable.

[0009] As a preferred embodiment of the present invention, the heating module includes a driving component, a ring-shaped upper heating sheet and at least four lower heating sheets; two of the lower heating sheets are symmetrically arranged around the output shaft of the centrifugation motor to form a first heating ring, and the other two lower heating sheets are symmetrically arranged outside the first heating ring to form a second heating ring, the first heating ring and the second heating ring are connected by a metal wire, and the ring-shaped upper heating sheet is installed on the upper cover and corresponds to the second heating ring; the driving component is fixed on the frame and is drivingly connected to the ring-shaped upper heating sheet and the second heating ring, and is used to drive the ring-shaped upper heating sheet and the second heating ring to move towards each other or away from each other.

[0010] Further preferably, the driving assembly includes an upper movable plate, a lower movable plate, a driving motor, a bracket, and two guiding blocks; the upper movable plate is movably mounted on the upper cover through an elastic member, the annular upper heating sheet is mounted in the upper movable plate, the lower movable plate is movably mounted on the frame through an elastic member, and the first heating ring and the second heating ring are mounted in the lower movable plate; the driving motor is mounted on the frame and its output shaft is connected to the bracket; the guiding blocks are symmetrically arranged on both sides of the frame through the bracket, and both the upper and lower end faces of the guiding blocks include a horizontal bearing surface and an inclined surface, and the inclined surfaces of the upper end face and the lower end face are correspondingly arranged and have opposite inclined directions; pulleys are fixedly provided on both sides of the upper movable plate and the lower movable plate corresponding to the positions of the guiding blocks; the pulleys can respectively abut against the upper and lower end faces of the guiding blocks and are used for rolling along the upper and lower end faces of the guiding blocks when the driving motor drives the guiding blocks to move back and forth along the frame.

[0011] As a preferred embodiment of the present invention, the laser module is arranged inside the annular upper heating sheet.

[0012] As a preferred embodiment of the present invention, the fluorescence detection module is arranged on the frame and is located outside the second heating ring.

[0013] As a preferred embodiment of the present invention, the fluorescence detection module includes an LED lamp, a filter, and a photodiode. The filter is arranged on the optical path of the LED lamp and is optically coupled with the LED lamp. The photodiode is arranged on the output optical path of the filter and is optically coupled with the filter. The electrical signal output end of the photodiode is electrically connected to the main control chip.

[0014] As a preferred embodiment of the present invention, an iron core is arranged on the upper cover, and an opening and closing electromagnet is arranged on the frame corresponding to the position of the iron core for controlling the opening and closing of the upper cover.

[0015] The present invention also provides a detection method based on the above portable nucleic acid extraction, amplification and detection integrated device, which includes the following steps: S1. Turn on the power of the integrated device, scan the QR code on the microfluidic chip or the reagent storage box through the scanning module, identify and read the information of the microfluidic chip or the reagent storage box on the QR code, and the Android all-in-one machine loads the corresponding detection program and parameters; S2. Open the upper cover through the Android all-in-one machine, load the microfluidic chip with the reagent storage box into the centrifugal turntable, add the sample and the sample lysate into the microfluidic chip through the sample injection port, close the upper cover, and the integrated device starts to run; S3. The annular upper heating sheet and the lower heating sheet clamp the microfluidic chip, and quickly heat the temperature to the set value through a closed-loop temperature control algorithm to heat the sample lysis area until the sample lyses and releases nucleic acid; S4. The centrifugal drive microfluidic chip rotates. After the lysis product is driven to the nucleic acid extraction area by centrifugal force, the laser module emits laser light to break the envelope in the reagent storage cartridge, sequentially releasing the first cleaning solution, the second cleaning solution, and the elution solution, and completing the multi-stage cleaning and elution of nucleic acids by centrifugal force; the laser module emits laser light to release the dilution solution, and the eluted nucleic acids and the dilution solution are driven to the nucleic acid dilution area by centrifugal force for dilution; S5. The diluted nucleic acids enter the quantitative dispensing area under the drive of centrifugal force and are quantitatively dispensed into multiple reaction chambers in the reaction detection area. The annular upper heating sheet and the lower heating sheet clamp the microfluidic chip again, and the temperature cycle is controlled according to the amplification program to perform nucleic acid amplification reaction; S5. After the cycle ends, the centrifugal module drives the microfluidic chip to move above the fluorescence detection module. While each reaction well passes through the fluorescence detection module in turn during the movement of the microfluidic chip, the fluorescence detection module emits excitation light, collects the excited fluorescence signal, converts the optical signal into an electrical signal, and sends it to the Android all-in-one machine; S6. The Android all-in-one machine generates an amplification curve and calculates the concentration according to the real-time fluorescence signal, displays the detection result, and outputs the detection result through the printing module.

[0016] Compared with the prior art, the beneficial effects of the present invention are as follows: By integrating components such as a microfluidic chip, a centrifugal module, a heating module, a fluorescence detection module, a barcode scanning module, and a printing module into an integrated device, and combining the loop-mediated isothermal amplification technology, the present invention uses a fluorescence probe to achieve low-cost, rapid, efficient, real-time, and highly sensitive nucleic acid analysis. While improving the degree of full-process automation, it integrates the functions of nucleic acid extraction, amplification, and detection, can simultaneously process multi-sample detection, reduces the sample addition operation steps, simplifies the operation process, significantly improves the detection speed, sensitivity, and result accuracy, thereby reducing the overall cost and improving the stability and reliability of the device. The reaction process is fully enclosed, with little pollution, and has a high detection throughput, small sample requirements, simple steps, easy operation, and good portability, and can well meet the clinical and on-site rapid detection needs. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 is a schematic structural diagram of the portable nucleic acid extraction, amplification, and detection integrated device of the present invention; Figure 2 is a schematic structural diagram of the portable nucleic acid extraction, amplification, and detection integrated device of the present invention with the housing removed; Figure 3 is a schematic structural diagram of the portable nucleic acid extraction, amplification, and detection integrated device of the present invention with the housing removed and the upper cover opened; Figure 4 is a partial structural diagram of the portable nucleic acid extraction, amplification, and detection integrated device of the present invention; Figure 5 This is a partial structural schematic diagram of another angle of the portable nucleic acid extraction, amplification and detection integrated device of the present invention; Figure 6 This is a partial structural schematic diagram of the heating module of the portable nucleic acid extraction, amplification and detection integrated device of the present invention; Figure 7 This is a structural schematic diagram of the microfluidic chip of the present invention; Figure 8 This is a cross-sectional view of the chip body of the present invention; Explanation of the reference numerals in the drawings: 100, housing; 101, frame; 102, upper cover; 103, iron core; 104, opening and closing electromagnet; 200, centrifugal module; 201, centrifugal motor; 202, centrifugal turntable; 300, heating module; 301, driving assembly; 3011, upper movable plate; 3012, lower movable plate; 3013, driving motor; 3014, bracket; 3015, guide block; 3016, elastic member; 3017, pulley; 302, annular upper heating sheet; 303, first heating ring; 304, second heating ring; 305, metal wire; 400, laser module; 500, fluorescence detection module; 600, printing module; 700, code scanning module; 800, main control chip; 900, Android all-in-one machine; 1000, chip body; 1010, sample lysis area; 1020, nucleic acid extraction area; 1021, mixing buffer pool; 1022, nucleic acid extraction column; 10221, extraction cavity; 10222, pressing ring; 10223, small hole; 10224, extraction membrane; 1030, nucleic acid dilution area; 1031, dilution liquid release pool; 1032, dilution pool; 1040, quantitative dispensing area; 1041, liquid channel; 1042, quantitative tank; 1050, reaction detection area; 1051, reaction cavity; 1052, paraffin tank; 1053, plugging groove; 1060, waste liquid tank; 1100, reagent storage box; 1101, reagent storage cavity; 1102, release hole; 1103, sample adding port. Detailed implementation manners

[0018] The present invention will be further described in detail below in conjunction with the accompanying drawings and specific implementation manners.

[0019] As Figure 1 shown, the portable nucleic acid extraction, amplification and detection integrated device of the present invention includes a housing 100, a frame 101 and an upper cover 102. A cavity is formed between the upper cover 102 and the housing 100. The frame 101 is fixedly installed in this cavity and is hinged to the upper cover 102. An iron core 103 is provided on the upper cover 102, and an opening and closing electromagnet 104 is provided on the frame 101 at a position corresponding to the iron core 103. The opening and closing control of the upper cover 102 is realized by powering off or on the opening and closing electromagnet 104. As Figure 2 andFigure 3 As shown in the figure, the frame 101 is used to install other components of the integrated device. The other components include a centrifugal module 200, a heating module 300, a laser module 400, a fluorescence detection module 500, a printing module 600, a barcode scanning module 700, a main control chip 800, an Android all-in-one computer 900, and a microfluidic chip with a reagent storage box 1100. The microfluidic chip is detachably installed in the centrifugal module 200. Among them, the centrifugal module 200 is used to drive the microfluidic chip to rotate to generate centrifugal force, which serves as the driving force for the liquid inside the microfluidic chip; the heating module 300 is used to perform contact double-sided heating on the upper and lower sides of the microfluidic chip to provide the uniform temperature required during the nucleic acid extraction and amplification process; the laser module 400 is used to emit laser light to rupture the coating on the reagent storage box 1100 to release the preset reagent; the fluorescence detection module 500 is used to perform real-time fluorescence signal detection on the isothermal amplification products in the microfluidic chip, convert the fluorescence signal into an electrical signal, and output the electrical signal; the main control chip 800 is installed on the frame 101 and is electrically connected to the centrifugal module 200, the heating module 300, the laser module 400, and the fluorescence detection module 500 respectively, and is used to control the operation of the centrifugal module 200, the heating module 300, the laser module 400, and the fluorescence detection module 500; the Android all-in-one computer 900 is installed on the casing 100 and is electrically connected or signal-connected to the main control chip 800, the printing module 600, and the barcode scanning module 700. The centrifugal module 200, the heating module 300, the fluorescence detection module 500, and the laser module 400 are controlled by the main control chip 800, and the main control chip 800, the printing module 600, and the barcode scanning module 700 are controlled by the Android all-in-one computer 900. By highly integrating the microfluidic chip, the centrifugal module 200, the heating module 300, the laser module 400, the fluorescence detection module 500, the printing module 600, the barcode scanning module 700, the main control chip 800, and the Android all-in-one computer 900, the present invention realizes the full-process automated operation of sample lysis, nucleic acid extraction, amplification reaction, and real-time detection, can achieve rapid detection of nucleic acids, the reaction process is fully enclosed, with little pollution, and has a high detection throughput, small sample demand, simple steps, easy operation, and good portability, and can well meet the clinical and on-site rapid detection requirements.

[0020] Specifically, the centrifugal module 200 is installed on the frame 101 as the centrifugal mechanism of the centrifugal microfluidic chip. The centrifugal module 200 includes a centrifugal motor 201 and a centrifugal turntable 202. One end of the centrifugal motor 201 is fixedly arranged on the bottom plate of the frame 101, and the other end with an output shaft is fixed on the top plate of the frame 101 and its output shaft penetrates through the top plate of the frame 101. The centrifugal motor 201 is electrically connected to the main control chip 800 and controlled by it. The centrifugal turntable 202 is of a circular structure, and its center position is drivingly connected to the output shaft of the centrifugal motor 201 through components such as bearings. At least two symmetrically arranged and hollowed-out card slots are provided on the centrifugal turntable 202, and the microfluidic chip is detachably fixed in the card slots of the centrifugal turntable 202.

[0021] Specifically, as Figure 7 and Figure 8As shown in the figure, the microfluidic chip includes a chip body 1000 and a reagent storage box 1100. The chip body 1000 has a fan-shaped structure. An accommodation cavity matching the reagent storage box 1100 is provided at a position of the chip body 1000 close to the center of the circle. The reagent storage box 1100 is arranged in this accommodation cavity. A sample lysis area 1010 is formed between the bottom wall of the accommodation cavity and the bottom wall of the reagent storage box 1100. The reagent storage box 1100 includes a box body and a sealing film covering the box body; a plurality of independent reagent storage cavities 1101 are recessed downward from the top of the box body. Release holes 1102 are provided at positions on the side walls of each reagent storage cavity 1101 close to the bottom wall. A coating film for controlling reagent release by laser is provided in the release holes 1102; a convex block extends outward from one side of the box body, and a sample addition port 1103 is provided on the convex block. The sample addition port 1103 communicates with the sample lysis area 1010. The other positions of the chip body 1000 except the accommodation cavity are covered with a transparent covering film to provide a closed space for nucleic acid extraction, amplification and detection and reduce contamination. A nucleic acid extraction area 1020, a nucleic acid dilution area 1030, a quantitative dispensing area 1040 and a reaction detection area 1050 are also provided on the chip body 1000. The sample lysis area 1010, the nucleic acid extraction area 1020, the nucleic acid dilution area 1030, the quantitative dispensing area 1040 and the reaction detection area 1050 are sequentially connected through microchannels and are arranged in sequence along the radial direction of the chip body 1000. The nucleic acid extraction area 1020 and the quantitative dispensing area 1040 are respectively connected to a waste liquid tank 1060 through microchannels; the nucleic acid dilution area 1030, the quantitative dispensing area 1040 and the waste liquid tank 1060 communicate with the atmosphere through microchannels respectively. The reaction detection area 1050 includes a plurality of reaction cavities 1051 with transparent areas to facilitate fluorescence detection of the amplification products. The above microfluidic chip integrates sample lysis, nucleic acid extraction and dilution, quantitative dispensing, amplification reaction and fluorescence detection on the chip body 1000 through modular design, and connects each area through microchannels. Relying on centrifugal force to drive the liquid flow, with highly integrated functions, it realizes the full-process integration, automation of nucleic acid detection and precise control of fluids. The operation is simple, and the detection process can be completed without external auxiliary equipment, greatly improving the detection efficiency and the accuracy of detection results while reducing the equipment procurement and maintenance costs, and also greatly reducing the operation difficulty, and can well meet the high-throughput and rapid detection requirements.

[0022] Among them, the nucleic acid extraction area 1020 includes a mixing buffer pool 1021 and a nucleic acid extraction column 1022 arranged successively from top to bottom, thereby ensuring that the flow path of the reagent under the action of centrifugal force is unidirectional flow, avoiding cross-contamination, and at the same time using the column extraction method can ensure the extraction efficiency and quality. The sample lysis area is directly connected to the mixing buffer pool 1021, the mixing buffer pool 1021 is connected to the nucleic acid extraction column 1022, and the nucleic acid extraction column 1022 is respectively connected to the nucleic acid dilution area 1030 and the waste liquid tank 1060 through microchannels. More specifically, the nucleic acid extraction column 1022 includes an extraction cavity 10221 with a cylindrical structure and a pressure ring 10222 hermetically installed in the extraction cavity 10221 and in interference fit with the extraction cavity 10221. The extraction cavity 10221 is integrally formed in the chip body 1000. A small hole 10223 communicating with the extraction cavity 10221 is opened on the bottom wall of the extraction cavity 10221. The inner diameter of the small hole 10223 is smaller than the inner diameter of the extraction cavity 10221 for allowing liquid to flow out. An extraction membrane 10224 is arranged on the bottom wall of the extraction cavity 10221, and the extraction membrane 10224 is arranged at the connection between the small hole 10223 and the extraction cavity 10221. The pressure ring 10222 abuts against the extraction membrane 10224 and is used to limit the position of the extraction membrane 10224 in the liquid flow direction to ensure the stability of the extraction membrane 10224 and the controlled fluid flow path. By embedding the nucleic acid extraction column 1022 into the microfluidic chip and combining it with the microfluidic channel, the present invention realizes the automatic flow and separation of liquids by using centrifugal force, avoiding sample loss and external interference, and ensuring high extraction efficiency and stable nucleic acid purity. The nucleic acid dilution area 1030 includes a dilution pool 1032 and a dilution release pool. The dilution pool 1032 is respectively connected to the diluent release pool 1031, the nucleic acid extraction area 1020 and the quantitative dispensing area 1040 through microchannels, and the dilution pool 1032 is connected to the atmosphere through a microchannel. The quantitative dispensing area 1040 includes a liquid channel 1041 and a plurality of equally sized quantitative slots 1042 arranged at intervals. The liquid channel 1041 is respectively connected to the nucleic acid dilution area 1030 and the waste liquid tank 1060 through microchannels. Each quantitative slot 1042 is respectively connected to the liquid channel 1041. By driving the nucleic acid sample solution to be measured into a plurality of equally sized quantitative slots 1042 by centrifugal force, accurate quantitative dispensing of the sample is realized, ensuring the quantitative consistency of the sample amplification reaction, and effectively guaranteeing the sensitivity, accuracy and reliability of the subsequent amplification reaction. The reaction detection area 1050 includes a plurality of reaction chambers 1051 arranged in one-to-one correspondence with the quantitative slots 1042. The reaction chambers 1051 are connected to the quantitative slots 1042 through microchannels, and the sample enters the reaction chambers 1051 from the quantitative slots 1042. Each reaction chamber 1051 is pre-loaded with freeze-dried microspheres of LAMP reagent (loop-mediated isothermal amplification reagent) and fluorescent probes. After the sample enters the reaction chamber 1051, the freeze-dried microspheres of LAMP reagent can be quickly and automatically redissolved to ensure the reaction sensitivity and specificity.Above the reaction chamber 1051, there is a paraffin wax tank 1052 filled with paraffin wax. On the microchannel between the reaction chamber 1051 and the metering tank 1042, there is a blocking groove 1053 communicating with the paraffin wax tank 1052. Before the amplification reaction, the paraffin wax is melted by heating, and then the microchannel at the entrance of the reaction chamber 1051 is blocked by centrifugal force to form a sealing layer, physically isolating the aerosol pollution source, effectively avoiding aerosol leakage and cross-contamination between samples during the amplification process, thus reducing the risk of false positives in the results, significantly improving the detection specificity, safety and accuracy, and being particularly suitable for the multi-sample high-throughput detection scenario.

[0023] As Figure 4 shown, the heating module 300 is arranged on the upper and lower sides of the microfluidic chip for contact-type double-sided uniform heating of the microfluidic chip. Specifically, as Figure 5 and Figure 6As shown in the figure, the heating module 300 includes a driving component 301, an annular upper heating sheet 302, and at least four lower heating sheets; two lower heating sheets are symmetrically arranged on the frame 101 with the output shaft of the centrifugal motor 201 as the center and form a first heating ring 303. The first heating ring 303 corresponds to the sample lysis area of the microfluidic chip. The first heating ring 303 heats the sample lysis area through the hollow centrifugal turntable 202. The other two lower heating sheets are symmetrically arranged on the frame 101 and form a second heating ring 304. The second heating ring 304 is located outside the first heating ring 303. The first heating ring 303 and the second heating ring 304 are connected by a metal wire 305. The annular upper heating sheet 302 is installed on the upper cover 102 and is arranged corresponding to the second heating ring 304. When the upper cover 102 is closed with the frame 101, the annular upper heating sheet 302 overlaps with the second heating ring 304 and clamps the reaction detection area 1050 of the microfluidic chip. Heating resistance wires are arranged inside the annular upper heating sheet 302, the first heating ring 303, and the second heating ring 304, and they are all heated by the heating resistance wires. The heating resistance wires are electrically connected to the main control chip 800 and are controlled by it. The driving component 301 is fixedly arranged on the frame 101 and is drivingly connected to the annular upper heating sheet 302 and the second heating ring 304, and is used to drive the annular upper heating sheet 302 and the second heating ring 304 to move in the direction of approaching or separating from each other. More specifically, the driving component 301 includes an upper movable plate 3011, a lower movable plate 3012, a driving motor 3013, a bracket 3014, and two guide blocks 3015. The upper movable plate 3011 is movably installed on the upper cover 102 through an elastic member 3016. The annular upper heating sheet 302 is installed in the upper movable plate 3011. The lower movable plate 3012 is movably installed on the frame 101 through an elastic member 3016. The first heating ring 303 and the second heating ring 304 are installed in the lower movable plate 3012. The elastic member 3016 includes a bolt and a spring. The lower end of the bolt's screw rod is provided with a thread for threaded connection with the frame 101 or the upper cover 102 to achieve fixed connection. The upper movable plate 3011 is sleeved on the upper end of the screw rod. The spring is sleeved on the bolt and is located between the upper movable plate 3011 and the frame 101 and between the lower movable plate 3012 and the upper cover 102. The driving motor 3013 is installed on the frame 101. The driving motor 3013 is electrically connected to the main control chip 800 and is controlled by it. The output shaft of the driving motor 3013 is connected to the bracket 3014. The bracket 3014 includes a first connecting portion and two second connecting portions perpendicular to the first connecting portion. The first connecting portion is horizontally arranged and is connected to the output shaft of the driving motor 3013, and an avoidance portion for avoiding the centrifugal motor 201 is provided on the first connecting portion. The second connecting portions are vertically and symmetrically arranged and are located on the left and right sides of the frame 101. The guide blocks 3015 are symmetrically arranged on the left and right sides of the frame 101 through the second connecting portions of the bracket 3014. Under the action of the driving motor 3013, the guide blocks 3015 can move along the front and back directions of the frame 101.More specifically, both the upper and lower end faces of the guiding block 3015 include a horizontal bearing surface and an inclined surface connected to the horizontal bearing surface. The inclined surfaces of the upper end face and the lower end face are correspondingly arranged and have opposite inclined directions: the inclined surfaces of both the upper and lower end faces are close to the front side of the frame 101, the inclined surface of the upper end face slopes downward, and the inclined surface of the lower end face slopes upward. Pulley 3017 is fixedly provided at positions on both sides of the upper movable plate 3011 and the lower movable plate 3012 corresponding to the guiding block 3015, and the central axis of the pulley 3017 is fixedly connected to the upper movable plate 3011 or the lower movable plate 3012. When the upper cover 102 is closed with the frame 101, the pulley 3017 can respectively abut against the upper and lower end faces of the guiding block 3015, and is used to roll along the upper and lower end faces of the guiding block 3015 when the driving motor 3013 drives the guiding block 3015 to move in the front-rear direction of the frame 101. The working process of the heating module 300 is as follows: In the natural state, the pulley 3017 of the upper movable plate 3011 and the lower movable plate 3012 respectively abuts against the horizontal bearing surface of the guiding block 3015, so that the horizontal bearing surface generates a squeezing force on the pulley 3017. The squeezing force drives the upper movable plate 3011 to compress the elastic member 3016 and move upward, and the lower movable plate 3012 to compress the elastic member 3016 and move downward, thereby driving the annular upper heating sheet 302 and the second heating ring 304 to move away from each other. In this state, the annular upper heating sheet 302 and the second heating ring 304 are not in contact with the microfluidic chip; when it is necessary to heat the microfluidic chip, the output shaft of the driving motor 3013 extends, driving the bracket 3014 and the guiding block 3015 to move backward, driving the pulley 3017 to roll onto the inclined surfaces of the upper and lower end faces of the guiding block 3015. As the relative position of the pulley 3017 on the guiding block 3015 moves, the acting force of the guiding block 3015 on the pulley 3017 gradually weakens to disappear, and the spring of the elastic member 3016 resets, driving the upper movable plate 3011 to move downward and the lower movable plate 3012 to move upward, driving the annular upper heating sheet 302 and the second heating ring 304 to move closer to each other, thereby clamping the microfluidic chip, and performing contact-type double-sided uniform heating on the microfluidic chip through the second heating ring 304 and the annular upper heating sheet 302, greatly improving the heat conduction efficiency and temperature uniformity, which is beneficial to improving the speed of sample lysis and nucleic acid amplification. When the heating is completed, the output shaft of the driving motor 3013 retracts, driving the guiding block 3015 to move to the front side of the frame 101, generating a squeezing force again, driving the annular upper heating sheet 302 and the second heating ring 304 to move away from each other, realizing the relaxation of the microfluidic chip. In some embodiments, a wrap-around edge is provided at the position where the second connecting portion of the bracket 3014 is connected to the guiding block 3015. The wrap-around edge is located at the front and rear ends of the guiding block 3015 and is used to limit the stroke of the guiding block 3015.In some embodiments, two symmetric slide bars are provided on both sides of the frame 101. A slider is slidably connected to the slide bar, and the slider is fixedly connected to the bottom of the second connecting portion of the bracket 3014, which is beneficial to improving the stability of the guide block 3015 when moving in the front-rear direction of the frame 101.

[0024] The laser module 400 is disposed on the upper cover 102 and inside the annular upper heating sheet 302, and is correspondingly disposed opposite to the reagent storage box 1100 of the microfluidic chip. The laser emitted by the laser module 400 irradiates the release hole 1102 to rupture the envelope in the release hole 1102, so as to release the reagent pre-placed inside the reagent storage box 1100. The laser intensity, irradiation time, and irradiation sequence can be adjusted according to the reagent requirements, effectively ensuring the independent, sequential, and precise release of multiple reagents to meet the complex requirements of multi-step reactions and being applicable to reactions sensitive to heating.

[0025] The fluorescence detection module 500 is disposed on the frame 101 and between the two lower heating sheets of the second heating ring 304, and is used for real-time detection of the fluorescence signal of the product after isothermal amplification in the reaction chamber 1051 and converting the fluorescence signal into an electrical signal, which is then transmitted to the Android all-in-one machine 900 through the main control chip 800. Specifically, the fluorescence detection module 500 includes an LED lamp, a filter, and a photodiode; wherein, the LED lamp serves as a light source for emitting excitation light; the filter is disposed on the optical path of the LED lamp and is optically coupled to the LED lamp, and is used for filtering out light of non-target wavelengths and transmitting the excitation light of the target wavelength; the photodiode is disposed on the output optical path of the filter and is optically coupled to the filter, and is used for receiving the fluorescence signal filtered by the filter and converting the fluorescence signal into an electrical signal; the electrical signal output terminal of the photodiode is electrically connected to the main control chip 800 to transmit the electrical signal generated by the photodiode to the main control chip 800 for processing, and the main control chip 800 is signal-connected to the Android all-in-one machine 900 through a data interface to transmit the processed data to the Android all-in-one machine 900 for display or further analysis.

[0026] The code scanning module 700 is installed on one side of the rack 101 and is used to identify the two-dimensional code information on the chip body 1000 or the reagent storage box 1100 and output the data information of the two-dimensional code. After receiving the data information of the two-dimensional code, the Android all-in-one machine 900 loads the corresponding detection program and executes the detection program. The printing module 600 is installed on one side of the rack 101 and is used to output the detection results of the Android all-in-one machine 900. The Android all-in-one machine 900 is sequentially connected to the main control chip 800, the printing module 600, and the code scanning module 700 through digital or analog interfaces; an operating software is installed in the Android all-in-one machine 900, and the operating software includes a temperature control unit, a motor drive control unit, an operation interface, a data storage unit, a closed-loop temperature control algorithm, and a fluorescence signal analysis algorithm. The operating software is used to control the integrated device to execute the isothermal amplification fluorescence detection process and quantitatively read the fluorescence value in the reaction chamber 1051. Preferably, the Android all-in-one machine 900 is a Raspberry Pi5 development board. Users can complete parameter setting and full-process monitoring through simple operations with the human-computer interaction system of the Android all-in-one machine 900. At the same time, the automatic identification and process loading of the microfluidic chip or the reagent storage box 1100 type are realized by using the two-dimensional code scanning function of the scanning module, greatly reducing the operation complexity. The fluorescence detection module 500 supports real-time fluorescence signal acquisition and dynamic monitoring to ensure the high sensitivity and accuracy of the amplification data. It can be seen that through the highly coordinated linkage and intelligent control among the modules of the present invention, the full-process integration and automation of nucleic acid detection are realized, which can ensure the efficiency of the detection process and the accuracy of the results, and is particularly suitable for the rapid detection requirements in multiple scenarios.

[0027] The detection method based on the above portable nucleic acid extraction, amplification and detection integrated device includes the following steps: S1. Turn on the power of the integrated device, scan the two-dimensional code on the microfluidic chip or the reagent storage box 1100 through the code scanning module 700, identify and read the information of the microfluidic chip or the reagent storage box 1100 on the two-dimensional code, and the Android all-in-one machine 900 loads the corresponding detection program and parameters; S2. By clicking the "open the upper cover 102" button on the Android all-in-one machine 900, the opening and closing electromagnet 104 is powered off and released, the upper cover 102 pops up, the microfluidic chip containing the reagent storage box 1100 is loaded into the centrifugal turntable 202, the sample and the sample lysate are added into the microfluidic chip through the sample addition port 1103, the upper cover 102 is closed, and the opening and closing electromagnet 104 is powered on to fix the upper cover 102, and the integrated device starts to run; S3. The annular upper heating sheet 302 and the second heating ring 304 clamp the microfluidic chip, and the temperature of the first heating ring 303 is quickly heated to the set value (such as 95 °C) through the closed-loop temperature control algorithm to heat the sample lysis area, maintain the high temperature until the sample is lysed and nucleic acid is released, and after the lysis is completed, the annular upper heating sheet 302 and the second heating ring 304 are loosened; S4. The centrifugally-driven microfluidic chip rotates. After the lysis product is driven to the nucleic acid extraction area 1020 by centrifugal force, the laser module 400 emits a laser to rupture the envelope in the reagent storage cartridge 1100, sequentially releasing the first cleaning solution, the second cleaning solution, and the elution solution. The liquid is driven to flow along a set path by centrifugal force to complete the multi-stage cleaning and elution of nucleic acids. The laser module 400 emits a laser to release the dilution solution, and the eluted nucleic acid and the dilution solution are driven to the nucleic acid dilution area 1030 by centrifugal force for dilution; S5. The diluted nucleic acid enters the quantitative dispensing area 1040 under the drive of centrifugal force and is quantitatively dispensed into multiple reaction chambers 1051 in the reaction detection area 1050. The annular upper heating sheet 302 and the second heating ring 304 clamp the microfluidic chip again, and the temperature cycle is controlled according to the amplification program to perform the nucleic acid amplification reaction; S5. After the cycle ends, the centrifugal module 200 drives the microfluidic chip to move above the fluorescence detection module 500. While each reaction well passes through the fluorescence detection module 500 in turn during the movement of the microfluidic chip, the fluorescence detection module 500 emits an excitation light, collects the excited fluorescence signal, converts the fluorescence signal into an electrical signal, and sends it to the Android all-in-one machine 900 for analysis; S6. The Android all-in-one machine 900 generates an amplification curve and calculates the concentration based on the real-time electrical signal, and displays the detection result. The user can output the detection result through the printing module 600.

[0028] The above embodiments are only the preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by those skilled in the art on the basis of the present invention belong to the scope of protection required by the present invention.

Claims

1. A portable integrated device for nucleic acid extraction, amplification and detection, characterized in that: including a frame for mounting other components of the integrated device, with an upper cover hinged on the frame; a microfluidic chip, which includes a chip body and a reagent storage box arranged in the chip body. On the chip body, there are a sample lysis area, a nucleic acid extraction area, a nucleic acid dilution area, a quantitative dispensing area, and a reaction detection area that are connected through microchannels and distributed radially along the chip body. The reaction detection area includes multiple reaction chambers; a centrifugation module installed on the frame, used to drive the microfluidic chip to rotate to generate centrifugal force, serving as the driving force for the liquid inside the microfluidic chip; a heating module arranged on the upper and lower sides of the microfluidic chip, used to heat the microfluidic chip to provide the required temperature during nucleic acid extraction and isothermal amplification processes; a laser module arranged on the upper cover, used to release the reagent pre-placed inside the reagent storage box; a fluorescence detection module, used to perform real-time detection of the fluorescence signal of the product after isothermal amplification in the reaction chamber and convert the fluorescence signal into an electrical signal for output; a printing module, used to output the detection result; a barcode scanning module, used to identify the two-dimensional code information on the chip body or the reagent storage box and output the data information of the two-dimensional code; a main control chip, which is electrically connected to the centrifugation module, the heating module, the laser module, and the fluorescence detection module respectively, and the centrifugation module, the heating module, the laser module, and the fluorescence detection module are controlled by the main control chip; an Android all-in-one machine, which is sequentially connected to the main control chip, the printing module, and the barcode scanning module through digital or analog interfaces.

2. The integrated portable nucleic acid extraction, amplification and detection device according to claim 1, characterized in that: The reagent storage box includes a box body and a sealing film covering the box body. The top of the box body is recessed downward to form a plurality of independent reagent storage cavities. On the side wall of each reagent storage cavity near the bottom wall, there is a release hole, and a coating film for controlling the release of the reagent is arranged in the release hole. One side of the box body extends outward to form a convex block, and a sample addition port is arranged on the convex block. The chip body is provided with a receiving cavity adapted to the reagent storage box, and the reagent storage box is arranged in the receiving cavity.

3. The integrated portable nucleic acid extraction, amplification and detection device according to claim 1 or 2, characterized in that: The nucleic acid extraction area includes a mixing buffer pool and a nucleic acid extraction column. The mixing buffer pool is respectively connected to the sample lysis pool and the nucleic acid extraction column, and the nucleic acid extraction column is respectively connected to the nucleic acid dilution area and the waste liquid tank through microchannels. The nucleic acid extraction column includes an extraction cavity in a cylindrical structure and a pressing ring hermetically installed in the extraction cavity. A small hole for liquid outflow is opened on the bottom wall of the extraction cavity, and an extraction membrane is arranged on the bottom wall of the extraction cavity. The pressing ring abuts against the extraction membrane and is used to limit the position of the extraction membrane in the liquid flow direction.

4. The integrated portable nucleic acid extraction, amplification and detection device according to claim 1, wherein: The centrifugation module includes a centrifugation motor fixed on the frame and a centrifugation turntable drivingly connected to the output shaft of the centrifugation motor. The centrifugation turntable includes at least two symmetrically arranged and hollowed-out card slots, and the microfluidic chip is installed in the card slots of the centrifugation turntable.

5. The integrated portable nucleic acid extraction, amplification and detection device according to claim 4, characterized in that: The heating module includes a driving component, a ring-shaped upper heating sheet, and at least four lower heating sheets; two of the lower heating sheets are symmetrically arranged around the output shaft of the centrifugal motor to form a first heating ring, and the other two lower heating sheets are symmetrically arranged outside the first heating ring to form a second heating ring. The first heating ring and the second heating ring are connected by a metal wire. The ring-shaped upper heating sheet is installed on the upper cover and is arranged corresponding to the second heating ring; the driving component is fixedly arranged on the frame and is drivingly connected to the ring-shaped upper heating sheet and the second heating ring, and is used to drive the ring-shaped upper heating sheet and the second heating ring to move in a direction close to or away from each other.

6. The integrated portable nucleic acid extraction, amplification and detection device according to claim 5, wherein: The driving component includes an upper movable plate, a lower movable plate, a driving motor, a bracket, and two guide blocks; the upper movable plate is movably installed on the upper cover through an elastic member, and the ring-shaped upper heating sheet is installed in the upper movable plate. The lower movable plate is movably installed on the frame through an elastic member, and the first heating ring and the second heating ring are installed in the lower movable plate; the driving motor is installed on the frame and its output shaft is connected to the bracket; the guide blocks are symmetrically arranged on both sides of the frame through the bracket. The upper and lower end faces of the guide block each include a horizontal bearing surface and an inclined surface. The inclined surfaces of the upper end face and the lower end face are correspondingly arranged and have opposite inclined directions; pulleys are fixedly arranged on both sides of the upper movable plate and the lower movable plate corresponding to the positions of the guide blocks; the pulleys can respectively abut against the upper and lower end faces of the guide block, and are used to roll along the upper and lower end faces of the guide block when the driving motor drives the guide block to move in the front-rear direction of the frame.

7. The integrated portable nucleic acid extraction, amplification and detection device according to claim 6, characterized in that: The fluorescence detection module is arranged on the frame and is located outside the second heating ring.

8. The integrated portable nucleic acid extraction, amplification and detection device according to claim 1 or 7, characterized in that: The fluorescence detection module includes an LED lamp, a filter, and a photodiode. The filter is arranged on the optical path of the LED lamp and is optically coupled to the LED lamp. The photodiode is arranged on the output optical path of the filter and is optically coupled to the filter. The electrical signal output end of the photodiode is electrically connected to the main control chip.

9. The integrated portable nucleic acid extraction, amplification and detection device according to claim 1, wherein: An iron core is arranged on the upper cover, and an opening and closing electromagnet is arranged on the frame corresponding to the position of the iron core, and is used to control the opening and closing of the upper cover.

10. A detection method for the portable nucleic acid extraction, amplification and detection integrated device according to any one of claims 1 to 9, characterized in that: It includes the following steps: S1. Turn on the power of the integrated device, scan the two-dimensional code on the microfluidic chip or the reagent storage box through the scanning module, identify and read the information of the microfluidic chip or the reagent storage box on the two-dimensional code, and the Android all-in-one machine loads the corresponding detection program and parameters; S2. Open the upper cover through the Android all-in-one machine, put the microfluidic chip with the reagent storage box into the centrifugal turntable, add the sample and the sample lysate into the microfluidic chip through the sample addition port, close the upper cover, and the integrated device starts to run; S3. The ring-shaped upper heating sheet and the lower heating sheet clamp the microfluidic chip, and quickly heat the temperature to the set value through a closed-loop temperature control algorithm to heat the sample lysis area until the sample lyses and releases nucleic acids; S4. The centrifugally-driven microfluidic chip rotates. After the lysis product is driven to the nucleic acid extraction area by centrifugal force, the laser module emits laser light to rupture the envelope in the reagent storage cartridge, sequentially releasing the first cleaning solution, the second cleaning solution, and the elution solution, and completing the multi-stage cleaning and elution of nucleic acids by centrifugal force; the laser module emits laser light to release the dilution solution, and the eluted nucleic acids and the dilution solution are driven to the nucleic acid dilution area by centrifugal force for dilution; S5. The diluted nucleic acids enter the quantitative dispensing area under the drive of centrifugal force and are quantitatively dispensed into multiple reaction chambers in the reaction detection area. The annular upper heating plate and the lower heating plate clamp the microfluidic chip again, and the temperature cycle is controlled according to the amplification program to perform nucleic acid amplification reaction; S5. After the cycle ends, the centrifugal module drives the microfluidic chip to move above the fluorescence detection module. While each reaction well passes through the fluorescence detection module in sequence during the movement of the microfluidic chip, the fluorescence detection module emits excitation light, collects the excited fluorescence signal, converts the optical signal into an electrical signal, and sends it to the Android all-in-one machine; S6. The Android all-in-one machine generates an amplification curve and calculates the concentration based on the real-time fluorescence signal, displays the detection result, and outputs the detection result through the printing module.

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