Centrifugal micro-fluidic chip for liquid drop digital nucleic acid amplification detection

Abstract

The invention discloses a centrifugal micro-fluidic chip for liquid drop digital nucleic acid amplification detection, the centrifugal micro-fluidic chip comprises a disc body and an adaptive pressure-sensitive adhesive film layer, and the disc body comprises a sample loading area arranged at the periphery of a centrifugal shaft hole; the front end of the extraction channel is connected to the sample loading area, and the rear end is connected with the switching valve; the magnetic bead nucleic acid extraction area is distributed on the outer edge of the extraction channel and is provided with an embedded magnet array; the collecting cavity is connected to the output end of the switching valve; a quantitative distribution channel; a gelatin-based thermosensitive valve; and the plurality of water-in-oil droplet generation and reaction cavities are annularly and uniformly distributed at the edge of the disc body and are communicated with the collection cavity. The chip realizes full-process automation of nucleic acid extraction, droplet generation and amplification detection through cooperation of centrifugal force and a functional structure, is simple and convenient to operate, high in sensitivity and accurate in result, and is suitable for the fields of gene diagnosis, pathogen detection and the like.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, and in particular relates to a centrifugal microfluidic chip for droplet digital nucleic acid amplification detection. Background Technology

[0002] Digital nucleic acid amplification detection, through sample partitioning and independent amplification, can achieve absolute quantification of mutated genes with a sensitivity of 0.005–0.1%, and is tolerable. PCR Inhibitors. However, their application is limited by (1) the contradiction between operational complexity and the applicability of point-of-care testing; (2) the difficulty in balancing robustness, cost and performance; and (3) the insufficient multiplex detection capabilities of miniaturized or portable platforms, which restricts large-scale clinical screening and rapid decision-making. There is an urgent need to develop simpler, more economical and faster digital nucleic acid testing technologies to meet the testing needs of multiple scenarios.

[0003] Microfluidic systems offer a promising platform for portable molecular diagnostics and have been applied in various clinical scenarios. Among them, the centrifugal microfluidic platform utilizes centrifugal force to automate fluid manipulation, enabling integrated nucleic acid detection in a compact design. This platform requires only a single servo motor to achieve precise liquid control using centrifugal, Coriolis, and Euler forces, significantly simplifying the miniaturization process and becoming a core technological architecture in the field of applied microfluidics.

[0004] Currently, the invention patent CN119955608A uses gel materials as a medium for the adsorption and desorption of nucleic acid molecules in nucleic acid extraction. However, in practice, it has been found that the gel modification process is complex, the feasibility of mass production is not high, and the pore size of the gel needs to be strictly controlled, otherwise the desorption of long-chain nucleic acid molecules is difficult. Secondly, the chip in patent CN119955608A needs to be used with lyophilized amplification reagents, but most manufacturers' amplification reagents contain glycerol, which is difficult to prepare in a lyophilized state. Therefore, it is necessary to design a chip that is compatible with liquid amplification reagents. Summary of the Invention

[0005] To address the technical problems existing in the background art described above, the present invention provides a centrifugal microfluidic chip and a droplet digital nucleic acid amplification and detection chip.

[0006] This invention employs the following technical solution: a centrifugal microfluidic chip for droplet digital nucleic acid amplification detection, wherein the centrifugal microfluidic chip has a chip structure layer, the chip structure layer includes a disk body, a centrifugal shaft hole is formed at the center of the structure layer, and further includes: The sample loading area is set in a designated area around the centrifugal shaft hole of the structural layer; An extraction channel is opened on the disc body according to a predetermined path. The front end of the extraction channel is connected to the sample loading area, and the rear end is connected to a switching valve. The magnetic bead nucleic acid extraction area is arranged in a designated area at the outer edge of the extraction channel, and the magnetic bead nucleic acid extraction area is equipped with an embedded magnet array to form a static magnetic field system. The collection chamber is connected to one of the output terminals of the switching valve; Several water-in-oil droplet generation and reaction chambers are uniformly arranged in a ring at the edge of the disk body; the collection chamber is connected to the several water-in-oil droplet generation and reaction chambers through a quantitative distribution channel; A thermistor valve is also provided between the quantitative dispensing channel and the water-in-oil droplet generation and reaction chamber to control the timing of the nucleic acid solution entering the water-in-oil droplet generation and reaction chamber.

[0007] In a further embodiment, the embedded magnet array of the magnetic bead nucleic acid extraction region includes: a single-row magnet array, a double-row magnet array, and a single-sided double-row magnet array.

[0008] In a further embodiment, the single-row magnet array is distributed as follows: it is evenly distributed on the outside of the extraction channel along the path of the extraction channel, and the magnetic beads are at the same vertical distance from the extraction channel.

[0009] In a further embodiment, the dual-row magnet array is distributed as follows: it is evenly distributed on the outer and inner sides of the extraction channel along the path of the extraction channel, and the magnetic beads are at the same vertical distance from the extraction channel.

[0010] In a further embodiment, the single-sided double-row magnet array is distributed as follows: it is distributed at equal intervals along the path of the extraction channel on the outside of the extraction channel and is set in two rows. The magnetic beads adjacent to the extraction channel are at the same vertical distance from the extraction channel, and the outermost magnetic bead is at the same vertical distance from the magnetic bead adjacent to the extraction channel.

[0011] In a further embodiment, the thermostatic valve is a gelatin thermostatic valve, which has precise phase change characteristics of gelling at less than 10°C and liquefying at more than 30°C.

[0012] In a further embodiment, the sample loading area includes a sample cavity connected to the front end of the extraction channel; A washing buffer reservoir is connected to the front end of the extraction channel via the first channel; The elution buffer reservoir is connected to the front end of the extraction channel via a second channel; The sample chamber, washing buffer reservoir, and elution buffer reservoir are arranged in a circumferential array around the centrifuge shaft hole of the structural layer. In a further embodiment, it also includes: a waste liquid chamber connected to another output end of the switching valve via a branch channel; each output end of the switching valve is provided with an air valve.

[0013] In a further embodiment, an amplification reagent reservoir and a quantitative chamber are sequentially arranged between the thermal valve and the quantitative dispensing channel; A residual liquid chamber is provided at the end of the quantitative distribution channel.

[0014] The beneficial effects of this invention are as follows: The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection provided by this invention successfully overcomes the key limitations of existing nucleic acid detection platforms, demonstrating excellent performance in terms of ease of operation, analytical performance, multiplexing capability, and cost-effectiveness. Specifically, this invention forms a static magnetic field system by arranging an embedded magnet array in the extraction area to achieve on-chip nucleic acid extraction; and for the first time, gelatin material is used in the thermistor valve of the microfluidic chip for switching liquid amplification reagents into droplet generation and the reaction chamber.

[0015] No external magnets or moving parts are required. The array of magnets achieves uniform wall distribution and bead deposition to prevent excessive aggregation. Strong adsorption capacity ensures no loss during washing and elution. Experiments show that 10% ( v / v The gelatin matrix exhibits ideal phase change properties: including gelation below 10°C and liquefaction above 30°C, further optimized to controllable gelation at 4°C and rapid liquefaction at 37°C, excellent biocompatibility, and reversible operation. In a fully automated processing flow, it provides optimal fluid control while ensuring... LAMP The reagent activity is unaffected, nor is the formation and stability of water-in-oil droplets affected. Attached Figure Description

[0016] Figure 1 This is a structural diagram of a centrifugal microfluidic chip for droplet digital nucleic acid amplification detection.

[0017] Figure 2 It is the adsorption kinetics of magnetic beads in microchannels. COMSOL Multiphysics numerical simulation diagram.

[0018] Figure 3 ( a () is a line graph showing the relationship between the maximum magnetic flux density and the distance between the magnets.

[0019] Figure 3 ( b ( ) is a scatter plot of the magnetic bead adsorption distribution.

[0020] Figure 4 ( a ( ) is a cloud map showing the distribution of magnetic field and fluid velocity in a single-row magnet array.

[0021] Figure 4 ( b ( ) is a cloud map showing the distribution of magnetic field and fluid velocity in a double-row magnet array.

[0022] Figure 4 ( c ( ) is a cloud map of the magnetic field and fluid velocity distribution of a single-sided double-row magnet array.

[0023] Figure 4 ( d ( ) is a scatter plot of magnetic bead adsorption under different magnet array configurations.

[0024] Figure 5 ( a )yes DNA Recycling efficiency graph.

[0025] Figure 5 ( b ) is the limit of detection for digital nucleic acid amplification (DNI) LOD Analysis diagram.

[0026] Figure 5 ( c () is the extraction under different experimental conditions gDNA Concentration comparison chart.

[0027] Figure 6 ( a () are different nucleic acid extraction protocols A 260 / A Box plot for 230 purity.

[0028] Figure 6 ( b () are different nucleic acid extraction protocols A 260 / A Box plot for 280 purity.

[0029] Figure 6 ( c ( ) is a graph showing the nucleic acid amplification kinetics of different nucleic acid extraction schemes.

[0030] Figure 6 ( d () is a bar chart showing the nucleic acid amplification yield of different nucleic acid extraction schemes.

[0031] Figure 7 This is a rheological characterization of gelatin solutions of different concentrations.

[0032] Figure 8 This is a comparison chart of the liquefaction effects of introducing a gelatin-based thermistor valve.

[0033] Figure 9 ( a () represents the rotational operation control state of the chip at different stages.

[0034] Figure 9 ( b )for mddLAMP - LoDA flowchart of the fluid handling process on the chip.

[0035] Figure 10 These are photos of each working node.

[0036] Figure 11 yes PIK 3 CA E 542 K The results of the mutation gene detection and the statistical results of droplet brightness are shown in the figure.

[0037] Figure 1 The following are labeled as follows: Sample chamber 1, Washing buffer reservoir 2, Elution buffer reservoir 3, Magnetic bead nucleic acid extraction area 4, Switching valve 5, Collection chamber 6, Waste liquid chamber 7, Quantitative dispensing channel 8, Quantitative chamber 9, Amplification reagent reservoir 10, Thermosensitive valve 11, Water-in-oil droplet generation and reaction chamber 12, Residual liquid chamber 13. Detailed Implementation

[0038] The present invention will now be further described with reference to the accompanying drawings and embodiments.

[0039] Example 1 To achieve integrated nucleic acid extraction without the need for a precision external magnet actuator, this invention proposes a static magnetic field system employing an embedded magnet array, and provides a centrifugal microfluidic chip for droplet digital nucleic acid amplification detection, named […]. multiplex droplet digital LAMP - based lab - on - a - disc , mddLAMP - LoD hereinafter referred to as mddLAMP - LoD chip.

[0040] The specific structure is as follows: Figure 1 As shown, the centrifugal microfluidic chip has a chip structure layer, which includes a disk body with a centrifugal shaft hole at its center. Correspondingly, the centrifugal microfluidic chip in this embodiment also has a pressure-sensitive adhesive film layer adapted to the disk body. Figure 1 The different colors in the diagram represent different radial lengths of the components, and the corresponding numerical examples are given in the diagram to facilitate a better understanding of the invention.

[0041] In this embodiment, a sample loading area is provided around the centrifugal shaft hole in the structural layer, such as... Figure 1The sample chamber 1, washing buffer reservoir 2, and elution buffer reservoir 3 are arranged in a circular array around the centrifuge shaft holes in the structural layer. Specifically, the sample chamber 1 is connected to the front end of the extraction channel; the washing buffer reservoir 2 is connected to the front end of the extraction channel via a first channel; and the elution buffer reservoir 3 is connected to the front end of the extraction channel via a second channel.

[0042] It also includes: an extraction channel opened on the disc body according to a predetermined path, the front end of the extraction channel being connected to the sample loading area and the rear end being connected to a switching valve 5; one of the output ends of the switching valve 5 is connected to a collection chamber.

[0043] A magnetic bead nucleic acid extraction area 4 is arranged in a designated area at the outer edge of the extraction channel. The magnetic bead nucleic acid extraction area 4 is equipped with an embedded magnet array to form a static magnetic field system, so as to realize integrated nucleic acid extraction without the need for a precision external magnet driver.

[0044] A plurality of water-in-oil droplet generation and reaction chambers 12 are uniformly arranged in a ring at the edge of the disc body, and the plurality of water-in-oil droplet generation and reaction chambers 12 are connected to the collection chamber.

[0045] This embodiment uses multiphysics simulation ( COMSOL Simulating the motion trajectory of a magnetic bead in a magneto-fluid coupled field, the system was successfully optimized to achieve three key performance indicators: i Uniform wall distribution of bead deposition prevents excessive aggregation; ii The strong adsorption capacity ensures no loss during washing and elution. This static design eliminates moving parts while guaranteeing high reproducibility of magnetic bead operation throughout the extraction process.

[0046] Based on this, the embedded magnet array of the magnetic bead nucleic acid extraction region 4 in this embodiment includes: a single-row magnet array, a double-row magnet array, and a single-sided double-row magnet array.

[0047] In a further embodiment, it also includes: a waste liquid chamber, which is connected to another output end of the switching valve 5 via a branch channel; each output end of the switching valve 5 is provided with an air valve.

[0048] This embodiment simulates the adsorption kinetics of magnetic beads within a microchannel by mimicking a static array of magnets. COMSOL Multiphysics numerical simulation research, such as Figure 2 As shown. Taking a single-row magnet array as an example, the influence of the magnetic gap distance, varying from 1 mm to 3 mm in increments of 0.25 mm, on the magnetic field distribution and magnetic bead adsorption efficiency is analyzed. The simulation adopts a coupled multiphysics framework: laminar flow field (incompressible Navier-Stokes flow, inlet velocity: 200 μL / min), magnetic field (neodymium iron boron permanent magnets) BMN-35°C is generated (fixed magnet - channel spacing 1 mm) and a particle tracking module (superparamagnetic iron oxide nanoparticles, diameter: 800 nm). Particles are continuously released at the channel entrance at 0.05-second release intervals for 1 second (a total of 20 particles).

[0049] The particle trajectory was calculated by solving the coupled motion equations under the combined effects of hydrodynamics and magnetism, with a focus on the magnetophoretic capture efficiency under different gap distances. The results show that... Figure 3 As shown, according to Figure 3 ( a The curve shows a clear downward trend, indicating that as the gap between magnets increases, the adsorption efficiency (or magnetic field strength) of the magnetic beads gradually decreases, which directly reflects the negative impact of the magnetic gap distance on performance. Figure 3 ( b The magnetic field strength gradually decreases with the spacing, and the magnetic beads are adsorbed and distributed in a 2 mm The most uniform time indicates that at 2 mm The lowest degree of dispersion in the magnetic gap distance indicates that the magnetic beads are more uniformly adsorbed.

[0050] To further analyze the optimal performance of single-row magnet arrays, double-row magnet arrays, and single-sided double-row magnet arrays, this embodiment simulates the magnetic bead adsorption modes under different magnet array configurations, such as... Figure 4 As shown. 4 ( a The single-row magnet array is distributed as follows: the magnets are evenly distributed along the path of the extraction channel on the outer side of the extraction channel, and the vertical distance between the magnets and the extraction channel is the same. The specific distribution parameters are: magnet spacing 2 mm, magnet-channel distance: fixed at 1 mm.

[0051] Figure 4 ( b The dual-row magnet array is distributed as follows: the magnets are evenly spaced along the extraction channel on both the outer and inner sides of the channel, with the vertical distance between the magnets and the extraction channel being the same. Specific parameters are: magnet spacing of 2 mm, symmetrically distributed on both sides of the microchannel, and magnet-channel distance fixed at 1 mm.

[0052] Figure 4 ( c The single-sided double-row magnet array is distributed as follows: magnets are evenly distributed along the extraction channel path on the outside of the extraction channel in two rows, with each row having the same vertical distance between the magnets and the extraction channel. Specific parameters are: arranged at 2mm intervals on one side of the channel; the first row of magnets is 1mm from the channel; and the second row has a 2.8mm magnet-channel offset distance.

[0053] A comparative analysis of the magnetic bead adsorption modes under three different magnet array configurations, such as... Figure 4 ( dAs shown in the figure, the uniformity of magnetic bead adsorption under a single-row magnet array is optimal.

[0054] In another embodiment, the multi-step rotation operation results in premature leakage of amplification reagents into the water-in-oil emulsion (... W / O Risks associated with the emulsification chamber. Initial attempts to use a cascaded siphon valve failed to maintain a consistent fluid velocity across all six parallel amplification zones, while conventional paraffin valves, due to their rapid solidification at room temperature (20-25°C) and melt transition temperature (approximately 50°C), could potentially damage the emulsification chamber. LAMP The reagent is reactive and therefore defective, and paraffin can cause... W / O Droplets are difficult to form.

[0055] To overcome these limitations, this embodiment introduces a specific feature: the collection chamber is connected to several water-in-oil droplet generation and reaction chambers 12 via a quantitative distribution channel 8; a gelatin thermosensitive valve 11 is also provided between the quantitative distribution channel 8 and the water-in-oil droplet generation and reaction chambers 12 to control the timing of nucleic acid solution entering the water-in-oil droplet generation and reaction chambers 12. An amplification reagent reservoir 10 and a quantitative chamber 9 are sequentially arranged between the thermosensitive valve 11 and the quantitative distribution channel 8. A residual liquid chamber 13 is provided at the end of the quantitative distribution channel 8.

[0056] Based on the above structural optimizations and improvements, the workflow of the centrifugal microfluidic chip in this embodiment is shown in Table 1.

[0057] Table 1 mddLAMP - LoD Chip Working Process Based on the above process, this embodiment further quantitatively evaluates and shows... mddLAMP - LoD On-chip magnetic bead extraction of the system DNA The recycling efficiency reaches 71.6%, such as Figure 5 ( a As shown in the figure, the recovery efficiency is comparable to that of commercial extraction kits. Using breast cancer cell lines... MCF -7 genome DNA (50-500) ng Input quantity) tests show that, as Figure 5 ( b As shown in the figure, a stable capture efficiency of over 70% was maintained in the 50-350 nanogram range, indicating that the binding capacity was not yet saturated. When the input volume increased to 400-500 nanograms, the efficiency dropped to approximately 65%, indicating that the system was approaching its saturation limit. On-chip extraction of clinical plasma and serum samples maintained high performance, and the binding capacity remained high. Figure 5 ( c ).

[0058] Quality indicators includeA 260 / A The 280 ratio is 1.8-2.0. A 260 / A A 230 ratio greater than 2.0 and good qPCR Amplification effect, such as Figure 6 As shown, this confirms the high purity of the nucleic acid and its compatibility with downstream molecular applications.

[0059] Example 2 Regarding the requirement in Example 1 for the thermal valve in the centrifugal microfluidic chip to precisely control fluid flow and ensure compatibility... LAMP To meet the requirements of reagent activity and compatibility with automated detection processes, the optimal gelatin concentration was screened through rheological characterization, and the formulation and application parameters of the core material of the thermosensitive valve were determined to ensure its functional stability and detection compatibility.

[0060] Prepare 5 gelatin solutions of different volume concentrations: 1% ( v / v ), 5% v / v ), 10% v / v ), 15% v / v ), 20% v / v The solvent was enzyme-free pure water (processed with 0.22...). μm (Membrane filtration to remove nuclease / protease contamination); medical-grade gelatin solution (molecular weight approximately 150) is used. kDa Gel strength ≥220 gBloom All solutions of various concentrations were tested using a 300-meter filtration system. rpm Stir magnetically for 15 minutes to ensure even mixing.

[0061] Combination Figure 7 , Figure 7 (in) a The figures show the viscosity changes of gelatin solutions (1%, 5%, 10%, 15%, and 20% by volume) during heating. The 10% gelatin solution exhibited the most significant viscosity change, indicating its optimal thermal response characteristics. Data for high-concentration samples (15% and 20%) below 20°C are incomplete due to brittle fracture occurring during measurement. Figure 7 (in) b The viscosity evolution during cooling confirms the existence of thermal hysteresis: gelatin needs to be cooled to below 10°C to fully solidify, while melting occurs above 30°C. 10% gelatin again exhibits the most significant phase transition characteristics. Figure 7 (in) c The temperature-dependent viscoelastic modulus of 10% gelatin ( G 'and GThe crossover point at 32°C marks the gel-sol transition, at which point the loss modulus ( G The dominant characteristic is indicated by the dominance of the '″' symbol, reflecting the fluid behavior. Based on this, 37°C was chosen as the activation temperature of the thermistor to ensure reliable melting.

[0062] Furthermore, such as Figure 8 As shown, the optimized 10% ( v / v The gelatin matrix exhibits ideal phase change properties, including controllable gelation at 4°C, rapid liquefaction at 37°C, excellent biocompatibility, and reversible operation. In a fully automated processing flow, it provides optimal fluid control while ensuring... LAMP The reagent activity is unaffected.

[0063] Example 3 Based on Example 1 mddLAMP - LoD The chip and the gelatin-based thermal valve of Example 2, in this embodiment mddLAMP - LoD The chip's workflow is as follows Figure 9 As shown, Figure 9 ( a ( ) represents the rotational operation control state of the chip at different stages. Figure 9 ( b )for mddLAMP - LoD The flowchart of the fluid handling process on the chip is consistent with Table 1 in Example 1. A photograph of each working node is shown below. Figure 10 As shown, digital nucleic acid amplification detection can be completed within one hour, from sample input to result output. This is specifically for... PIK 3 CAE 542 K The detection results of the mutated gene and the statistical results of droplet brightness are as follows: Figure 11 As shown.

[0064] Figure 11 (in) a The image shows a fluorescence imaging pattern: from left to right, the negative control (…). NC ) and different copy numbers (50 copies / μL 199 copies / μL 1588 copies / μL 6362 copies / μL 12724 copies / μL )of PIK 3 CAE 542 KFluorescence imaging of the mutated gene sample. It is evident that the negative control showed almost no fluorescence signal. As the copy number of the mutated gene increased, the number of fluorescent droplets significantly increased and the fluorescence intensity gradually strengthened, clearly demonstrating the concentration dependence of the detection.

[0065] Figure 11 (in) b The following is a scatter plot of fluorescence intensity: each subplot corresponds to the fluorescence intensity distribution of different samples. The fluorescence intensity of the negative control (leftmost) is concentrated in the low range, while as the copy number of the mutated gene increases, the number of droplets with high fluorescence intensity increases significantly. The range and density of the scatter plot distribution are positively correlated with the gene copy number, reflecting the detection's ability to distinguish samples of different concentrations.

[0066] Figure 11 (in) c The histogram shows the fluorescence intensity count: the peak value and distribution range of the histogram change with the copy number of the mutant gene. The negative control peak is concentrated in the low-intensity region. As the copy number increases, the droplet count peak of high-intensity fluorescence gradually increases and the distribution range shifts to the right, further quantifying the difference in fluorescence signal at different concentrations, indicating that this detection method can effectively detect the fluorescence intensity count. PIK 3 CAE 542 K Sensitive and quantitative analysis of mutated genes.

[0067] In summary, this detection method is effective for... PIK 3 CAE 542 K Mutant genes have good sensitivity and quantification capabilities, enabling accurate detection over a wide concentration range.

Claims

1. A centrifugal microfluidic chip for droplet digital nucleic acid amplification detection, the centrifugal microfluidic chip having a chip structure layer, the chip structure layer comprising a disk body, wherein a centrifugal shaft hole is formed at the center of the structure layer, characterized in that, Also includes: The sample loading area is set in a designated area around the centrifugal shaft hole of the structural layer; An extraction channel is opened on the disc body according to a predetermined path. The front end of the extraction channel is connected to the sample loading area, and the rear end is connected to a switching valve. The magnetic bead nucleic acid extraction area is located in a designated area at the outer edge of the extraction channel, and the magnetic bead nucleic acid extraction area is equipped with an embedded magnet array to form a static magnetic field system. The collection chamber is connected to one of the output terminals of the switching valve; Several water-in-oil droplet generation and reaction chambers are uniformly arranged in a ring at the edge of the disk body; the collection chamber is connected to the several water-in-oil droplet generation and reaction chambers through a quantitative distribution channel; A thermistor valve is also provided between the quantitative dispensing channel and the water-in-oil droplet generation and reaction chamber to control the timing of the nucleic acid solution entering the water-in-oil droplet generation and reaction chamber.

2. The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 1, characterized in that, The embedded magnet array in the nucleic acid extraction region of the magnetic beads includes: a single-row magnet array, a double-row magnet array, and a single-sided double-row magnet array.

3. The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 2, characterized in that, The single-row magnet array is distributed as follows: it is evenly distributed on the outside of the extraction channel along the path of the extraction channel, and the magnetic beads are at the same vertical distance from the extraction channel.

4. The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 2, characterized in that, The double-row magnet array is distributed as follows: it is evenly distributed on the outer and inner sides of the extraction channel along the path of the extraction channel, and the magnetic beads are at the same vertical distance from the extraction channel.

5. A centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 2, characterized in that, The single-sided double-row magnet array is distributed as follows: it is distributed at equal intervals along the path of the extraction channel on the outside of the extraction channel and is set in two rows. The magnetic beads adjacent to the extraction channel are at the same vertical distance from the extraction channel, and the outermost magnetic bead is at the same vertical distance from the magnetic bead adjacent to the extraction channel.

6. The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 1, characterized in that, The thermostatic valve is a gelatin thermostatic valve, which has precise phase change characteristics of gelling at less than 10°C and liquefaction at more than 30°C.

7. The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 1, characterized in that, The sample loading area includes a sample cavity, which is connected to the front end of the extraction channel; A washing buffer reservoir is connected to the front end of the extraction channel via the first channel; The elution buffer reservoir is connected to the front end of the extraction channel via a second channel; The sample chamber, washing buffer reservoir, and elution buffer reservoir are arranged in a circumferential array around the centrifuge shaft hole of the structural layer.

8. The centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 1, characterized in that, It also includes: a waste liquid chamber, which is connected to the other output end of the switching valve via a branch channel; each output end of the switching valve is equipped with an air valve.

9. A centrifugal microfluidic chip for droplet digital nucleic acid amplification detection according to claim 1, characterized in that, An amplification reagent reservoir and a quantitative dispensing chamber are sequentially arranged between the thermal valve and the quantitative dispensing channel; A residual liquid chamber is provided at the end of the quantitative distribution channel.

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