Laser-induced graphene and use thereof in PCR detection

Abstract

The present invention relates to a laser-induced graphene, the use thereof in PCR detection, and a disc-type microfluidic chip for nucleic acid detection. The laser-induced graphene has efficient electrothermal conversion capability, rapid and accurate temperature response when an external voltage is applied thereto, rapid temperature change capability and efficient heat transfer attributes, thus achieving accurate regulation and control of liquid temperatures, and satisfying temperature accuracy requirements of PCR processes. The present invention further relates to the disc-type microfluidic chip for nucleic acid detection, which has the following advantages: 1, said chip has a high degree of integration and involves low reagent and sample consumptions, and can achieve "sample-in and result-out" simply by using one drop of blood or other body fluid of a subject; 2, said chip has a small size, is portable, only needs simply rotation of a handle by fingers without the need of any instruments and devices, and therefore said chip is easy to operate and involves low requirements on skills of laboratory personnel; 3, by means of immune test strips and only requiring a couple of minutes, personnel can then read detection results, achieving direct result interpretation without needing any instrument; and 4, said chip has low cost, and is convenient to process and suitable for mass production.

Description

Laser-induced graphene and its application in PCR detection Technical Field

[0001] The present invention belongs to the technical field of determination or inspection of nucleic acids, pathogens, microorganisms, etc., and specifically relates to laser-induced graphene (LIG) and its application in PCR detection, as well as a disc-type nucleic acid detection microfluidic chip. Background Art

[0002] Polymerase chain reaction (PCR) is a molecular biology technique used to amplify specific DNA fragments. Due to its rapidity, sensitivity, specificity, and accuracy, it is widely used for the rapid detection and identification of bacteria, fungi, viruses, and parasites, as well as for early disease screening and diagnosis. PCR utilizes the fact that DNA denatures into single strands at 95°C in vitro. At lower temperatures (typically around 60°C), primers bind to the single strands based on complementary base pairing. The temperature is then adjusted to the optimal reaction temperature of DNA polymerase (around 72°C), where the enzyme synthesizes complementary strands along the phosphate-to-pentose (5′-3′) chain. Based on the PCR reaction principle, a complete PCR reaction proceeds through three stages: denaturation (90-95°C), annealing (40-60°C), and extension (70-75°C), which are repeated 45 times. Therefore, the rate at which the temperature control module is ramped up and down during the PCR reaction determines the reaction speed. Improving the ramping speed of the temperature control module can effectively increase the PCR reaction speed.

[0003] Traditional PCR instruments require the use of dedicated thermal cycling equipment and auxiliary cooling methods such as fans to provide the temperature required for the PCR reaction. For example, CN110551622A discloses a rapid PCR reaction chip and a rapid fluorescence quantitative detector, which includes a panel and a bottom film. At least one reaction pool is provided on the bottom surface of the panel, and the reaction pool does not pass through the panel. Sample addition holes and exhaust holes are provided on both sides of the reaction pool and are connected to the top surface of the panel. A bottom film is attached to the bottom surface of the panel, and the panel is made of a light-transmitting material. When the PCR chip is placed on the temperature control module, the bottom film is in close contact with the temperature control module. After the sample is added, the PCR reaction liquid in the reaction pool above the bottom film is separated from the temperature control module by only one layer of bottom film. The cooling capacity or heating capacity of the temperature control module can be quickly transferred to the PCR reaction liquid through the bottom film. CN107603874A discloses a microfluidic PCR detection system, comprising a microfluidic PCR chip, a sample loading device, a thermal cycler, and a fluorescence collection device, wherein the microfluidic PCR chip comprises a base portion, a chamber portion, a sealing cover, and a gas inlet; the sample loading device is used to add a sample to be detected at the hydrophilic unit, form a sealing oil layer above the added sample to be detected, and inject gas into the reaction chamber through the gas inlet; the thermal cycler is used to heat the base to allow the sample to be detected to undergo thermal cycle amplification; the fluorescence collection device is used to collect fluorescence signals from the sample to be detected after thermal cycle amplification, and can output the collected signals.

[0004] This type of thermal cycler is expensive, bulky, and has a complex system. It requires a 220V power supply and has a slow heating and cooling rate (4-5°C / s). As a result, PCR technology is almost entirely used in large centralized clinical laboratories or testing centers and cannot be effectively applied in the field of rapid testing.

[0005] Pathogen detection, on the other hand, has widespread applications in infection diagnosis and management. Early diagnosis of certain infectious diseases, in particular, is crucial for identifying illness and providing appropriate treatment. Immediate and on-site diagnostic decisions also help prevent the spread of epidemics and pandemics. Nucleic acid testing technology enables early diagnosis of disease, taking only a few hours, and provides molecular information about the pathogen. Its wide range of applications, high sensitivity, strong specificity, and rapidity have made it an increasingly popular alternative to traditional culture and immunoassay methods.

[0006] CN112126587B discloses a nucleic acid detection chip device, a nucleic acid detection chip, and a preparation method thereof, comprising preparing a modified chip, a reaction chip, and a valve chip, and sealing the reaction chip and the valve chip with a modified bottom plate to form a nucleic acid detection chip. CN115651807B discloses a nucleic acid detection chip and a nucleic acid detection method, comprising a flow channel layer, an elastic film layer, and an airway layer stacked in sequence; the flow channel layer is provided with a microfluidic channel, an inlet group, and an outlet group, and the flow channel layer is further provided with a virus separation area, a nucleic acid lysis area, a nucleic acid distribution area, a LAMP amplification area, and a CRISPR detection area connected by the microfluidic channel; the elastic film layer is correspondingly provided with a connection inlet group and a connection outlet group; the airway layer is provided with an airway channel, and the airway channel corresponds to the microfluidic channel and the nucleic acid lysis area, the nucleic acid distribution area, the LAMP amplification area, and the CRISPR detection area respectively; the airway layer is correspondingly provided with an inlet group and an outlet group.

[0007] However, nucleic acid testing is currently performed almost exclusively in large, centralized clinical laboratories. This limited coverage stems largely from the complexity and high cost of traditional nucleic acid technology. Commercial nucleic acid testing primarily utilizes polymerase chain reaction (PCR) to amplify and detect specific nucleic acid targets. This system requires not only cumbersome specialized thermal cycling equipment and fluorescence detection instruments, but also highly trained personnel to operate. Effective point-of-care (POCT) devices for rapid on-site nucleic acid processing, cleaning, amplification, and detection are lacking.

[0008] Microfluidic chip technology leverages microchannels to enable controllable fluid flow, consolidating the entire testing process—sampling, dilution, reagent addition, reaction, separation, and detection—on a single microplatform. Combining this with isothermal nucleic acid amplification technology enables integrated and automated "sample in, result out" processing. However, due to the complexity of primary samples such as blood, sputum, urine, and tissue, sample preparation lacks standardized standards. To overcome this challenge, improvements are needed not only in the design and fabrication of microfluidic devices, but also in the fundamental chemistry and materials for nucleic acid capture and extraction. Furthermore, microfluidic chip design typically requires controlled, directional, and sequential flow, requiring centrifugation or externally driven pumps. Eliminating the need for instrumentation and simplifying the internal design of microfluidic chips is also crucial in resource-limited settings. Therefore, the development of low-cost, integrated, simple-to-use, instrument-free nucleic acid analysis devices that can be used by minimally trained personnel is crucial to enable widespread adoption of nucleic acid testing.

[0009] Summary of the Invention

[0010] Based on the above problems, the present invention proposes a laser-induced graphene PCR detection device and method, which adopts an ultra-fast temperature ramp chip based on laser-induced graphene material to solve the problems that the current PCR instrument thermal cycler equipment is expensive, large in size, has a complex system, requires a 220V power supply, and has a slow temperature ramp rate (4-5°C / s).

[0011] In addition, in order to solve the above problems, the present invention integrates the sample processing function with the nucleic acid amplification detection function to develop a low-cost, integrated disc-type nucleic acid detection microfluidic chip, which imitates the appearance of a rotary dial telephone and uses cellulose filter paper as the nucleic acid adsorption material. It only requires a drop of blood or other body fluid of the test subject. After the sample is injected, the cells in the body fluid are automatically lysed and combined with the nucleic acid. After a simple wash, it reacts with the nucleic acid amplification reaction liquid in the liquid storage tank to achieve nucleic acid amplification. Finally, the results are observed and read through immune test strips, color development reaction or fluorescent color development reaction. The conversion of different functions can be achieved through simple rotation, thereby realizing on-site instant detection of pathogens. It has the advantages of high integration, low cost, simple operation, and visible test results. It has important practical application value and social and economic benefits for on-site instant detection of nucleic acids.

[0012] The complete technical solution of the present invention includes:

[0013] A method for preparing laser-induced graphene comprises: reducing a precursor material layer by laser-induced processing.

[0014] Furthermore, the method includes providing a carbon-containing precursor material layer, which is located on a base material layer, scanning the surface of the carbon-containing precursor material layer with a laser, and the local heat generated by laser irradiation carbonizes the surface of the precursor material, thereby forming graphene.

[0015] Furthermore, the precursor material layer and the base material layer are made of the same material or are separate different materials;

[0016] Preferably, the precursor material is selected from polyimide, phenolic resin, polyethyleneimine, lignocellulose or wood;

[0017] Preferably, the laser used is selected from a carbon dioxide laser or a femtosecond laser;

[0018] Preferably, the laser scanning rate is 0.5-500 mm / s, preferably 20-300 mm / s, more preferably 50-150 mm / s;

[0019] Preferably, the laser flux of the laser induced processing is greater than or equal to 5.5 J / cm 2 , preferably greater than 5.5 J / cm 2, more preferably 5.8-80 J / cm 2 , more preferably 5.8-60 J / cm 2 , most preferably 5.8-50J / cm 2 ;

[0020] Preferably, the substrate material is selected from polyimide material, polyimide film, polyimide tape, phenolic resin, polyethyleneimine, wood cellulose or wood.

[0021] Laser-induced graphene obtained by the above method.

[0022] The above-mentioned application of laser-induced graphene in PCR detection.

[0023] A laser-induced graphene PCR detection device comprises a PCR reaction chamber and a graphene heating device.

[0024] Furthermore, the graphene heating device comprises a laser-induced graphene layer and a substrate material layer. Preferably, the laser-induced graphene layer is located on the surface of the substrate material layer and has a thickness of 1-120 μm, preferably 2-100 μm, 3-80 μm, or 5-60 μm.

[0025] Furthermore, the laser-induced graphene layer is prepared by laser-induced processing to reduce a precursor material layer, and the precursor material layer is located on the surface of the substrate material layer. Preferably, the laser flux formed at the beginning of the laser-induced processing is 5.5 J / cm 2 .

[0026] Furthermore, the laser used includes but is not limited to a carbon dioxide laser and a femtosecond laser, and the base material layer includes but is not limited to a polyimide material, a polyimide film, a polyimide tape, a phenolic resin, polyethyleneimine, wood cellulose, and wood.

[0027] Furthermore, the detection device also includes a power supply and a power control module, and the power control module controls the power supply to perform power on / off cycle operations on the graphene heating device.

[0028] Furthermore, the graphene heating device is in contact with the PCR reaction chamber, and heats the PCR reaction chamber when powered on, and cools the PCR reaction chamber when powered off, so as to achieve a heating / cooling cycle of the PCR reaction chamber.

[0029] A laser-induced graphene PCR detection device includes a PCR reaction chamber, a graphene heating device, a power supply, and a power control module. The graphene heating device is connected to the power supply, which is controlled by the power control module. The power control module controls the power supply to perform power-on / power-off cycling operations on the graphene heating device. The graphene heating device contacts the PCR reaction chamber and heats the PCR reaction chamber when powered on and cools the PCR reaction chamber when powered off, thereby achieving a heating / cooling cycle for the PCR reaction chamber.

[0030] Furthermore, the power supply is a low-voltage direct current power supply.

[0031] Furthermore, the power source includes but is not limited to a battery, a rechargeable battery, a mobile power source, a mobile phone, a power supply, and a 220V power source.

[0032] Furthermore, the graphene heating device is in sheet shape.

[0033] Furthermore, the graphene heating device includes a laser-induced graphene layer and a base material layer. Preferably, the laser-induced graphene layer is located on the surface of the base material layer and has a thickness of 1-120 μm, preferably 2-100 μm, 3-80 μm, or 5-60 μm.

[0034] Furthermore, the laser-induced graphene layer is prepared by laser-induced processing to reduce a precursor material layer, the precursor material layer is located on the surface of the substrate material layer, and the laser flux at the beginning of the laser-induced processing is 5.5 J / cm 2 .

[0035] Furthermore, the laser used includes but is not limited to a carbon dioxide laser and a femtosecond laser, and the base material layer includes but is not limited to a polyimide material, a polyimide film, a polyimide tape, a phenolic resin, polyethyleneimine, wood cellulose, and wood.

[0036] Furthermore, the PCR detection device is an instant rapid detection device or a high-throughput desktop detection device.

[0037] Furthermore, the PCR reaction chamber is a disc-type PCR detection chip, and the disc-type PCR detection chip includes an upper sample injection layer and a lower detection layer.

[0038] Furthermore, the sample introduction layer is included in the top cover layer, and the detection layer is included in the liquid reservoir layer.

[0039] The method for performing the PCR detection using the detection device is to use a power control module to control the power supply to perform power on / off cycles on the graphene heating device, so that the thermal cycle of the PCR reaction chamber is 1-120 times, preferably 1-80 times, more preferably 1-50 times, for example 45 times.

[0040] A PCR detection chip comprises a top cover layer located above and a liquid reservoir layer located below, wherein the top cover layer is rotatable relative to the liquid reservoir layer, the top cover layer comprises an injection port for adding a test sample, and an adsorbent for adsorbing the test sample is provided below the injection port;

[0041] The liquid storage pool layer includes at least a sample addition pool, an amplification reaction pool, and a product detection pool. When the top cover layer rotates, the adsorbent can enter the sample addition pool, the amplification reaction pool, and the product detection pool in sequence. A test strip injection hole is set in the product detection pool.

[0042] Furthermore, the top cover layer and the liquid reservoir layer are buckled together to form a sealed space as a whole, and both the top cover layer and the liquid reservoir layer are circular.

[0043] Furthermore, the top cover layer is provided with a rotating handle which can be turned to realize its rotation.

[0044] Furthermore, the product detection pool is sealed with a sealant that can melt at a certain temperature and allow the liquid in the product detection pool to fall. Preferably, the sealant is paraffin wax that can melt at 60-90°C.

[0045] Furthermore, a test strip groove for adding a test strip is provided below the liquid reservoir layer, and a through hole is provided at one end of the test strip groove as an observation window.

[0046] Furthermore, at least one washing pool is included between the sample loading pool and the amplification reaction pool.

[0047] Furthermore, there are two washing tanks, including a first washing tank and a second washing tank.

[0048] Furthermore, the top cover layer and the liquid reservoir layer are relatively rotated by a circular slideway.

[0049] Furthermore, the adsorbent is cellulose filter paper.

[0050] Furthermore, the sealant is paraffin, and the paraffin can melt at 60-90°C.

[0051] Furthermore, the materials of the top cover layer and the liquid reservoir layer include but are not limited to polymethyl methacrylate (PMMA), epoxy resin, PVC, and PC. The concave and convex structures of the slide and the reaction pool are made by laser etching technology, or by etching the structure on the substrate and then casting it with polydimethylsiloxane (PDMS).

[0052] Furthermore, the first washing pool and the second washing pool are loaded with washing buffer respectively, the amplification reaction pool is loaded with nucleic acid amplification reaction buffer or PCR reaction reagent, and the product detection pool is loaded with running buffer.

[0053] Furthermore, the surface of the top cover layer is marked accordingly according to the sample loading pool, the first washing pool, the second washing pool, and the amplification reaction pool of the liquid reservoir layer.

[0054] Furthermore, the top cover layer and the reservoir layer are subjected to surface hydrophobic treatment, that is, the top cover layer and the reservoir layer are treated with a trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane reagent in a vacuum for three hours, so that the trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane forms a self-assembled monolayer on the surface of the top cover layer and the reservoir layer.

[0055] A disc-shaped nucleic acid detection microfluidic chip comprises a top cover layer located above and a liquid reservoir layer located below. The top cover layer and the liquid reservoir layer are buckled together to form a sealed space as a whole. Both the top cover layer and the liquid reservoir layer are circular, and the top cover layer can rotate relative to the liquid reservoir layer. The top cover layer includes an inlet for adding a test sample, and an adsorbent for adsorbing the test sample is provided below the inlet. The top cover layer is provided with a rotating handle that can be turned to achieve rotation.

[0056] The liquid reservoir layer includes at least a sample loading pool, an amplification reaction pool, and a product detection pool. During the rotation of the top cover layer, the adsorbent can sequentially enter the sample loading pool, the amplification reaction pool, and the product detection pool. The product detection pool is provided with a test strip sampling hole and is sealed with a sealant. The sealant can melt at a certain temperature and allow the liquid in the product detection pool to fall out.

[0057] A test strip groove for adding a test strip is provided below the liquid reservoir layer, and a through hole is provided at one end of the test strip groove as an observation window.

[0058] Furthermore, at least one washing pool is included between the sample loading pool and the amplification reaction pool.

[0059] Furthermore, there are two washing tanks, including a first washing tank and a second washing tank.

[0060] Furthermore, the top cover layer and the liquid reservoir layer are relatively rotated by a circular slideway.

[0061] Furthermore, the adsorbent is cellulose filter paper.

[0062] Furthermore, the sealant is paraffin, and the paraffin can melt at 60-90°C.

[0063] Furthermore, the materials of the top cover layer and the liquid reservoir layer include but are not limited to polymethyl methacrylate (PMMA), epoxy resin, PVC, and PC. The concave and convex structures of the slide and the reaction pool are made by laser etching technology, or by etching the structure on the substrate and then casting it with polydimethylsiloxane (PDMS).

[0064] Furthermore, the first washing pool and the second washing pool are loaded with washing buffer respectively, the amplification reaction pool is loaded with nucleic acid amplification reaction buffer or PCR reaction reagent, and the product detection pool is loaded with running buffer.

[0065] Furthermore, the surface of the top cover layer is marked accordingly according to the sample loading pool, the first washing pool, the second washing pool, and the amplification reaction pool of the liquid reservoir layer.

[0066] Furthermore, the top cover layer and the reservoir layer are subjected to surface hydrophobic treatment, that is, the top cover layer and the reservoir layer are treated with a trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane reagent in a vacuum for three hours, so that the trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane forms a self-assembled monolayer on the surface of the top cover layer and the reservoir layer.

[0067] Furthermore, the method comprises the following steps:

[0068] Step 1: Rotate the sampling port of the top cover layer above the sample pool of the liquid reservoir layer, use a pipette to inject a certain amount of body fluid onto the surface of the adsorbent through the sampling port, and wait for a certain period of time for the body fluid to dry;

[0069] Step 2: Use your finger to move the rotating handle of the top cover layer to move the adsorbent to the first washing tank to wash away impurities on the surface of the adsorbent. In this step, moving the rotating handle left and right can speed up the washing process.

[0070] Step 3: Move the handle of the top cover layer again to move the adsorbed material to the second washing tank for further washing. In this step, moving the handle left or right can speed up the washing process.

[0071] Step 4: Turning the rotating handle of the top cover layer to rotate the adsorbent into the amplification reaction pool; then turning on the heating device to raise the temperature to a certain level, so that the reaction buffer in the amplification reaction pool and the nucleic acid on the adsorbent undergo an amplification reaction, wherein the amplification reaction includes but is not limited to a constant temperature amplification reaction and a PCR reaction;

[0072] Step 5: After the amplification reaction is completed, the adsorbent is rotated again to move to the product detection pool; the amplification product on the adsorbent is absorbed by the test paper through the connecting hole; and the result is observed with the naked eye through the observation window on the groove of the test strip.

[0073] The detection device with the chip includes a graphene heating device, a power supply, and a power control module. The graphene heating device is connected to the power supply, and the power supply is controlled by the power control module. The power control module controls the power supply to perform power on / off cycle operations on the graphene heating device. The graphene heating device contacts the disc-type nucleic acid detection microfluidic chip and heats the disc-type nucleic acid detection microfluidic chip when powered on and cools the disc-type nucleic acid detection microfluidic chip when powered off, thereby realizing a heating / cooling cycle of the disc-type nucleic acid detection microfluidic chip.

[0074] Compared with the existing technology, the laser-induced graphene PCR detection device of the present invention has the following advantages:

[0075] 1. This invention provides a novel laser-induced graphene temperature ramp chip. This chip is fabricated using laser induction, eliminating the need for a mask. The fabricated pattern can be freely designed based on actual needs, with controllable morphology and adjustable composition. The simple fabrication method allows for batch production at a low cost, significantly reducing the cost of current PCR thermal cycling equipment and making it suitable for rapid on-site testing.

[0076] 2. The laser-induced graphene material prepared by the present invention has good mechanical strength and thermal stability, excellent electrical conductivity and thermal conductivity, and can quickly generate and conduct heat through the thermal effect of electric current; at the same time, it has good porosity, which is conducive to heat dissipation and cooling. Therefore, the laser-induced graphene temperature adjustment chip can achieve ultra-fast temperature adjustment and cooling simply by controlling the voltage. By applying a low voltage, ultra-fast temperature increase (>10°C / s) can be achieved; after turning off the voltage, it can rely on good porosity to independently achieve ultra-fast temperature reduction (>10°C / s), without the need for other auxiliary cooling methods, reducing the volume of the amplification chip and equipment, and effectively applied to the field of instant rapid detection.

[0077] Compared with the existing technology, the disc-type nucleic acid detection microfluidic chip of the present invention has the following advantages:

[0078] 1. High degree of integration, low consumption of reagents and samples, only a drop of blood or other body fluids from the examinee is needed to input the sample and output the result.

[0079] 2. Small size, easy to carry, no need for instruments or equipment, only need to turn the handle with your fingers, easy to operate, and low technical requirements for the experimenter.

[0080] 3. With the help of immune test strips, the test results can be read manually in just a few minutes. The results are intuitive and do not require any instruments.

[0081] 4. Low cost, easy to process and mass produce. BRIEF DESCRIPTION OF THE DRAWINGS

[0082] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the specific embodiments or the description of the prior art. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0083] FIG1 is the C peak of the XPS of the graphene material prepared by the method of the present invention (the laser flux is 36 J / cm 2 ).

[0084] Figure 2 shows the results of a 5.5 J / cm 2 , 5.8J / cm 2 , 10J / cm 2 , 15J / cm 2 , 20J / cm 2 , 25J / cm 2 、30J / cm 2 、36J / cm 2 、38J / cm 2 , 40J / cm 2 , 46J / cm 2 , 50J / cm 2 , 60J / cm 2 , 70J / cm 2 , 80J / cm 2 Surface morphology of laser-induced graphene (No. 1-15).

[0085] Figure 3 shows the results of a 40 J / cm 2 Voltage-temperature variation diagram of laser-induced graphene material.

[0086] Figure 4 shows the results of a 36 J / cm 2 Voltage-temperature variation diagram of laser-induced graphene material.

[0087] FIG5 is a graph showing the relationship between the maximum temperature and heating rate of the laser-induced graphene surface and the input power.

[0088] Figure 6 is a comparison of the surface temperature of laser-induced graphene under the same input power.

[0089] FIG7 is a graph showing the temperature change of the graphene surface induced by laser under step-by-step increase in applied voltage.

[0090] Figure 8 is a comparison of surface temperature under the same applied voltage under starting heating and step heating.

[0091] Figure 9 is a comparison of the laser-induced cooling rate of graphene and other materials.

[0092] Figure 10 is a temperature cycle diagram of laser-induced graphene (35°C-120°C).

[0093] FIG11 is a graph showing the temperature change of the laser-induced graphene heat source and the heated liquid.

[0094] FIG12 is a schematic diagram of the preparation process of the laser-induced graphene temperature rise and fall chip of the present invention.

[0095] FIG13 is a schematic diagram of the connection of the laser-induced graphene temperature rise and fall chip.

[0096] FIG14 is a schematic diagram of the detection process using a laser-induced graphene temperature ramp chip.

[0097] Figure 15 is a graph showing the actual temperature rise and fall performance of the laser-induced graphene temperature rise and fall chip.

[0098] FIG16 is a comparison of the heat conduction pathway of the laser-induced graphene temperature ramp chip of the present invention and a conventional PCR instrument.

[0099] FIG17 is a schematic structural diagram of a PCR reaction chamber.

[0100] FIG18 is a schematic diagram of the structure of a disc-type nucleic acid detection microfluidic chip.

[0101] FIG19 is a schematic diagram of the upper surface structure of the top cover layer.

[0102] FIG20 is a schematic diagram of the lower surface structure of the top cover layer.

[0103] FIG21 is a schematic structural diagram of the liquid reservoir layer.

[0104] Figure 22 is a schematic diagram of the immune test strip.

[0105] Figure 23 is a schematic diagram of the test results of the immune test strips.

[0106] In the figure: 1-laser; 2-laser-induced graphene temperature adjustment chip; 3-substrate material layer; 4-power supply; 5-power control module; 6-PCR reaction chamber, 7-sample injection layer, 8-detection layer, 9-test strip, 10-sealing plug, 11-spin column, 12-injection port, 13-annular slide, 14-sample injection chamber, 15-front wash chamber, 16-back wash chamber, 17-amplification reaction chamber, 18-detection chamber, 19-observation window, 20-test strip groove;

[0107] 21-top cover layer, 22-liquid reservoir layer, 23-test paper strip, 24-lid, 25-rotating handle, 26-injection port, 27-first outer slide, 28-first inner slide, 29-nucleic acid filter paper fixing hole, 30-filter paper, 31-second outer slide, 32-second inner slide, 33-sample loading pool, 34-first washing pool, 35-second washing pool, 36-amplification reaction pool, 37-product detection pool, 38-test paper strip injection hole, 39-observation window, 40-test paper strip groove, 41-sample pad, 42-nitrocellulose membrane, 43-gold label pad, 44-water absorbent pad, 45-bottom plate, 46-detection line, 47-quality control line. DETAILED DESCRIPTION

[0108] The following will clearly and completely describe the technical solutions in the embodiments of the present application in conjunction with the drawings in the embodiments of the present application. Obviously, the described embodiments are only for illustration and are not intended to limit the present application.

[0109] Laser-induced graphene The present invention first discloses a method for preparing graphene as a PCR detection heating device using laser induction, and the graphite flake material obtained thereby, as shown in FIG12 . The method comprises: reducing a precursor material layer using laser-induced processing.

[0110] In one embodiment of the present invention, a method for preparing laser-induced graphene includes: providing a carbon-containing precursor material layer, scanning the surface of the carbon-containing precursor material layer with a laser, and carbonizing the surface of the precursor material by local heat generated by laser irradiation, thereby forming graphene.

[0111] Specifically, during the preparation process, a special carbon-containing precursor material layer is provided. The precursor material layer can be located on the base material layer 3. The precursor material layer and the base material layer can be the same material or different materials. The precursor material includes but is not limited to polyimide, phenolic resin, polyethyleneimine, lignocellulose, wood, etc. The surface of the special carbon-containing precursor material layer is directly scanned by a laser 1. The local heat generated by the laser irradiation causes the surface of the precursor material to be carbonized. During the carbonization process, carbon atoms are converted from sp 3 Key reorganization into sp 2 The bond is formed, and the oxygen and nitrogen groups of the precursor material are decomposed to obtain the laser-induced graphene temperature rise and fall chip 2. The laser used includes but is not limited to a carbon dioxide laser, a femtosecond laser, and the like.

[0112] In order to meet the rapid temperature rise and fall requirements of the PCR detection device, the laser flux at the beginning of the formation of the laser-induced graphene material in this embodiment is 5.5 J / cm 2By adjusting the laser parameters, the physical and chemical properties of the laser-induced graphene material can be controlled. By increasing the laser power, the thickness of the laser-induced graphene material can be increased, the conductivity can be enhanced, and the resistance can be reduced. According to the current thermal effect (Q = V 2 / R*t), under the same input voltage conditions, the smaller the resistance, the more heat is generated in the same time, and the faster the heating rate. At the same time, increasing the laser power will increase the gas release rate during the preparation process, thereby increasing the number of pores, increasing the porosity and pore size of the laser-induced graphene material, and achieving a faster cooling rate. However, if the laser power exceeds the threshold, the structure of the laser-induced graphene material will be destroyed and lose its function. Therefore, after design and actual verification, this embodiment optimizes the laser flux at which the laser-induced graphene material begins to form, and selects 5.5J / cm 2 The laser flux can not only obtain a complete graphene structure, but also obtain a large number of pores and porosity, thereby improving the heat transfer efficiency and obtaining better heating and cooling performance.

[0113] Surprisingly, it was found that when the laser fluence of the laser induced processing is greater than or equal to 5.5 J / cm 2 , preferably greater than 5.5 J / cm 2 , more preferably 5.8-80 J / cm 2 , more preferably 5.8-60 J / cm 2 , most preferably 5.8-50J / cm 2 Therefore, in one embodiment of the present invention, the laser flux of the laser induced processing is greater than or equal to 5.5 J / cm 2 , preferably greater than 5.5 J / cm 2 , more preferably 5.8-80 J / cm 2 , more preferably 5.8-60 J / cm 2 , most preferably 5.8-50J / cm 2 More surprisingly, it was found that the laser flux gradually increased from 0 to less than 5.5J / cm 2 When the flux reaches 5.5 J / cm 2 When the flux increases to more than 5.5 J / cm 2 As the laser flux continues to increase, the graphene degree of the laser-induced graphene material formed on the surface becomes more complete. 2 , especially 5.8-50J / cm 2Within the laser flux range, the quality of laser-induced graphene formation gradually improves, the surface resistance gradually decreases, and the heating rate gradually increases. However, when the laser flux exceeds 50J / cm 2 After that, the laser-induced graphene formed on the polyimide substrate becomes difficult to effectively adhere, easily peels off under slight external force, and its mechanical strength decreases and its brittleness increases.

[0114] In order to adapt to the rapid heating and cooling requirements of the PCR detection device, the heating and cooling chip in the present invention adopts a sheet-like laser-induced graphene structure. By controlling the power, the laser-induced graphene material is concentrated on the surface of the substrate precursor material layer, with a thickness of generally 1-120 μm, preferably 2-100 μm, 3-80 μm, or 5-60 μm.

[0115] The laser-induced graphene of the present invention is produced by laser scanning on a material with a high molecular weight polymer as a substrate. Compared with traditional graphene materials, it has the characteristics of simple preparation process and strong product performance reliability.

[0116] Laser-induced graphene (LIG) exhibits an extremely high electrothermal conversion efficiency (>99%), allowing it to rapidly reach high temperatures even with relatively low input power. Under conditions of an applied voltage of 8V and an input power of less than 2.4W, it achieves a maximum surface temperature approaching 300°C, with a maximum heating rate of 68.5°C. Compared to other electrothermal materials, both maximum temperature and heating rate exhibit significant advantages. Furthermore, the maximum surface temperature and heating rate of LIG exhibit a clear functional relationship with the input power. Under the same input power, independent LIG samples can achieve the same surface temperature. This demonstrates that the temperature of LIG can be easily and rapidly controlled by varying the input electrical energy density. Due to its extremely high electrothermal conversion efficiency, the material's inherently low specific heat capacity, and its multi-layered porous structure, the electrical energy is converted to thermal energy at a rapid rate, making LIG temperature control both convenient and rapid. Furthermore, LIG ​​possesses inherently stable electrical power control capabilities.

[0117] Laser-induced graphene also has the ability to cool down quickly. Due to the low input power and the loose and porous surface structure (laser-induced graphene has unique microstructural advantages over graphene formed by traditional chemical deposition or surface growth), heat can be dissipated quickly after the power supply is stopped. The maximum natural cooling rate reaches 36°C / s, far exceeding traditional electric heating materials.

[0118] Laser-induced graphene can maintain stable performance during long-term heating and cooling processes. Experiments have shown that during 100 heating and cooling cycles, the heating and cooling rates and the temperature levels that can be achieved remain stable in each cycle.

[0119] In addition, the contact thermal resistance between graphene and a variety of materials (silicon dioxide, high molecular polymers such as PMMA, polyethylene, etc.) is very low. This feature helps graphene transfer heat to external objects quickly and efficiently, and through hard contact, it can effectively heat and cool the target object.

[0120] In summary, with the help of laser-induced graphene's efficient electrothermal conversion ability, fast and accurate temperature response under external applied voltage, rapid temperature change ability and efficient heat transfer properties, precise control of liquid temperature can be achieved to meet the temperature accuracy requirements of the PCR process.

[0121] Therefore, the laser-induced graphene of the present invention can be well applied in PCR detection.

[0122] Therefore, in another aspect, the present invention also discloses the application of the laser-induced graphene in PCR detection.

[0123] Laser-induced graphene PCR detection device

[0124] The present invention also discloses a PCR detection device prepared using the graphene material prepared above, as shown in Figures 13-14, comprising a PCR reaction chamber 6, a graphene heating device, a low-voltage DC power supply 4, and a power control module 5, wherein the graphene heating device is the laser-induced graphene temperature rise and fall chip 2 obtained above, the graphene heating device is close to the PCR reaction chamber, and the low-voltage DC power supply 4 powers the graphene heating device to achieve ultra-fast temperature rise (>10°C / s); after turning off the voltage, it can independently achieve ultra-fast temperature drop (>10°C / s) by relying on the heat dissipation of the surrounding environment, without the need for other auxiliary cooling methods. The measured temperature rise and fall performance is shown in Figure 15, where the solid line is the set temperature and the dotted line is the actual temperature. It can be seen from the figure that the actual temperature is in good agreement with the set temperature. In addition, the voltage supply method includes but is not limited to batteries, rechargeable batteries, mobile power supplies, mobile phones, power supplies, and 220V power supplies.

[0125] The volume of the above-mentioned PCR reaction microchamber is 20-100 μL, preferably 25-100 μL, which can be loaded with fluorescent quantitative PCR reaction reagents (generally 20 μL, including enzymes, substrates, primers, fluorescent molecules and target detection objects). As shown in Figure 16, in the present invention, the graphene temperature rise and fall chip is close to the PCR reaction chamber, and heat conduction is directly transferred from the graphene temperature rise and fall chip to the PCR reaction chamber. Compared with the traditional PCR instrument, heat conduction needs to be transferred from the traditional PCR instrument heating material to the aluminum base plate to the thin layer of air, and finally to the PCR reaction chamber. The heat conduction efficiency is high, the heat loss is small, and the temperature rise and fall speed is fast. The multi-step heat conduction path in the traditional PCR instrument is reduced, the heat conduction efficiency is effectively improved, and the reaction speed is further improved.

[0126] The present invention also provides a control method for the above-mentioned detection. Specifically, the control method includes controlling the power-on / off time (power supply voltage) of the graphene heating device by the power supply during the thermal cycle. The power-on / off time (power supply voltage) control is implemented by the control module of the power supply, and precise control of temperature rise and fall under different conditions is achieved according to environmental conditions (temperature, wind speed, etc.), and the cycle is 1-120 times, preferably 1-80 times, more preferably 1-50 times, for example 45 times.

[0127] As described above, the present invention provides a novel laser-induced graphene temperature control chip that can autonomously achieve ultra-fast temperature control. This chip is manufactured using laser-induced processing, allowing for batch production at a low cost. This chip can improve the temperature control module of current PCR instruments, reducing costs and increasing PCR reaction speed. Furthermore, this laser-induced graphene temperature control chip can be effectively applied to the field of instant rapid testing, thereby promoting PCR technology to truly achieve rapid on-site testing. It can also be used in high-throughput desktop detection devices.

[0128] Optionally, the PCR reaction chamber is a disc-type PCR detection chip having an upper and lower layer structure, which includes, from top to bottom, a sample injection layer 7 and a detection layer 8 .

[0129] As shown in Figure 17 , the sample injection layer 7 of the present invention has a diameter of 60 mm and a height of 3 mm. A rotating column 11 with a 5 mm raised projection is located on its upper surface, allowing for finger-operated rotation of the sample injection layer. An injection port 12 with a diameter of 4 mm and a height of 2 mm is located on one side of the rotating column 11, along with a sealing plug 10 that matches the size of the injection port 12. This plug seals the injection port after sample addition to prevent contamination.

[0130] The detection layer 8 has a diameter of 60 mm and a height of 7 mm. An annular slide 13 is provided on the outer surface of the upper surface of the detection layer 8, and a corresponding annular slider is provided on the lower surface of the sample feeding layer. These slides enable the sample feeding layer to rotate within the detection layer, allowing the cellulose filter paper in the sample feeding layer to enter different reaction chambers.

[0131] Below the injection port 12 is a filter paper mounting hole for inserting cellulose filter paper. The cellulose filter paper is cut into a 6mm diameter circle and taped to the bottom of the injection port with 3M tape, completely covering the port. This allows the sample to be directly absorbed by the cellulose filter paper after injection. When the sample is adsorbed onto the cellulose filter paper, the negatively charged macromolecular DNA curls and tightly binds to the cellulose filter paper matrix.

[0132] The detection layer 8 is cut into circular rings with an outer diameter of 50 mm and an inner diameter of 40 mm. These chambers include, in order, a sample injection chamber 14, a pre-wash chamber 15, a post-wash chamber 16, an amplification reaction chamber 17, and a detection chamber 18. Each reaction chamber is independent to prevent leakage. A test strip injection port is provided within the detection chamber and sealed with paraffin wax.

[0133] The front wash chamber 15 and the back wash chamber 16 hold 500 μL of wash buffer, such as Tris buffer, TE buffer, or FTA card purification reagents. The amplification reaction chamber 17 holds 50 μL of reaction buffer, including RPA reagents and LAMP reagents. The detection chamber 18 holds 100 μL of running buffer, such as Tris buffer or TE buffer. The test strip inlet is sealed with paraffin wax. Paraffin wax with a melting point of approximately 60°C is selected. After the amplification reaction is completed, the cellulose filter paper 10 is rotated into the detection chamber, allowing the product in the cellulose filter paper 10 to mix with the reaction buffer in the detection chamber 18. The heating device in this area is then heated to approximately 60°C, melting the paraffin and allowing the liquid to drip onto the test strip 9. The test strip groove 20 is located on the back of the detection layer 8 and communicates with the test strip inlet. An elliptical observation window 19, located within the groove, facilitates visual inspection of the immunoassay results.

[0134] The sampling layer and the detection layer were treated with surface hydrophobicity, that is, the two-layer structure was incubated with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane reagent in a vacuum for three hours, so that the silane formed a self-assembled monolayer on the inorganic surface, increasing its hydrophobicity and preventing leakage.

[0135] The upper and lower layers of the PCR reaction chamber snap together to form a sealed space, preventing liquid leakage. The front is labeled with numbers ①, ②, ③, and ④, corresponding to the sample injection chamber, pre-wash chamber, post-wash chamber, and amplification reaction chamber on the detection layer. This design mimics the appearance of a rotary dial telephone. Rotating the dial forces cellulose filter paper into different reaction chambers, enabling the three processes of sample injection, washing, and amplification.

[0136] When nucleic acid detection is performed using PCR, the present invention uses cellulose filter paper as an adsorbent material for nucleic acid detection, which can quickly bind nucleic acids from complex biological samples, retain them after a simple washing step, and can be directly released into the amplification reaction system. The present invention explores the ability of cellulose filter papers made of different materials to bind nucleic acids in blood. It was found that FTA cards can quickly capture nucleic acids in body fluids such as saliva, urine, blood, and serum. Therefore, the use of a rapid and economical method for purifying nucleic acids from different biological samples based on cellulose filter paper provides a key technical means for the PCR detection chip of the present invention to perform rapid and instant detection in resource-limited environments.

[0137] After the nucleic acid extraction is completed, when detecting the nucleic acid amplification product, an immune test strip is used to detect the product after nucleic acid amplification. The immune test strip is loaded in the test strip groove, and the sample pad is placed below the test strip injection hole. The detection line and quality control line are located below the observation window in the test strip groove.

[0138] Take the isothermal amplification of nucleic acid to detect EGFR gene in blood as an example. Cellulose filter paper and immune test strips are applied to the PCR detection chip of the present invention. 500 μL of Tris buffer is added to the pre-wash sample chamber and the post-wash sample chamber respectively. 50 μL of loop-mediated isothermal amplification reaction buffer is added to the amplification reaction chamber. 100 μL of TE buffer is added to the detection chamber as the running buffer. A certain concentration of EGFR plasmid is added to normal human blood to make the final concentration reach 10 5 copies / mL, 104copies / mL, 103copies / mL, 102copies / mL. Take 15μL of this series of samples and inject them into the injection port of the PCR detection chip of the present invention respectively, so that the nucleic acid plasmid is adsorbed on the cellulose filter paper. After washing twice, the cellulose filter paper is placed in the nucleic acid amplification reaction buffer, and the heating device is turned on to raise the temperature of the amplification reaction chamber to 65°C for nucleic acid amplification. After 40 minutes, the reaction is completed. The cellulose filter paper is rotated to the detection chamber, and the amplification product on the cellulose filter paper is diluted with running buffer, and the temperature of the reaction chamber is raised to 80°C to melt the paraffin in the detection chamber, and the paraffin is absorbed by capillary force through the test strip injection hole to the immune test strip for color reaction. The reaction results can be visually interpreted through the observation window. The immune test strips ①, ②, ③, ④, and ⑤ are 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 The results were as follows: 1) PCR detection chip 10 copies / mL and blank control, wherein immune test strips ①, ②, ③ all showed red strips on the test line and the quality control line, and immune test strips ④ and ⑤ only showed red strips on the quality control line, indicating that the PCR detection chip of the present invention can reach 10 3 The detection limit was 20 copies / mL.

[0139] Therefore, the present invention designs a low-cost, portable, on-site rapid nucleic acid PCR detection chip that, through a simple rotation operation, can meet the needs of rapid, on-site nucleic acid detection, especially in resource-limited areas. Its advantages are:

[0140] (1) A disc-shaped PCR detection chip was designed, imitating the appearance of a rotary dial telephone. The entire process, including nucleic acid extraction, washing, and amplification, can be completed by simply rotating the column with your fingers, requiring low technical skills from the experimental operator. The chip has a high degree of integration, consumes little reagents and samples, and only requires a drop of blood or other body fluid from the examinee to complete the sample input and result output. Finally, the test results can be directly read manually using an immunoassay strip, without the need for any instrument.

[0141] (2) The disc-type PCR detection chip is easy to produce and has low cost, including all elements such as the preparation of the PCR detection chip, nucleic acid amplification reaction buffer, enzymes and primers.

[0142] (3) The detection time is less than 60 minutes, including nucleic acid extraction and nucleic acid amplification, which is conducive to rapid and immediate screening of pathogens.

[0143] A nucleic acid detection method based on a disc-type PCR detection chip includes the following five steps:

[0144] Step 1: Rotate the injection port of the sample layer over the injection chamber of the detection layer to inject the sample. Use a pipette to inject a certain amount of body fluid (such as blood, serum, urine, or saliva) through the injection port onto the surface of the cellulose filter paper. Wait 3-5 minutes for the liquid to dry. Special chemicals in the cellulose filter paper can autonomously lyse cells in the body fluid and bind to negatively charged nucleic acids.

[0145] Step 2: Use your fingers to move the spin column in the sample injection chamber to rotate the cellulose filter paper to the front wash chamber. Wait for 2 minutes to wash away impurities on the surface of the cellulose filter paper, such as cells, proteins, and phenols. Gently moving the spin column left and right can speed up the washing process.

[0146] Step 3: Move the spin column in the sample chamber again to move the cellulose filter paper to the backwash chamber. Wait another 2 minutes to allow for more thorough washing. Gently moving the spin column left or right can speed up the washing process.

[0147] Step 4: Move the rotating column in the sample injection chamber to rotate the cellulose filter paper into the amplification reaction chamber. Then, turn on the heating device. When the temperature rises to a certain level, the reaction buffer in the amplification reaction chamber will initiate an amplification reaction with the nucleic acid on the cellulose filter paper, which is not limited to isothermal amplification and PCR reactions.

[0148] Step 5: After the reaction is complete, rotate the cellulose filter paper again to move it into the detection chamber. The amplification product on the cellulose filter paper is absorbed by the sample pad on the immunoassay strip through the connecting holes, and the results are displayed on the test line and the quality control line. The results can be observed visually through the observation window in the groove of the test strip.

[0149] In summary, it is demonstrated that the above-mentioned device has great socioeconomic benefits for expanding the fields of clinical pathogen molecular detection, epidemiological investigation, food inspection and quarantine in resource-limited environments.

[0150] Disc-type nucleic acid detection microfluidic chip

[0151] The following will be combined with the drawings in the embodiments of this application to clearly and completely describe the technical solutions in the embodiments of the disc-type nucleic acid detection microfluidic chip of this application. Obviously, the described embodiments are only for illustration and are not intended to limit this application.

[0152] As shown in FIG18 , the disc-shaped nucleic acid detection microfluidic chip of the present invention has a two-layer structure, including, from top to bottom, a top cover layer 21 and a liquid reservoir layer 22 .

[0153] The top cover 21 of the present invention has a diameter of 60 mm and a height of 3 mm. A rotating handle 25 with a 5 mm raised height is provided on the top surface, allowing the top cover to be rotated by pushing it with a finger. A sample inlet 26 with a diameter of 4 mm and a height of 2 mm is located on one side of the rotating handle 25, along with a cap 24 that matches the size of the sample inlet 26 and seals the inlet after sample addition to prevent contamination, as shown in Figure 19.

[0154] A circular first outer slide 27 and a first inner slide 28 are designed below the top cover layer 21. The first outer slide 27 and the first inner slide 28 are designed to facilitate the rotation of the top cover layer in the liquid reservoir layer. The inner diameter of the first outer slide 27 is 54 mm, the outer diameter is 56 mm, and the height is 3 mm. The diameter of the first inner slide 28 is 5 mm and the height is 3 mm. A nucleic acid filter paper fixing hole 29 with a diameter of 6 mm and a height of 1 mm is provided below the injection port 26 for embedding cellulose filter paper 30. The cellulose filter paper 30 is cut into a circle with a diameter of 6 mm and affixed to the bottom of the injection port with 3M tape, and can completely cover the injection port so that the liquid is directly absorbed by the cellulose filter paper after injection, as shown in Figure 20. When the sample is adsorbed onto the cellulose filter paper, the negatively charged macromolecular DNA curls and tightly binds to the cellulose filter paper matrix.

[0155] As shown in Figure 21, the reservoir layer 22 has a diameter of 60 mm and a height of 7 mm. A 3mm-deep, annular second outer slideway 31 is cut into the upper surface of the reservoir layer in the form of a ring with an outer diameter of 56 mm and an inner diameter of 54 mm. A 5mm-diameter groove serves as a cylindrical second inner slideway 32. These grooves match the height of the raised annular first outer slideway 27 and first inner slideway 28 in the top cover layer 21, facilitating the rotation of the top cover layer. That is, when the top cover layer rotates, the cellulose filter paper in the top cover layer can enter different reaction tanks through rotation.

[0156] Each reaction chamber is formed by cutting a 2.5mm deep circular slideway in the liquid reservoir layer, with an outer diameter of 50mm and an inner diameter of 40mm. This includes, in order, a sample loading reservoir 33, a first wash reservoir 34, a second wash reservoir 35, an amplification reaction reservoir 36, and a product detection reservoir 37. Each reaction reservoir is independent to prevent leakage. A test strip sampling port 38 is provided in the product detection reservoir and sealed with paraffin wax.

[0157] The bottom area of ​​the sample pool 33 is 68 mm 2 The bottom area of ​​the first washing tank 34 and the second washing tank 35 are both 235mm 2 , accommodating 500 μL of washing buffer, such as Tris buffer, TE buffer, FTA card purification reagent, etc.; the bottom area of ​​the amplification reaction pool 36 is 30 mm 2 , accommodating 50 μL of reaction buffer, including RPA reagent, LAMP reagent, etc.; the bottom area of ​​the product detection pool 37 is 40 mm 2 , accommodating 100μL running buffer, such as Tris buffer, TE buffer, etc. The test strip sampling hole 38 is a through hole with a diameter of 3mm, which is sealed by paraffin. Select paraffin with a melting point of about 60℃. After the amplification reaction is completed, rotate the cellulose filter paper to the product detection pool to mix the product in the cellulose filter paper 30 with the reaction buffer in the product detection pool 37. Then, increase the temperature of the heating device at this part to about 60℃ to melt the paraffin and let the liquid fall onto the test strip 23; the test strip groove 40 is located on the back of the liquid storage tank layer 22, with a length of 60mm, a width of 6mm, and a height of 2mm, and is connected to the test strip sampling hole; the observation window 39 is located in the test strip groove, which is an elliptical through hole with a long axis of 10mm and a short axis of 5mm, which is convenient for visually checking the results of the immune test strip.

[0158] The device's upper and lower layers utilize low-cost, easily processable, and biocompatible chip materials such as polymethyl methacrylate (PMMA), epoxy resin, PVC, and PC as substrates. The channels and reaction tanks are fabricated directly using laser etching, or the structure is etched onto the PMMA substrate and then molded with polydimethylsiloxane (PDMS). The top cover and reservoir layers undergo a hydrophobic treatment. The two-layer structure is treated with trichloro(1H,1H,2H,2H-perfluorooctyl)silane in a vacuum for three hours, allowing the silane to form a self-assembled monolayer on the surface, increasing its hydrophobicity and preventing leakage.

[0159] The chip's upper and lower layers snap together to form a sealed space, preventing liquid leakage. The front is labeled 1, 2, 3, and 4, corresponding to the sample reservoir, first wash reservoir, second wash reservoir, and amplification reaction reservoir. This design mimics the appearance of a rotary dial telephone. Rotating the dial forces cellulose filter paper into different reaction reservoirs, enabling the three processes of sample injection, washing, and amplification.

[0160] The present invention uses cellulose filter paper as a nucleic acid adsorption material, which can rapidly bind nucleic acids from complex biological samples. After a simple washing step, these nucleic acids are retained and released directly into the amplification reaction system. The present invention investigated the ability of cellulose filter paper made of different materials to bind nucleic acids in blood. 15 μL of blood from a lung cancer patient was dripped onto cellulose filter paper made of different materials. The blood was allowed to dry for 3-5 minutes, then washed twice in 0.1 M Tris buffer for 2-3 minutes each. Finally, the blood was amplified in LAMP reaction buffer for 40 minutes. EGFR gene detection was performed, and the amplified products were detected by agarose gel electrophoresis. A4 paper, toilet paper, FTA card, paper towel, and a test kit were used. Results showed that, with the exception of A4 paper, nucleic acids in the cellulose of the toilet paper, FTA card, and paper towel were rapidly captured and retained during the washing step. The capture efficiency was consistent with the test kit extraction method. In particular, the FTA card, soaked in a special denaturant and chelating agent, was able to adsorb more nucleic acids. When the FTA card was used to adsorb various biological samples, it was found that the card could rapidly capture nucleic acids from various body fluids, including saliva, urine, blood, and serum. Therefore, the rapid and cost-effective method of purifying nucleic acids from various biological samples using cellulose filter paper provides a key technical solution for the microfluidic chip of the present invention to enable rapid, immediate testing in resource-limited environments.

[0161] After the nucleic acid extraction is completed, when detecting the nucleic acid amplification product, an immune test strip is used for the detection of the product after nucleic acid amplification. The immune test strip is low-cost, fast and simple, and the result interpretation is intuitive. The immune test strip is loaded in the test strip groove, and the sample pad is placed below the test strip injection hole. The detection line and quality control line are located below the observation window in the test strip groove.

[0162] An immunoassay strip utilizes the principle of specific binding between antibodies and antigens to detect the substance being tested in a sample. Its basic structure is shown in Figure 22. A sample pad 41, a gold label pad 43, a nitrocellulose membrane 42, and an absorbent pad 44 are affixed to a base plate 45 in this order. The gold label pad contains a primary antibody labeled with colloidal gold. The nitrocellulose membrane is coated with streptavidin on the test line and a secondary antibody (goat anti-mouse IgG) on the control line. Forward primer 5 is modified with biotin at its end, while reverse primer 5 is modified with an antigen at its end. Table 1 shows examples of primer sequences used to detect the EGFR gene in the blood of lung cancer patients.

[0163] During the test, after a sample containing the substance to be detected is added to the sample pad, the sample moves toward the absorbent pad under the action of chromatography, first passing through the gold-labeled pad, specifically binding to the gold-labeled antibody therein, and continuing to move toward the absorbent paper end. When the sample moves to the test line 46, the substance to be detected in the sample (which has been bound to the gold-labeled antibody) specifically binds to the streptavidin in the test line in a double-sandwich manner, displaying a red strip. The gold-labeled antibody that has not been bound to the substance to be detected continues chromatography, moves to the quality control line 47, and specifically binds to the secondary antibody therein, displaying a red strip, as shown in Figure 23.

[0164] Take the isothermal amplification of nucleic acid to detect EGFR gene in blood as an example. Cellulose filter paper and immune test strips are applied to the microfluidic chip of the present invention. 500μl of Tris buffer is added to the first washing pool and the second washing pool, respectively. 50μl of loop-mediated isothermal amplification reaction buffer is added to the amplification reaction pool. 100μl of TE buffer is added to the product detection pool as running buffer. A certain concentration of EGFR plasmid is added to normal human blood to make the final concentration reach 10 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 copies / mL. Take 15μ5 of this series of samples and inject them into the injection port of the microfluidic chip of the present invention respectively, so that the nucleic acid plasmid is adsorbed onto the cellulose filter paper. After washing twice, the cellulose filter paper is placed in the nucleic acid amplification reaction buffer, and the heating device is turned on to raise the temperature of the amplification reaction pool to 65°C for nucleic acid amplification. After 40 minutes, the reaction is completed. The cellulose filter paper is rotated to the product detection pool, and the amplification product on the cellulose filter paper is diluted with running buffer, and the temperature of the reaction pool is raised to 80°C to melt the paraffin in the product detection pool, and the paraffin is absorbed by capillary force through the test strip injection hole to the immune test strip for color reaction. The reaction results can be visually interpreted through the observation window. The immune test strips 1, 2, 3, 4, and 5 are 10 respectively. 5 copies / mL, 10 4 copies / mL, 10 3 copies / mL, 10 2 The results were as follows: 10 copies / mL and blank control, among which immune test strips 1, 2, and 3 all showed red strips on the detection line and the quality control line, and immune test strips 4 and 5 only showed red strips on the quality control line, indicating that the microfluidic chip of the present invention can reach 10 3 The detection limit was 20 copies / mL.

[0165] Therefore, the present invention designs a low-cost, portable, on-site rapid nucleic acid detection chip that can meet the needs of rapid, on-site nucleic acid detection through a simple rotation operation, especially in resource-limited areas. Its advantages are:

[0166] A disc-type microfluidic chip was designed by imitating the appearance of a rotary dial telephone. The entire process including nucleic acid extraction, washing and amplification can be completed by just turning the handle with fingers, and low technical requirements are placed on the experimental operators.

[0167] The system is highly integrated, consumes minimal reagents and samples, and requires only a drop of blood or other bodily fluid from the test subject to deliver the results. Finally, the test results can be read directly using an immunoassay strip, without the need for any other instrument.

[0168] (3) Disc-type microfluidic chips are easy to produce and have low cost, including all elements such as the preparation of microfluidic chips, nucleic acid amplification reaction buffer, enzymes, and primers.

[0169] (4) The detection time is less than 60 minutes, including nucleic acid extraction and nucleic acid amplification, which is conducive to rapid and immediate screening of pathogens.

[0170] A disc-type microfluidic chip operation process based on nucleic acid detection includes the following five steps:

[0171] Step 1: Rotate the sample inlet of the top cover layer over the sample reservoir of the reservoir layer to inject the sample. Use a pipette to inject a certain amount of body fluid (such as blood, serum, urine, or saliva) through the sample inlet onto the surface of the cellulose filter paper. Wait 3-5 minutes for the liquid to dry. Special chemicals in the cellulose filter paper can autonomously lyse cells in the body fluid and bind to negatively charged nucleic acids.

[0172] Step 2: Use your finger to turn the handle on the top cover to move the cellulose filter paper to the first washing tank. Wait for 2 minutes to wash away impurities on the surface of the cellulose filter paper, such as cells, proteins, phenols, etc. Slightly turning the handle left and right can speed up the washing process.

[0173] Step 3: Move the top cover handle again to move the cellulose filter paper to the second washing tank and wait for 2 minutes to allow for a more thorough washing. Slightly moving the handle left or right can speed up the washing process.

[0174] Step 4: Move the handle on the top cover to rotate the cellulose filter paper into the amplification reaction pool. Then, turn on the heating device. When the temperature rises to a certain level, the reaction buffer in the amplification reaction pool and the nucleic acid on the cellulose filter paper will undergo an amplification reaction, which is not limited to isothermal amplification reactions and PCR reactions.

[0175] Step 5: After the reaction is complete, rotate the cellulose filter paper again to move it into the product detection reservoir. The amplification product on the cellulose filter paper is absorbed by the sample pad on the immunoassay strip through the connecting holes, and the results are displayed on the test and control lines. The results can be visually observed through the observation window in the groove of the test strip.

[0176] In summary, it is demonstrated that the proposed device has great socioeconomic benefits for expanding our clinical pathogen molecular detection, epidemiological investigation, food inspection and quarantine and other fields in resource-limited settings.

[0177] Table 1. Primer sequences used to detect EGFR gene in the blood of lung cancer patients

[0178] The present invention also discloses a detection device with the disc-type nucleic acid detection microfluidic chip, comprising a graphene heating device, a power supply, and a power control module. The graphene heating device is connected to the power supply, and the power supply is controlled by the power control module. The power control module controls the power supply to perform power on / off cycle operation on the graphene heating device. The graphene heating device contacts the disc-type nucleic acid detection microfluidic chip and heats the disc-type nucleic acid detection microfluidic chip when powered on, and cools the disc-type nucleic acid detection microfluidic chip when powered off, thereby realizing a heating / cooling cycle of the disc-type nucleic acid detection microfluidic chip.

[0179] The graphene heating device includes a laser-induced graphene layer and a substrate material layer. The laser-induced graphene layer is located on the surface of the substrate material layer and has a thickness of 1-120 μm, preferably 2-100 μm, 3-80 μm, or 5-60 μm. During the preparation process, a special carbon-containing precursor material is provided. The precursor material is placed on a material substrate. The precursor material includes but is not limited to polyimide, phenolic resin, polyethyleneimine, lignocellulose, wood, etc. A laser is used to directly scan the surface of the above-mentioned special carbon-containing precursor material. The local heat generated by laser irradiation causes the surface of the precursor material to be carbonized. During the carbonization process, carbon atoms are converted from sp 3 Key reorganization into sp 2 The bonds are broken, and the oxygen and nitrogen groups of the precursor material are decomposed to obtain a laser-induced graphene heating device.

[0180] In order to meet the rapid temperature rise and fall requirements of the nucleic acid detection chip, the laser flux at the beginning of the laser-induced graphene material formation in this embodiment is 5.5 J / cm 2 By adjusting the laser parameters, the physical and chemical properties of the laser-induced graphene material can be controlled. By increasing the laser power, the thickness of the laser-induced graphene material can be increased, the conductivity can be enhanced, and the resistance can be reduced. According to the current thermal effect (Q = V 2 / R*t), under the same input voltage conditions, the smaller the resistance, the more heat is generated in the same time, and the faster the heating rate. At the same time, increasing the laser power will increase the gas release rate during the preparation process, thereby increasing the number of pores, increasing the porosity and pore size of the laser-induced graphene material, and achieving a faster cooling rate. However, if the laser power exceeds the threshold, the structure of the laser-induced graphene material will be destroyed and lose its function. In this embodiment, the laser flux selected to start the formation of the laser-induced graphene material is 5.5J / cm 2 .

[0181] The graphene heating device is placed close to the disc-shaped nucleic acid detection microfluidic chip. After the low-voltage DC power supply powers the graphene heating device, ultra-fast heating (>10°C / s) is achieved. After the voltage is turned off, ultra-fast cooling (>10°C / s) can be achieved independently by relying on the heat dissipation of the surrounding environment, without the need for other auxiliary cooling methods. Compared with traditional PCR instruments, heat conduction needs to go from the traditional PCR instrument heating material to the aluminum base plate to a thin layer of air, and finally to the disc-shaped nucleic acid detection microfluidic chip. The heat conduction efficiency is high, the heat loss is small, and the temperature rise and fall speed is fast. The multi-step heat conduction path in the traditional PCR instrument is reduced, the heat conduction efficiency is effectively improved, and the reaction speed is further increased.

[0182] The present invention also provides an optimal control method for the above-mentioned detection. Specifically, the control method includes controlling the power-on / off time (power supply voltage) of the graphene heating device by the power supply during the thermal cycle. The power-on / off time (power supply voltage) control is implemented by the control module of the power supply, and precise control of temperature rise and fall under different conditions is achieved according to environmental conditions (temperature, wind speed, etc.), and the cycle is repeated 45 times.

[0183] As described above, the present invention provides a novel ultra-fast temperature control chip based on laser-induced graphene materials that can autonomously achieve ultra-fast temperature control. This chip, fabricated using laser-induced processing, can be manufactured in batches at a low cost. This improves the temperature control module of current PCR instruments, reducing costs and increasing PCR reaction speed. Furthermore, this ultra-fast temperature control chip based on laser-induced graphene materials can be effectively applied in the field of rapid point-of-care testing, thereby promoting the realization of rapid on-site PCR technology.

[0184] Example

[0185] The following examples illustrate the present invention by way of example only, but the present invention is not limited thereto. The scope of the present invention is limited only by the claims.

[0186] General preparation procedure of laser-induced graphene

[0187] In air atmosphere, 5.5 J / cm 2 , 5.8J / cm 2, 10J / cm 2 , 15J / cm 2 , 20J / cm 2 , 25J / cm 2 、30J / cm 2 、36J / cm 2 、38J / cm 2 , 40J / cm 2 , 46J / cm 2 , 50J / cm 2 , 60J / cm 2 , 70J / cm 2 , 80J / cm 2 Lasers with different laser fluxes were scanned on a polyimide film substrate with a thickness of 100 μm at a rate of 100 mm / s. The local heat generated by the laser irradiation carbonized the surface of the precursor material, thereby forming graphene.

[0188] FIG1 shows the C peak of the XPS of the graphene material prepared by the method of the present invention (laser flux is 36 J / cm 2 ). As can be seen from Figure 1, the obtained material shows significant sp 2 peak, indicating the formation of graphene structure.

[0189] Figure 2 shows the results of a 5.5 J / cm 2 , 5.8J / cm 2 , 10J / cm 2 , 15J / cm 2 , 20J / cm 2 , 25J / cm 2 、30J / cm 2 、36J / cm 2 、38J / cm 2 , 40J / cm 2 , 46J / cm 2 , 50J / cm 2 , 60J / cm 2 , 70J / cm 2 , 80J / cm 2 Surface morphology of laser-induced graphene (No. 1-15).

[0190] Figure 3 shows the results of the experiment with a flux of 40 J / cm 2 The temperature rise curves of the laser-induced graphene material at different voltages show that the maximum surface temperature can reach 398.5°C ((All temperature curve values ​​in Figures 3-5, 7-11 are obtained by OMEGA TM Ultra-fine bare thermocouples are used to directly measure the surface temperature of the heated material / internal temperature of the liquid).

[0191] Figure 4 shows the results of the experiment with a flux of 36 J / cm 2 The laser-induced heating curve of graphene material under different voltages has the largest heating rate, reaching 68.5℃ / S.

[0192] Figure 5 shows the laser-induced graphene (laser flux of 36 J / cm 2 ) The maximum surface temperature and heating rate have a clear functional relationship with the input power. Under the same input power, three independent laser-induced graphene (all at 36J / cm 2 The same laser flux and the same conditions induce the same surface temperature (Figure 6). This shows that the temperature of laser-induced graphene can be quickly and conveniently regulated by changing the input electrical energy density. Due to the extremely high electrothermal conversion efficiency, as well as the very low specific heat capacity and multi-layer porous structure of the material itself, the rate of conversion of electrical energy into thermal energy is very fast, which also makes the temperature regulation of laser-induced graphene not only convenient but also fast. Figure 7 shows that during the continuous heating process, the input voltage is increased in steps of 1V, and the laser-induced graphene (laser flux is 36J / cm 2 ) The surface temperature rapidly rises in response (the infrared images in Figure 6 and the maximum and average temperatures of the heated material surface were captured using a FLIR infrared camera). Furthermore, under dynamic temperature changes, the actual surface temperature is nearly identical to that achieved by heating from scratch (Figure 8), demonstrating the inherently stable electrical power control capability of laser-induced graphene.

[0193] Laser-induced graphene also has a rapid cooling capability. Due to the low input power and the loose porous structure of the surface (a unique microstructural advantage of laser-induced graphene over graphene formed by traditional chemical deposition or surface growth), heat can be dissipated quickly after power is turned off. The maximum natural cooling rate reaches 36°C / s, far exceeding that of traditional electrothermal materials (Figure 9, laser flux is 36J / cm 2 ).

[0194] Laser-induced graphene can maintain stable performance during long-term heating and cooling. Figure 10 shows the heating and cooling rate and the temperature level that can be achieved during 100 heating and cooling cycles. The temperature remains stable in each cycle (the laser flux is 36J / cm 2 ).

[0195] In summary, with the help of the efficient electrothermal conversion capability of laser-induced graphene, the rapid and accurate temperature response under external applied voltage, the rapid temperature change capability and the efficient heat transfer properties, the liquid temperature can be precisely controlled to meet the temperature accuracy requirements of the PCR process. Figure 11 shows the laser-induced graphene (laser flux of 36J / cm2 ) The temperature (red) is regularly raised and lowered, so that the PCR solution temperature (blue) changes rapidly between 60°C and 95°C, completing the PCR.

[0196] The above applications are only some embodiments of the present application. For those skilled in the art, without departing from the inventive concept of the present application, several modifications and improvements can be made, which all fall within the scope of protection of the present application.

Claims

1. A method for preparing laser-induced graphene, comprising: Laser induced processing is used to reduce the precursor material layer.

2. The method according to claim 1, comprising: Providing a carbon-containing precursor material layer, scanning the surface of the carbon-containing precursor material layer with a laser, and carbonizing the surface of the precursor material by local heat generated by laser irradiation, thereby forming graphene; Preferably, the precursor material layer is located on the base material layer; Preferably, the precursor material layer and the base material layer are integrally made of the same material, or are separate different materials; Preferably, the precursor material is selected from polyimide, phenolic resin, polyethyleneimine, lignocellulose or wood; Preferably, the laser used is selected from a carbon dioxide laser or a femtosecond laser; Preferably, the laser scanning rate is 0.5-500 mm / s, preferably 20-300 mm / s, more preferably 50-150 mm / s; Preferably, the laser flux of the laser induced processing is greater than or equal to 5.5 J / cm 2 , preferably greater than 5.5 J / cm 2 , more preferably 5.8-80 J / cm 2 , more preferably 5.8-60 J / cm 2 , most preferably 5.8-50J / cm 2 ; Preferably, the substrate material is selected from polyimide material, polyimide film, polyimide tape, phenolic resin, polyethyleneimine, wood cellulose or wood.

3. Laser induced graphene obtained by the method according to claim 1 or 2.

4. Use of laser induced graphene in PCR detection according to claim 3.

5. A laser-induced graphene PCR detection device, comprising a PCR reaction chamber and a graphene heating device.

6. The laser-induced graphene PCR detection device according to claim 5, wherein the graphene heating device comprises a laser-induced graphene layer and a substrate material layer; Preferably, the laser-induced graphene layer is located on the surface of the base material layer, and has a thickness of 1-120 μm, preferably 2-100 μm, 3-80 μm, or 5-60 μm.

7. The laser-induced graphene PCR detection device according to claim 5 or 6, wherein the laser-induced graphene layer is prepared by laser-induced processing to reduce a precursor material layer, and the precursor material layer is located on the surface of the substrate material layer. Preferably, the laser flux formed at the beginning of the laser-induced processing is 5.5 J / cm 2 .

8. The laser-induced graphene PCR detection device according to any one of claims 5 to 7, wherein the laser used is selected from a carbon dioxide laser and a femtosecond laser, and the substrate material layer is selected from a polyimide material, a polyimide film, a polyimide tape, a phenolic resin, polyethyleneimine, lignocellulose or wood; Preferably, the detection device further comprises a power supply and a power control module, and the power control module controls the power supply to perform power on / off cycle operation on the graphene heating device; Preferably, the graphene heating device is in contact with the PCR reaction chamber, and heats the PCR reaction chamber when powered on, and cools the PCR reaction chamber when powered off, so as to achieve a heating / cooling cycle of the PCR reaction chamber.

9. A laser-induced graphene PCR detection device, characterized in that: The invention comprises a PCR reaction chamber, a graphene heating device, a power source, and a power control module. The graphene heating device is connected to the power source, and the power source is controlled by the power control module. The power control module controls the power source to perform power-on / power-off cycle operation on the graphene heating device. The graphene heating device is in contact with the PCR reaction chamber, and heats the PCR reaction chamber when powered on, and cools the PCR reaction chamber when powered off, so as to realize the heating / cooling cycle of the PCR reaction chamber.

10. The laser-induced graphene PCR detection device according to claim 9, characterized in that: The graphene heating device comprises the laser induced graphene layer according to claim 4; Preferably, the power supply is a low voltage DC power supply; Preferably, the power source includes but is not limited to a battery, a rechargeable battery, a mobile power source, a mobile phone, a power supply, a 220V power supply; Preferably, the graphene heating device is in sheet shape; Preferably, the graphene heating device comprises a laser induced graphene layer and a base material layer, wherein the laser induced graphene layer is located on the surface of the base material layer and has a thickness of 1-120 μm, preferably 2-100 μm, 3-80 μm, or 5-60 μm; Preferably, the PCR detection device is an instant rapid detection device or a high-throughput desktop detection device; Preferably, the PCR reaction chamber is a disc-type PCR detection chip, and the disc-type PCR detection chip comprises an upper sample injection layer and a lower detection layer; Preferably, the sample injection layer is included in the top cover layer, and the detection layer is included in the liquid reservoir layer.

11. A method for performing PCR detection using the detection device according to claim 9 or 10, characterized in that: The power control module is used to control the power supply to perform power on / off cycles on the graphene heating device, so that the thermal cycle of the PCR reaction chamber is 1-120 times, preferably 1-80 times, more preferably 1-50 times, for example 45 times.

12. A PCR detection chip, comprising a top cover layer located at the top and a liquid reservoir layer located at the bottom, wherein the top cover layer can rotate relative to the liquid reservoir layer, the top cover layer comprises an injection port for adding a test sample, and an adsorbent for adsorbing the test sample is provided below the injection port; The liquid storage pool layer at least includes a sample addition pool, an amplification reaction pool, and a product detection pool. During the rotation of the top cover layer, the adsorbent can enter the sample addition pool, the amplification reaction pool, and the product detection pool in sequence. A test strip injection hole is arranged in the product detection pool.

13. The PCR detection chip according to claim 12, characterized in that: The top cover layer and the liquid reservoir layer are buckled together to form a sealed space as a whole, and both the top cover layer and the liquid reservoir layer are circular; Preferably, the top cover layer is provided with a rotating handle which can be turned to achieve rotation; Preferably, the product detection pool is sealed with a sealant, which can melt at a certain temperature and make the liquid in the product detection pool fall; preferably, the sealant is paraffin, which can melt at 60-90°C; Preferably, a test strip groove for adding a test strip is provided below the liquid reservoir layer, and a through hole is provided at one end of the test strip groove as an observation window; Preferably, at least one washing pool is further included between the sample loading pool and the amplification reaction pool; preferably, there are two washing pools, including a first washing pool and a second washing pool; Preferably, the top cover layer and the liquid reservoir layer are relatively rotated by a circular slideway; Preferably, the adsorbent is cellulose filter paper; Preferably, the material of the top cover layer and the liquid reservoir layer is selected from polymethyl methacrylate PMMA, epoxy resin, PVC, PC, and the concave-convex structure of the slideway and the reaction pool is made by laser etching technology, or etching the structure on the substrate and then using polydimethylsiloxane PDMS for molding; Preferably, the first washing pool and the second washing pool are respectively loaded with washing buffer, the amplification reaction pool is loaded with nucleic acid amplification reaction buffer or PCR reaction reagent, and the product detection pool is loaded with running buffer; Preferably, the surface of the top cover layer is marked accordingly according to the sample loading pool, the first washing pool, the second washing pool, and the amplification reaction pool of the liquid reservoir layer; Preferably, the top cover layer and the reservoir layer are subjected to surface hydrophobic treatment, that is, the top cover layer and the reservoir layer are treated with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane reagent in a vacuum for three hours, so that trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane forms a self-assembled monolayer on the surface of the top cover layer and the reservoir layer.

14. A method for nucleic acid detection using the chip according to claim 12 or 13, characterized in that: The following steps are involved: Step 1, rotate the injection port of the top cover layer above the sample pool of the liquid reservoir layer, use a pipette to inject a certain amount of body fluid onto the surface of the adsorbent through the injection port, and wait for a certain period of time to allow the body fluid to dry; Step 2, use your fingers to move the rotating handle of the top cover layer to rotate the adsorbent to the first washing pool to wash away impurities on the surface of the adsorbent; in this step, moving the rotating handle left and right can speed up the washing speed; Step 3, turn the rotating handle of the top cover layer again to rotate the adsorbed object to the second washing tank for further washing; in this step, turning the rotating handle left and right can speed up the washing speed; Step 4, turning the rotating handle of the top cover layer to rotate the adsorbent to the amplification reaction pool; then turning on the heating device to raise the temperature to a certain level, so that the reaction buffer in the amplification reaction pool and the nucleic acid on the adsorbent undergo an amplification reaction, and the amplification reaction includes but is not limited to a constant temperature amplification reaction and a PCR reaction; Step 5, after the amplification reaction is completed, rotate again to rotate the adsorbent to the product detection pool; the amplification product on the adsorbent is absorbed by the test paper through the connecting hole; and the result is observed with the naked eye through the observation window on the groove of the test strip.

15. A disc-type nucleic acid detection microfluidic chip, characterized in that: The invention comprises a top cover layer located at the top and a liquid reservoir layer located at the bottom, the top cover layer and the liquid reservoir layer are buckled together to form a sealed space as a whole, the top cover layer and the liquid reservoir layer are both circular, and the top cover layer can rotate relative to the liquid reservoir layer, the top cover layer comprises an injection port for adding a test sample, and an adsorbent for adsorbing the test sample is provided below the injection port; the top cover layer is provided with a rotating handle that can be turned to realize its rotation; The liquid storage pool layer at least includes a sample loading pool, an amplification reaction pool, and a product detection pool. During the rotation of the top cover layer, the adsorbent can sequentially enter the sample loading pool, the amplification reaction pool, and the product detection pool. The product detection pool is provided with a test strip injection hole and sealed with a sealant. The sealant can melt at a certain temperature and allow the liquid in the product detection pool to fall down. A test strip groove for adding a test strip is provided below the liquid storage tank layer, and a through hole is provided at one end of the test strip groove as an observation window.

16. The disc-type nucleic acid detection microfluidic chip according to claim 15, characterized in that: At least one washing pool is further included between the sample loading pool and the amplification reaction pool; preferably, there are two washing pools, including a first washing pool and a second washing pool; Preferably, the top cover layer and the liquid reservoir layer are relatively rotated by a circular slideway; Preferably, the sealant is paraffin, which can melt at 60-90°C; Preferably, the first washing pool and the second washing pool are respectively loaded with washing buffer, the amplification reaction pool is loaded with nucleic acid amplification reaction buffer or PCR reaction reagent, and the product detection pool is loaded with running buffer; Preferably, the surface of the top cover layer is marked accordingly according to the sample loading pool, the first washing pool, the second washing pool, and the amplification reaction pool of the liquid reservoir layer; Preferably, the top cover layer and the reservoir layer are subjected to surface hydrophobic treatment, that is, the top cover layer and the reservoir layer are treated with trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane reagent in a vacuum for three hours, so that trichloro(1H, 1H, 2H, 2H-perfluorooctyl)silane forms a self-assembled monolayer on the surface of the top cover layer and the reservoir layer.

17. A method for nucleic acid detection using the chip according to claim 15 or 16, characterized in that: The following steps are involved: Step 1, rotate the injection port of the top cover layer above the sample pool of the liquid reservoir layer, use a pipette to inject a certain amount of body fluid onto the surface of the adsorbent through the injection port, and wait for a certain period of time to allow the body fluid to dry; Step 2, use your fingers to move the rotating handle of the top cover layer to rotate the adsorbent to the first washing pool to wash away impurities on the surface of the adsorbent; in this step, moving the rotating handle left and right can speed up the washing speed; Step 3, turn the rotating handle of the top cover layer again to rotate the adsorbed object to the second washing tank for further washing; in this step, turning the rotating handle left and right can speed up the washing speed; Step 4, turning the rotating handle of the top cover layer to rotate the adsorbent to the amplification reaction pool; then turning on the heating device to raise the temperature to a certain level, so that the reaction buffer in the amplification reaction pool and the nucleic acid on the adsorbent undergo an amplification reaction, and the amplification reaction includes but is not limited to a constant temperature amplification reaction and a PCR reaction; Step 5, after the amplification reaction is completed, rotate again to rotate the adsorbent to the product detection pool; the amplification product on the adsorbent is absorbed by the test paper through the connecting hole; and the result is observed with the naked eye through the observation window on the groove of the test strip.

18. A detection device with the disc-type nucleic acid detection microfluidic chip according to claim 15 or 16, characterized in that: The invention comprises a graphene heating device, a power supply, and a power supply control module. The graphene heating device is connected to the power supply, and the power supply is controlled by the power supply control module. The power supply control module controls the power supply to perform power-on / power-off cycle operation on the graphene heating device. The graphene heating device contacts a disc-type nucleic acid detection microfluidic chip, heats the disc-type nucleic acid detection microfluidic chip when powered on, and cools the disc-type nucleic acid detection microfluidic chip when powered off, so as to realize the heating / cooling cycle of the disc-type nucleic acid detection microfluidic chip.

19. The detection device according to claim 18, wherein the graphene heating device comprises the laser induced graphene according to claim 4.

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