A digital nucleic acid detection method based on a hydrogel self-water-absorption microfluidic chip

Abstract

The application discloses a kind of digital nucleic acid detection methods based on hydrogel self-absorbing water microfluidic chip, belong to the field of biotechnology.The detection method includes: first, hydrogel monomer is mixed with nucleic acid amplification reagent containing specific detection target nucleic acid primer, fluorescent dye to obtain mixed solution, gel is formed to obtain the hydrogel microfluidic chip with the thickness of 150-250 μm and the average pore size of 20-90 nm in gel interior;Then, the sample solution to be measured is added dropwise to the surface of the hydrogel microfluidic chip, and after sealing, it is placed for 8-12 min;After nucleic acid amplification reaction, fluorescence imaging is carried out, and quantitative analysis is carried out by counting fluorescent spots.The application uses the water absorption and swelling characteristics of hydrogel to absorb nucleic acid in the sample solution to be measured, without complex pump-in system design and fluid control;By embedding specific primers in different positions of the reaction unit in the chip array, digital nucleic acid detection of multiple targets can be carried out.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, specifically to a digital nucleic acid detection method based on a hydrogel self-absorbing microfluidic chip. Background Technology

[0002] With the rapid development of the field of life and health analysis, the accurate analysis of nucleic acids is of paramount importance for disease diagnosis, pathogen detection, and gene sequencing. Digital PCR (dPCR) is a powerful technique that can achieve absolute quantification of nucleic acids without the need for a standard curve. By dividing the sample into thousands to millions of microreaction units, it enables highly sensitive analysis at the single-molecule level.

[0003] In recent years, especially in resource-constrained areas, the demand for point-of-care testing (POCT) has grown rapidly, but testing technologies such as dPCR, which rely on laboratory conditions, are often difficult to apply in these scenarios. Isothermal amplification technologies, such as loop-mediated isothermal amplification reaction (LAMP) and recombinase polymerase amplification reaction (RPA), have shown good application potential in on-site nucleic acid testing because they do not require complex temperature cycling control and are easy to integrate with portable devices.

[0004] Currently, microfluidic chips based on droplet or microcavity partitions are the main platform for digital nucleic acid detection. To dispense sample solutions into these microreaction units, researchers have developed various liquid-driven methods, such as syringe pumps, centrifugal force, vacuum aspiration, and finger pressure aspiration. Anal. Chem 2025, 97, 18469.; Biosens. Bioelectron 2024, 255, 116240.; ACS Nano (2020, 14, 10385). While these methods can achieve relatively precise liquid manipulation, they all rely on external mechanical forces and are prone to problems such as liquid backflow, pumping asynchrony, and cross-contamination. In addition, droplet generation and the construction of microcavity structures usually require complex microchannel designs, which increases the risk of droplet breakage, fusion, and uneven distribution.

[0005] Current dPCR systems rely heavily on bulky and complex instruments, which are insufficient for the needs of point-of-care testing (POCT). Therefore, it is necessary to develop self-driven sample separation strategies that do not require droplet generation or external drive. This will help simplify the operation process, reduce system complexity, and promote the application of digital nucleic acid testing in POCT.

[0006] Multiplex nucleic acid detection is another significant challenge for point-of-care testing (POCT) analysis. An ideal digital nucleic acid detection platform should be able to simultaneously detect multiple targets in a single reaction to increase throughput and reduce costs. Current main strategies for multiplex digital detection include spectral encoding methods based on fluorescent probes, which distinguish different targets by different fluorescence colors or melting curves. However, this method is limited by fluorescence signal crosstalk and requires high-resolution optical detection equipment. Another commonly used strategy is spatial partitioning encoding, which encodes different targets in different regions and uses vacuum-driven sample loading. However, these dPCR chips require distributing different samples to specific, high-density microcavity regions, increasing the complexity of microchannel design, sample pumping, and dispensing control.

[0007] Therefore, there is an urgent need to build a simpler, lower-cost, and self-driven multiplex digital nucleic acid quantification platform to meet the needs of practical POCT applications. Summary of the Invention

[0008] The purpose of this invention is to provide a digital nucleic acid detection method based on a hydrogel self-absorbing microfluidic chip for non-diagnostic purposes. This method can achieve rapid, simple, and accurate quantitative detection of target nucleic acid molecules without the need for complex droplet generation and microchannel chip preparation, thus meeting the needs of practical POCT applications.

[0009] To achieve the above objectives, the present invention adopts the following technical solution: This invention provides a digital nucleic acid detection method based on a hydrogel self-absorbing microfluidic chip, comprising the following steps: (1) The hydrogel monomer is mixed with a nucleic acid amplification reagent containing primers and fluorescent dyes for specific detection target nucleic acids to obtain a mixture, and the mixture is gelled to form a hydrogel microfluidic chip with a thickness of 150~250 μm and an average pore size of 20~90 nm inside the gel. (2) Drop the sample solution to be tested onto the surface of the hydrogel microfluidic chip, seal it and let it stand for 8-12 min; then place it under nucleic acid amplification conditions to carry out nucleic acid amplification reaction. (3) After the amplification is completed, fluorescence imaging is performed on the hydrogel microfluidic chip, and the fluorescence spots are counted for quantitative analysis.

[0010] This invention utilizes the inherent water-absorbing properties of hydrogels to achieve self-driven sample solution transport without the need for any external pumps or power sources. As a highly biocompatible self-propelled pump, hydrogels can rapidly absorb aqueous samples and distribute them within their internal network, thus eliminating the need for bulky equipment and complex operations. During isothermal amplification, each nucleic acid molecule is amplified in situ within the cross-linked hydrogel network, generating a series of fluorescent amplicon spots for digital counting. The final fluorescent spots are counted using a fluorescence imaging system, thereby achieving absolute digital quantification of nucleic acids.

[0011] This invention demonstrates that hydrogels with an average pore size in the range of 20–90 nm can spontaneously absorb and distribute nucleic acids from the sample throughout the hydrogel matrix within 8–12 minutes. The suitable pore size provides an effective entry channel for nucleic acid molecules, ensuring sample loading can be completed without external pumping or complex pretreatment, thus significantly simplifying the sample loading process. Furthermore, it restricts the diffusion of macromolecular amplification products, promoting in-situ accumulation of nucleic acid amplification products within the nanopores. This ultimately results in fluorescent amplification spots of uniform size, clear boundaries, and concentrated brightness, improving the resolvability and counting accuracy of the fluorescent spots, and achieving precise digital quantitative detection of nucleic acids.

[0012] Preferably, the average pore size inside the hydrogel microfluidic chip is 30~40 nm.

[0013] Specifically, the sample to be tested is a solution sample containing nucleic acid molecules, which can be a medical clinical sample, an environmental sample, or a food sample, and the sample solution to be tested is prepared by nucleic acid extraction.

[0014] In step (1), the prepared mixture is a single hydrogel microfluidic chip reaction unit containing specific nucleic acid amplification primers for a certain target. The hydrogel monomers are photocured or chemically cross-linked to form a hydrogel in the mixture system, resulting in a ready-to-use hydrogel microfluidic chip for subsequent nucleic acid amplification reactions.

[0015] To ensure the uniformity of the prepared hydrogel microfluidic chip, the mixture was added to the microfluidic chip array mold and then solidified into a gel by photocuring or chemical cross-linking.

[0016] In one specific embodiment of the present invention, the mold is made by bonding a glass slide and an incubation chamber. A cover plate is used to tightly bond the mold to the frame of the incubation chamber to form a sealed chamber, in which gel can be formed and subsequent nucleic acid amplification reactions can be completed.

[0017] In another specific embodiment of the present invention, a positive film is first manufactured using a photolithography machine, and then polydimethylsiloxane is used for molding to create a microfluidic chip array mold containing multiple reaction chambers. The resulting gel is then deposited within the reaction chambers to complete the subsequent nucleic acid amplification reaction.

[0018] In this invention, the hydrogel can be a polyethylene glycol-based hydrogel or a polyacrylamide hydrogel.

[0019] In one specific embodiment of the present invention, the hydrogel monomer is poly(ethylene glycol) diacrylate (PEGDA). PEGDA is solidified into a gel under light irradiation mediated by a photoinitiator.

[0020] Preferably, the molecular weight of the poly(ethylene glycol) diacrylate is 700 MW, and the final volume percentage concentration of the poly(ethylene glycol) diacrylate in the mixture is 5-8%. This invention demonstrates that the hydrogel chip formed using the above ratio has high water absorption and swelling properties, and can absorb nucleic acid molecules from the sample solution within 1-10 minutes, distributing them evenly within the porous hydrogel.

[0021] More preferably, the final volume percentage concentration of poly(ethylene glycol) diacrylate in the mixture is 6.8%, the template size of a single hydrogel chip is 9×9 mm, and a hydrogel chip with a thickness of 200 μm is formed.

[0022] The photoinitiator can be an ultraviolet photoinitiator, and can be, but is not limited to, photoinitiator 1173.

[0023] Preferably, the mixture contains photoinitiator 1173, and the mixture is heated at an intensity of 20 mW / cm². 2 The hydrogel is irradiated with ultraviolet light at a wavelength of 365 nm for 2-6 minutes to solidify into a gel. This invention demonstrates that the hydrogel formed under these conditions exhibits appropriately sized and clearly spaced fluorescent dots in nucleic acid amplification reactions. More preferably, the irradiation time is 4 minutes.

[0024] In another specific embodiment of the present invention, the hydrogel monomers are polyethylene glycol-acrylate and polyethylene glycol-mercapto. A hydrogel can be formed after standing at room temperature for 3-5 minutes.

[0025] Preferably, the molecular weight of polyethylene glycol-acrylate is 5000~10000 MW, the molecular weight of polyethylene glycol-mercapto-acrylate is 2000~3400 MW, the molar ratio of the two is 1:2, and the concentration of polyethylene glycol-acrylate in the mixed solution is 3~9 mM. This invention demonstrates that the hydrogel chip formed using the above-mentioned hydrogel precursor solution exhibits high water absorption and swelling properties, capable of absorbing nucleic acid molecules from the sample solution within 1~10 min and uniformly distributing them within the porous hydrogel.

[0026] More preferably, the amount of polyethylene glycol-acrylate added to the mixture is 7.68 mM, the amount of polyethylene glycol-mercapto-thiol added is 15.36 mM, the template size of a single hydrogel chip is 9×9 mm, forming a hydrogel chip with a thickness of 200 μm.

[0027] In this invention, the nucleic acid amplification reaction can be, but is not limited to, loop-mediated isothermal amplification (LAMP), recombinase polymerase amplification (RPA), or rolling circle amplification (RCA).

[0028] According to the specific nucleic acid amplification method, the present invention selects the appropriate reagents to prepare the mixture. When the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, as a specific embodiment of the present invention, the mixture comprises: 1× isothermal amplification buffer, 2~6 mM MgSO4, 1~1.8 mM dNTPs, 120~360 U Bst 2.0 DNA polymerase, 0.5~2× fluorescent dye, 5~8% (v / v) poly(ethylene glycol) diacrylate, 0.03~0.05% (v / v) photoinitiator 1173 and 1× LAMP primer mixture, wherein the 1× LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM F3 and B3, and 0.8 μM LF and LB.

[0029] Preferably, the mixture comprises: 1× isothermal amplification buffer, 4.76 mM MgSO4, 1.67 mM dNTPs, and 286 U Bst 2.0 DNA polymerase, fluorescent dye, 6.8% by volume of poly(ethylene glycol) diacrylate, 0.04% by volume of photoinitiator 1173, and a 1×LAMP primer mixture, wherein the 1×LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM F3 and B3, and 0.8 μM LF and LB.

[0030] In another specific embodiment of the present invention, the mixture comprises: 1× isothermal amplification buffer, 2~6 mM MgSO4, 1~1.8 mM dNTPs, and 120~360 U / mL. Bst 2.0 DNA polymerase, 0.5–2× fluorescent dye, 3–9 mM polyethylene glycol-acrylate, 6–18 mM polyethylene glycol-mercapto, and a 1× LAMP primer mixture, wherein the 1× LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM F3 and B3, and 0.4 μM LF and LB.

[0031] Preferably, the mixture comprises: 1× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTPs, 240 U / mL Bst 2.0 DNA polymerase, fluorescent dye, 7.68 mM polyethylene glycol-acrylate, 15.36 mM polyethylene glycol-mercapto, and a 1× LAMP primer mixture, wherein the 1× LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM MF3 and B3, and 0.4 μM LF and LB.

[0032] In this invention, when the target analyte is RNA, 200-400 U / mL of reverse transcriptase is added to the reaction system. Preferably, 300 U / mL of reverse transcriptase is added to the reaction system.

[0033] The fluorescent dye is either Eva Green or SYBR Green.

[0034] The present invention demonstrates that the hydrogel microfluidic chip prepared by the present invention has excellent reagent preservation capabilities. Multiple hydrogel microfluidic chip arrays for detecting analytes can be prepared in advance as needed and stored under refrigeration for 30 days, ready for immediate detection at any time.

[0035] As a preferred option, the pre-prepared ready-to-use hydrogel microfluidic chip array is stored at 4°C.

[0036] In step (2), the sample solution to be tested is dropped onto the center of the hydrogel microfluidic chip. Utilizing the water absorption and swelling properties of the hydrogel, the sample solution is automatically absorbed and evenly distributed to each reaction unit under the action of capillary drive and swelling drive.

[0037] Preferably, after the sample solution is dropped into the center of the hydrogel chip, the entire incubation chamber is immediately sealed with a cover plate and left to stand at room temperature for 10 minutes. The target nucleic acid molecules in the sample solution are loaded into the porous hydrogel through passive aspiration, thus completing the sample loading.

[0038] This invention demonstrates that hydrogel chips possess abundant nanopore structures, which can effectively limit the diffusion of macromolecular nucleic acid amplicon during nucleic acid amplification. This allows the amplification of target nucleic acids to occur in situ, accumulating to form nucleic acid clusters within the hydrogel nanopores. After binding with fluorescent dyes, a series of fluorescent spots can be observed under specific fluorescence imaging conditions, thereby achieving digital absolute quantitative detection of target nucleic acid molecules.

[0039] This invention demonstrates that hydrogel microfluidic chips possess excellent resistance to matrix interference. Based on the molecular sieve effect of the porous structure of hydrogels, large particles in the sample solution are trapped outside the hydrogel and cannot participate in or inhibit nucleic acid amplification reactions. Target nucleic acid molecules and small molecules are drawn into the porous hydrogel through water absorption, but due to the different diffusion depths, the inhibitory effect on nucleic acid amplification is greatly reduced.

[0040] In this invention, when the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, the conditions for the loop-mediated isothermal amplification reaction are heating at 63~68℃ for 10~30 min.

[0041] As a preferred method, the ring-mediated isothermal amplification reaction is performed under the condition of heating at 65°C for 20 min.

[0042] In step (3), the hydrogel microfluidic chip after nucleic acid amplification is imaged and photographed under the fluorescence imaging conditions corresponding to the fluorescent dye.

[0043] Furthermore, the fluorescent spots are counted manually or identified by software, and the absolute quantitative detection of target nucleic acid molecules is achieved based on the number of fluorescent spots.

[0044] Preferably, ImageJ software is used to binarize the fluorescence imaging images of the hydrogel chip, and the watershed function is used to segment adjacent amplification points and count them accurately.

[0045] Another objective of this invention is to provide a simple, rapid, and low-cost method for on-site multiplex nucleic acid testing based on a hydrogel-based self-absorbing platform, thereby promoting the development of practical digital point-of-care testing (POCT) systems.

[0046] To achieve multiplex analysis, this invention fabricates an array composed of several hydrogel microfluidic chips, in which different hydrogel reaction units are preloaded with specific primers for different targets, thereby realizing spatially localized sample loading and multi-target digital amplification.

[0047] Specifically, an array of hydrogel microfluidic chips is fabricated on the same substrate. Each hydrogel microfluidic chip contains specific primers targeting different targets, serving as reaction chambers for multiplex digital nucleic acid detection. During nucleic acid detection, the same sample is dropped into different hydrogel reaction units. After the nucleic acid amplification reaction is complete, fluorescence images of the hydrogel microfluidic chip array are acquired. Based on the spatial location of the fluorescent dots on the hydrogel chips in the array, the detection results of the corresponding target nucleic acids are determined. Furthermore, the fluorescent dots in the images are identified, counted, and statistically analyzed to determine the quantitative detection results of different target nucleic acids.

[0048] The beneficial effects of this invention are as follows: (1) This invention provides a droplet-free digital nucleic acid detection method utilizing the water absorption and swelling properties of hydrogels. Hydrogels possess abundant three-dimensional porous structures, and due to their rich hydrophilic groups, they can absorb aqueous solutions under non-equilibrium, unsaturated conditions through capillary action and swelling. Utilizing the water absorption and swelling properties of hydrogels to absorb nucleic acids from the sample solution eliminates the need for complex pumping system design and fluid control, avoiding the cumbersome steps of on-site reagent preparation. During detection, due to the spatial confinement effect of the hydrogel nanoporous structure, the amplicon of the large nucleic acid molecules after amplification is restricted within the hydrogel nanoporous structure. After binding with fluorescent dyes, a series of fluorescent spots can be observed under specific fluorescence imaging conditions. Each fluorescent spot represents a single original target nucleic acid molecule in the sample solution, providing a simple, sensitive, and inexpensive digital nucleic acid detection method.

[0049] (2) The method provided by this invention can be used for point-of-care testing and has excellent resistance to sample matrix interference. By mixing the hydrogel precursor solution, nucleic acid amplification reagent, and fluorescent dye, and adding them to the chip array mold, a prefabricated ready-to-use hydrogel microfluidic chip is obtained after gelation. This chip can be stored for a long time and used for on-site nucleic acid detection at any time. The hydrogel has a high internal surface area, porosity, and biocompatibility, which can provide an ideal environment for biomolecular interaction and effectively maintain the activity of nucleic acid amplification reagent in the prefabricated hydrogel chip. The molecular sieve effect provided by the porous structure of the hydrogel chip can effectively trap large particulate impurities in the sample solution and draw target nucleic acids and small molecules into different depths inside the hydrogel, which can effectively avoid the inhibitory effect of inhibitors on nucleic acid amplification.

[0050] (3) The hydrogel microfluidic chip array provided by this invention can detect different targets, including pathogens, viruses, and specific genes. By changing the specific nucleic acid amplification primers for different targets, multiple targets can be digitally detected. This method can be used to detect multiple targets simultaneously by pre-embedding specific primers in the reaction units at different positions in the chip array, and the detection results of different targets can be determined by the spatial position of the chip array. Attached Figure Description

[0051] Figure 1 This is a physical image of a hydrogel self-absorbing microfluidic chip array. Images a and b are taken from different viewpoints.

[0052] Figure 2 Optimization results for the photocurable hydrogel system (BSA, hydrogel concentration).

[0053] Figure 3 The results show the optimization of the photocurable hydrogel system under different UV irradiation times.

[0054] Figure 4 The results are for the photocurable hydrogel system at different amplification times.

[0055] Figure 5 The results of digital nucleic acid detection using a photocurable hydrogel microfluidic chip at different template concentrations are shown. In the image, a is a fluorescence imaging image; b is a comparison of the number of samples detected versus the number of spiked samples (measured using a commercial droplet digital chip).

[0056] Figure 6 It is a mask for microfluidic chip arrays.

[0057] Figure 7 Characterization of the nucleic acid inhalation hydrogel chip. In image a, fluorescence characterization of small molecule FITC and nucleic acids of different lengths in the hydrogel; in image b, quantitative fluorescence image of the corresponding inhalation depth.

[0058] Figure 8 The effect of hydrogel chip concentration on nucleic acid uptake is shown. a) is a scanning electron microscope image of hydrogel chips with concentrations of 6%, 12%, and 18%; b) is a fluorescence characterization image of nucleic acid uptake; c) is a quantitative fluorescence image of the corresponding uptake depth; d, e, and f are statistical distribution maps of pore size for hydrogels with concentrations of 6%, 12%, and 18%, respectively.

[0059] Figure 9 Results are shown for the hydrogel microfluidic chip system under different template concentrations. In the figure, a represents the endpoint quantification plot; b represents the spiked statistics.

[0060] Figure 10This study compares the reagent preservation and matrix interference resistance of hydrogel microfluidic chips. Figure a shows a schematic diagram of reagent preservation in aqueous solution and hydrogel; figure b shows the quantitative detection fluorescence images after one day and one month of preservation in the hydrogel microfluidic chip; figure c shows the real-time fluorescence quantitative amplification curves after one day and one month of reagent preservation in both aqueous solution and hydrogel systems; and figure d shows the quantitative detection fluorescence images of the mixed amplification system and the self-absorbing amplification system.

[0061] Figure 11 This image shows the results of simultaneous detection of Staphylococcus aureus, Salmonella typhi, and Vibrio parahaemolyticus using a hydrogel self-absorbing microfluidic chip array. Image a shows a photograph of the hydrogel self-absorbing microfluidic chip array; images be show the fluorescence spectra of Staphylococcus aureus, Salmonella typhi, Vibrio parahaemolyticus, and a mixture of the three bacteria, respectively; and image f shows the hydrogel fluorescent dot count results.

[0062] Figure 12 The results represent actual sample testing. Where a represents the spiked recovery rate of the method of this invention and the commercial digital nucleic acid detection chip; b represents a photograph of a fresh fruit and vegetable sample solution; and c represents the spiked recovery rate of different fruit and vegetable samples detected by the method of this invention and the commercial digital nucleic acid detection chip. Detailed Implementation

[0063] The present invention will be further described below with reference to specific embodiments. These embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the present invention without departing from the spirit and essence of the invention are within the scope of the invention.

[0064] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available unless otherwise specified.

[0065] The targets for testing in the following examples are Staphylococcus aureus, Salmonella typhi, and Vibrio parahaemolyticus. The bacterial genomic DNA was extracted from the kit and stored at -20°C for later use.

[0066] The primer sequences for Staphylococcus aureus LAMP amplification are as follows, from left to right: 5' end to 3' end: F3: TGCAAAGAAAATTGAAGTCGA; B3: CGTTGTCTTCGCTCCAAAT; FIP: CGTTTACCATTTTTCCATCAGCATATTTGACAAAGGTCAAAGAACT; BIP: TCAAGGCTTGGCTAAAGTTGCTTATTCGCTTGTGCTTCACTT; LF: ACGCTAAGCCACGTCCATAT; LB: CAAACCTAACAATACACATGAACA.

[0067] The primer sequences for LAMP amplification of Vibrio parahaemolyticus are as follows, from left to right: 5' end to 3' end: F3: GTGCGAAGAACTTCATGTTG; B3: GATGAGCGGTTGATGTCC; FIP:ACATCGCTTGTGCCTTGATGAACTCAACACAAGAAGAGATCG; BIP: GCGCAAGGTTACAACATCACGGCAGAAGTTAGCGTCTCG; LF: ACTCGTTCATCTCAAGCACTT; LB: TGTTTGATACTCACGCCTTGT.

[0068] The primer sequences for LAMP amplification of Salmonella Typhi are as follows, from left to right: 5' end to 3' end: F3: CCAACAATCCATCAGCAAG; B3:AGCATATGTTTTGTTTCCTGAA; FIP: AACACATAGCCAAGCTCCCGCAGTCAGTATTTCTGGGTAAC; BIP: GAACGCGCTTGATGAGCTTTCGAAATATTCATTGACGTTGC; LF: GAGTTTCTCCCCCTCTTCATGC; LB: ACCACTGTCTGGCGGTGA.

[0069] Example 1 This embodiment employs a photopolymerization strategy to prepare a PEGDA-based hydrogel microfluidic chip array. Specifically, a mixture of hydrogel precursor solution and LAMP amplification reagent is added to the chip array chamber and crosslinked under ultraviolet light to form a 200 μm thick hydrogel microfluidic chip array. This self-absorbing hydrogel microfluidic chip array is then used for multiplex digital nucleic acid detection.

[0070] 1. Fabrication of hydrogel microfluidic chip arrays Step 1: Prepare hydrogel precursor solution and LAMP amplification reagents The LAMP amplification reagent (19 μL) consists of the following components: 4.76 mM MgSO4, 1.67 mM dNTPs, 286 U Bst 2.0 DNA polymerase, 1×LAMP buffer, 1×SYBR Green I and primer mixture (1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.8 μM LF and LB).

[0071] The PEGDA hydrogel precursor solution is composed of the following components: poly(ethylene glycol) diacrylate (PEGDA) monomer is dissolved in 20 mM HEPES buffer saline (pH=7.4), and 0.1% (v / v) 2-hydroxy-2-methylphenylacetone solution (photoinitiator 1173) is added as a photoinitiator to obtain a PEGDA precursor solution with a concentration of 17% (v / v).

[0072] Step 2: Fabrication of hydrogel microfluidic chip array A simple microfluidic chip chamber was fabricated by attaching incubation chambers to the surface of a glass slide. LAMP amplification reagents containing different specific primers were mixed with PEGDA hydrogel precursor solution to a final PEGDA concentration of 6.8% (v / v). This mixture was then added to different microfluidic chip chambers. A photomask was then placed over the chambers, and a light source with an intensity of 20 mW / cm² was used. 2 The mixture was irradiated with ultraviolet light (wavelength 365 nm) for 4 min, and solidified under ultraviolet light. Then, the unsolidified liquid around the gelled chip was blown away with nitrogen gas. A cover was then placed on the surface of the microfluidic chip array to seal it, thus obtaining a ready-to-use hydrogel self-absorbing microfluidic chip array. Figure 1 ).

[0073] 2. Multiplex digital nucleic acid testing For nucleic acid testing, the sealed cap of the pre-fabricated, ready-to-use hydrogel self-absorbing microfluidic chip array is torn open, and the sample solution is dropped onto the surface of the hydrogel chip. The array is then immediately resealed and allowed to stand for 10 minutes to allow the sample solution to be fully absorbed into the array. The sealed array is then placed at 65°C for 30 minutes for a LAMP reaction. After nucleic acid amplification, the hydrogel microfluidic chip array is imaged using a blue LED (488 nm) or a fluorescence microscope, and the fluorescent spots in the array are counted. Each fluorescent spot corresponds to a target nucleic acid molecule in the original sample solution, and the spatial position of each hydrogel chip in the microfluidic chip array corresponds to a different target, thus enabling multiplex digital nucleic acid detection.

[0074] Example 2 This embodiment optimizes the preparation conditions of the hydrogel microfluidic chip array in Example 1 by adjusting the concentration of LAMP reagents (BSA concentration and hydrogel content) through single-factor adjustment. Specifically, Staphylococcus aureus genomic DNA extracted from the kit is used as the detection sample, and tests are conducted under the following different preparation conditions of hydrogel microfluidic chip arrays.

[0075] 1. BSA concentration BSA is a common nucleic acid amplification stabilizer that can protect enzymes in the reaction system. Two BSA concentration groups were set up: 0× and 1×. The LAMP amplification reagent composition for the 0× BSA concentration group consisted of the following components: 4.76 mM MgSO4, 1.67 mM dNTPs, and 286 U... Bst 2.0 DNA polymerase, 1×LAMP buffer, 1×SYBR Green I and primer mixture (1.6 μM FIB and BIP, 0.2 μM F3 and B3, 0.8 μM LF and LB); the LAMP amplification reagent for the 1×BSA concentration group consisted of the following components: 1×BSA (1 mg / mL), 4.76 mM MgSO4, 1.67 mM dNTPs, 286 U Bst 2.0 μM DNA polymerase, 1×LAMP buffer, 1×SYBR Green I, and primer mixture (1.6 μM FIB and BIP, 0.2 μM MF3 and B3, 0.8 μM LF and LB). PEGDA hydrogel precursor solution, hydrogel microfluidic chip array, and digital nucleic acid detection were performed according to Example 1. For result analysis, the optimal concentrations were determined based on two factors: the number of amplification points and the brightness-to-dark ratio.

[0076] 2. Hydrogel content The hydrogel concentration affects nucleic acid absorption and digital amplification. The optimal hydrogel concentration was optimized based on two factors: the number of amplification points and the brightness-to-dark ratio. The LAMP amplification reagent and PEGDA hydrogel precursor solution were prepared according to Example 1. When preparing the hydrogel microfluidic chip array, PEGDA hydrogel precursor solution with volume percentages of 28%, 34%, 40%, 46%, and 52% was added to the mixture to obtain hydrogel microfluidic chips with different hydrogel concentrations. Digital nucleic acid detection was then performed according to Example 1.

[0077] 3. Results Analysis The results are as follows Figure 2 As shown, the optimal reaction conditions were determined (the final concentration of BSA in the system was 0×, and the volume percentage of the PEGDA hydrogel precursor solution in the system was 40% (v / v)) to ensure that the size of the fluorescent dots used for visualization and counting was appropriate and the spacing was clear.

[0078] Example 3 This embodiment optimizes the UV irradiation time in step two of Example 1, using four gradients: 0 min, 2 min, 4 min, and 6 min, while other conditions remain the same as in Example 1. Staphylococcus aureus genomic DNA extracted using the kit was used as the detection sample.

[0079] The results are as follows Figure 3 As shown, the number of amplification spots initially increases and then decreases with increasing UV irradiation time, reaching its maximum at 4 min. This is likely because when irradiation time is too short, such as 0 min or 2 min, the PEGDA hydrogel does not gel completely. This means the hydrogel pores are too large and the PEGDA monomers are not fully activated by the photoinitiator to form large molecular chains. Consequently, the large pore size causes some amplified nucleic acid clusters to disperse, preventing them from aggregating into observable fluorescent spots, thus reducing the number of fluorescent spots. The inhibitory effect of the PEGDA monomers themselves on the amplification reaction is also amplified under insufficient irradiation time. Short irradiation time means low excitation of the photoinitiator, resulting in less activated PEGDA monomers, which triggers a chain reaction of the remaining PEGDA monomers in the system, also reducing the number of fluorescent spots. As irradiation time exceeds 4 min, further increases in irradiation time also reduce the number of fluorescent spots. This is because the small pore size of the hydrogel restricts the free movement of the LAMP reagents, inhibiting amplification efficiency. Therefore, the optimal UV irradiation time is 4 min.

[0080] Example 4 This embodiment optimizes the LAMP reaction time in Example 1 by using four time gradients: heating in a metal bath at 65°C for 20 min, 25 min, 30 min, and 35 min, with other conditions remaining the same as in Example 1. Staphylococcus aureus genomic DNA extracted using the kit was used as the detection sample.

[0081] The results are as follows Figure 4 As shown, fluorescent spots began to appear after 20 minutes of amplification. Before reaching 30 minutes, longer heating times resulted in a greater number of amplified spots. After 30 minutes, despite further increases in heating time, the number of amplified spots did not change significantly. Regarding the brightness ratio, there was no significant difference between the 20-minute and 25-minute gradients, but both were significantly lower than the ratios at 30 and 35 minutes. Therefore, the optimal heating time for amplification is 30 minutes, which will allow for the observation of clearly defined, moderately bright fluorescent spots suitable for counting.

[0082] Example 5 This embodiment explores the accuracy verification of the hydrogel self-absorbing microfluidic chip prepared in Example 1 for digital nucleic acid detection. Specifically, Staphylococcus aureus genomic DNA extracted from the kit was used as the test sample, and its spike concentration was determined using a commercial digital PCR chip (QuantStudio 3D Digital PCR System platform). Subsequently, the same sample was tested using the hydrogel self-absorbing microfluidic chip prepared in Example 1. The testing method for the hydrogel self-absorbing microfluidic chip is as follows: (1) During the test, tear open the pre-made ready-to-use hydrogel chip sealing cap, add 5 μL of sample solution to the surface of the hydrogel chip, and seal it immediately. After standing for 10 min, the sample can be spontaneously absorbed into its nanoporous structure through the water absorption of the hydrogel.

[0083] (2) During the nucleic acid amplification reaction, the hydrogel chip sealed on the surface of the glass slide was placed on a 65°C heating plate and incubated for 30 min for LAMP reaction. During this process, the target nucleic acid was amplified in situ, and the amplification product was confined in the cross-linked hydrogel network to form discrete fluorescent nucleic acid spots for digital counting.

[0084] (3) After amplification, fluorescent spot imaging is observed using a fluorescence microscope (Leica DMi8, Leica, Wetzlar, Germany) or the spots are counted visually using a blue LED flashlight. For microscope-based fluorescent spot imaging, ImageJ software can be used for counting and analysis. Each fluorescent spot corresponds to one nucleic acid molecule in the sample, thus achieving visualized absolute quantitative analysis of nucleic acids.

[0085] like Figure 5 As shown, the hydrogel-based digital nucleic acid detection method of this invention has high accuracy and reliability, and a good linear relationship (R0). 2 =0.9992), which shows that visual counting can effectively replace quantitative counting using traditional fluorescence imaging.

[0086] Example 6 In this embodiment, a master mold for a hydrogel microfluidic chip array is fabricated using SU-8 photolithography, and the chip array structure is formed by polydimethylsiloxane (PDMS) molding. The specific method is as follows: (1) Chip array master mold preparation: First, the silicon wafer was pretreated by cleaning it sequentially with acetone, deionized water, and piranha solution. After cleaning, it was dried with nitrogen. Then, the silicon wafer was placed in an oven at 120°C for 30 min to remove moisture. After removing the dry and clean silicon wafer, SU-8 2075 photoresist was poured onto the silicon wafer in a dark environment, manually spin-coated, and then treated with a spin coater at 1250 rpm for 30 s. Then, pre-baking was performed, first baking the silicon wafer at 65°C for 7 min, and then treating it at 95°C for 40 min. After the treatment, the silicon wafer was placed in an Aligner 12016042 lithography machine (MA6-BSA), and a pre-designed mask with a partitioned pattern (such as...) was applied. Figure 6 The sample is placed on top of the silicon wafer, and the instrument is set to an exposure time of 10 seconds. It is then dried again, first at 65°C for 5 minutes, then at 95°C for 15 minutes. After drying, the silicon wafer is developed using SU-8 developer for 20 minutes. Finally, the developed silicon wafer is rinsed with isopropanol to remove unexposed areas, and then dried to create the SU-8 positive mold. The SU-8 positive mold can be reused multiple times, hundreds of times, while remaining undamaged.

[0087] (2) Chip array fabrication: Polydimethylsiloxane (PDMS) monomer and curing agent are mixed at a mass ratio of 10:1 and stirred thoroughly to prepare a PDMS solution. Pour the PDMS onto the prepared SU-8 positive mold and then dry it at 75°C for 2 hours. Carefully remove the PDMS chip, which has several independent chambers formed on its surface.

[0088] By adding a mixture containing specific primers for different targets, LAMP nucleic acid amplification reagents, fluorescent dyes, and hydrogel precursor solution to different independent chambers, and then sealing the chambers, a ready-to-use hydrogel self-absorbing microfluidic multiplex detection chip array is formed.

[0089] This invention can be used to create a multiplex detection platform that supports the detection of different targets by changing the primer type, or it can be used to create a multiplex detection platform that supports the simultaneous detection of more targets by changing the SU-8 cation membrane.

[0090] Example 7 This embodiment investigates the absorption capacity of a polyethylene glycol (PEG)-based hydrogel microfluidic chip for nucleic acids in solution. Specifically, a mixture containing PEG hydrogel monomers and LAMP amplification reagents was added to the chip array chamber and placed at room temperature to form a 200 μm thick hydrogel microfluidic chip array. This self-absorbing hydrogel microfluidic chip array was then used for the LAMP reaction.

[0091] 1. Fabrication of hydrogel microfluidic chip arrays The LAMP amplification hydrogel system consists of the following components: 1× isothermal amplification buffer, 6 mM MgSO4, 1.4 mM dNTPs, and 640 U / mL. Bst 2.0 DNA polymerase, 1×SYBR Green I, 12% (v / v) PEG hydrogel monomer and 1×LAMP primer mixture, wherein the 1×LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM F3 and B3, and 0.4 μM LF and LB.

[0092] LAMP amplification reagents containing different specific primers were added to different chambers of the microfluidic chip and placed at room temperature for 3-5 minutes to form hydrogels, thus obtaining a ready-to-use hydrogel self-absorbing microfluidic chip array.

[0093] 2. Absorption capacity of PEG-based hydrogel microfluidic chip for nucleic acids of different lengths in solution. Synthetically synthesized nucleic acid fragments of 47 nt and 222 nt lengths were mixed with TE buffer and SYBR Green II dye, respectively. 5 μL of this mixture (2 μL TE buffer, 2 μL nucleic acid, and 1 μL SYBR Green II dye) was then dropped onto the surface of a PEG hydrogel chip, followed by the addition of mineral oil to prevent evaporation. A FITC aqueous solution (a green fluorescent dye derivative used to observe the absorption of water molecules in the hydrogel) was added as a control. Finally, the longitudinal fluorescence intensity distribution of the hydrogel was measured using a laser scanning confocal microscope (LSCM) at 0.5, 5, and 10 min. This longitudinal fluorescence distribution reflects the presence of nucleic acids at different depths within the hydrogel.

[0094] The results are as follows Figure 7 As shown in Figure a, both FITC and 47 nt nucleic acid fragments were rapidly absorbed and uniformly distributed within the hydrogel, with a maximum penetration depth of approximately 200 µm. With increasing nucleic acid length, both the diffusion rate and penetration depth decreased significantly. Figure 7 (b) This may be due to the steric hindrance effect generated by the cross-linked hydrogel network. The pore size of the hydrogel has a significant impact on the nucleic acid absorption process, while the hydrogel concentration directly determines its cross-linking density and pore structure.

[0095] 4. Effect of hydrogel concentration on nucleic acid absorption in solution Hydrogel microfluidic chips of different concentrations (6%, 12%, and 18% by volume) were prepared. They were frozen at -80°C overnight and then freeze-dried in a vacuum freeze dryer for 36 hours to achieve a moisture-free state. The prepared samples were sputter-coated with gold and then observed for morphology and pore size using a thermal field emission scanning electron microscope (SEM) (Zeiss G300).

[0096] A mixture containing either a 47 nt or 222 nt nucleic acid fragment (2 μL TE buffer, 2 μL nucleic acid, and 1 μL SYBR Green II dye) was dropped onto the surface of the PEG hydrogel chips of different concentrations, and then mineral oil was added to prevent evaporation. Finally, the longitudinal distribution fluorescence intensity of the hydrogel was measured using a laser scanning confocal microscope at 0.5, 5, and 10 min.

[0097] Scanning electron microscope (SEM) images ( Figure 8 As shown in a), with increasing hydrogel concentration, the hydrogel chip forms a denser three-dimensional network structure, and the average pore size significantly decreases from 82.82 nm to 39.15 nm and 22.59 nm. Figure 8 (d, e, f). In high-concentration hydrogels, their dense surface structure significantly slows down the penetration of nucleic acids into deeper regions; in contrast, in hydrogels with low cross-linking density, nucleic acids can be rapidly absorbed and distributed throughout the hydrogel matrix. Figure 8 (bc). This phenomenon is mainly attributed to the greater resistance to the diffusion of nucleic acid molecules caused by the smaller pore size. The above results indicate that the nucleic acid uptake process can be effectively controlled by adjusting the hydrogel concentration.

[0098] Fluorescence characterization results confirmed that nucleic acid molecules can be spontaneously absorbed into the three-dimensional porous hydrogel structure without external driving, strongly supporting the feasibility of a hydrogel-based self-water-absorbing nucleic acid absorption mechanism. Based on these results, to ensure that the nucleic acids in the sample solution can be fully absorbed into the hydrogel matrix, the absorption time was set to 10 min in subsequent experiments.

[0099] Example 8 This embodiment explores the accuracy verification of the hydrogel self-absorbing microfluidic chip prepared in Example 7 for digital nucleic acid detection. Specifically, Staphylococcus aureus genomic DNA extracted from a commercially available kit was used as the test sample, and the method is as follows: (1) During the test, tear open the pre-made ready-to-use hydrogel chip sealing cap, add 5 μL of sample solution to the surface of the hydrogel chip, and seal it immediately. After standing for 10 min, the sample can be spontaneously absorbed into its nanoporous structure through the water absorption of the hydrogel.

[0100] (2) During the nucleic acid amplification reaction, the hydrogel chip sealed on the surface of the glass slide was placed on a 65°C heating plate and incubated for 20 min for LAMP reaction. During this process, the target nucleic acid was amplified in situ, and the amplification products were confined in the cross-linked hydrogel network to form discrete fluorescent nucleic acid spots for digital counting.

[0101] (3) After amplification, fluorescent spot imaging is observed using a fluorescence microscope (Leica DMi8, Leica, Wetzlar, Germany) or the spots are counted visually using a blue LED flashlight. For microscope-based fluorescent spot imaging, ImageJ software can be used for counting and analysis. Each fluorescent spot corresponds to one nucleic acid molecule in the sample, thus achieving visualized absolute quantitative analysis of nucleic acids.

[0102] The results are as follows Figure 9 As shown, reliable digital nucleic acid detection was achieved in the concentration range of 1–400 copies / μL. Figure 9 (a). The detection results are highly consistent with those obtained from commercial digital PCR (QuantStudio 3D Digital PCR System platform). Figure 9 b, R 2 = 0.997), indicating that the method has high absolute quantitative accuracy.

[0103] Example 9 This embodiment investigates the reagent preservation ability and matrix interference resistance of the hydrogel microfluidic chip prepared in Example 7. Specifically, the detection performance of the pre-fabricated ready-to-use hydrogel microfluidic chip was compared after one day and one month of storage, and the detection results of gel amplification by directly mixing the sample solution with the hydrogel precursor solution and self-absorption amplification were compared. The methods are as follows: 1. Reagent preservation capability Self-absorption hydrogel amplification: The prefabricated self-absorption hydrogel microfluidic chip was stored at 4°C in the dark. On the first day and the first month of storage, the same concentration of the test sample solution was added according to the method described in Example 8, and the endpoint fluorescence images were captured and recorded. The number of amplified fluorescent spots was statistically analyzed.

[0104] Aqueous solution amplification: The LAMP reaction system without hydrogel monomers was stored at 4°C in the dark. On the first day of storage and at the first month of storage, positive (with DNA template added) and negative (without DNA template added) real-time fluorescence amplification of the aqueous solution system was performed using a real-time fluorescence quantitative PCR instrument (Applied Biosystems™).

[0105] A schematic diagram of nucleic acid amplification reagent storage in aqueous solutions and hydrogels is shown below. Figure 10 As shown in Figure a, the test results indicate that the reagents stored in the hydrogel maintained stable amplification curves and Ct values ​​even after one month, demonstrating excellent reagent preservation capabilities and long-term stability. Figure 10 (c). In contrast, the reagent stored in the aqueous solution showed significant false positive results after the same amount of time.

[0106] like Figure 10 As shown in b, the hydrogel chip can still achieve digital amplification and absolute quantification of nucleic acids after one month of storage, and the number of fluorescent spots does not change significantly, further verifying its stability.

[0107] 2. Resistance to matrix interference Holly leaves were harvested, 1 g of leaves were chopped, and 1 mL of sterile water was added and thoroughly ground with a glass rod. The liquid containing the holly juice was sterilized and used as an amplification inhibitor, added to both the control and experimental groups. In the control group, 0.5 μL of the juice was added to the *Vibrio parahaemolyticus* DNA sample to be tested. This nucleic acid sample mixed with the inhibitor was then thoroughly mixed with other reagents in the LAMP reaction system. Finally, hydrogel monomers were added, and the mixture was allowed to stand at room temperature until complete gel formation before incubation at 65°C for 20 min to amplify, simulating digital quantitative detection without the hydrogel absorption nucleic acid process. In the experimental group, 0.5 μL of the juice was also added to the *Vibrio parahaemolyticus* DNA sample to be tested for premixing. The DNA sample containing the inhibitor was then added to a pre-fabricated hydrogel self-absorption microfluidic chip, followed by incubation at 65°C for 20 min to amplify, thus simulating the detection method using the hydrogel self-absorption nucleic acid process. The endpoint fluorescence images after amplification were compared between the two groups, and the effect of the hydrogel self-absorption nucleic acid process on the anti-interference performance of the system was statistically analyzed.

[0108] The results are as follows Figure 10 As shown in Figure d, no fluorescent spots were observed when plant juice was directly mixed with the LAMP reaction system, indicating severe inhibition of amplification. In contrast, clear and countable fluorescent spots were observed when the sample was added to the self-absorbing hydrogel chip. These results indicate that the LAMP reaction is significantly inhibited in the mixed system, while the self-absorbing hydrogel chip can effectively maintain the amplification reaction in the presence of inhibitors.

[0109] Example 10 This embodiment explores multiplex digital nucleic acid detection using a hydrogel self-absorption microfluidic chip array. Specifically, different primers were pre-embedded in different regions of the hydrogel microfluidic chip array prepared in Example 7 to detect Staphylococcus aureus, Salmonella typhimurium, and Vibrio parahaemolyticus, respectively, achieving simultaneous detection of three targets with a single sample loading. The method is as follows: (1) Tear off the sealing cover on the pre-made ready-to-use hydrogel microfluidic chip array, drop a sample solution containing genomic DNA of a single pathogen of Staphylococcus aureus, Salmonella typhimurium, or Vibrio parahaemolyticus, or a mixture of the three, onto the multi-hydrogel chip, and then seal it again.

[0110] (2) Let stand for 10 min to allow the hydrogel to fully exert its self-absorption effect and absorb the nucleic acid in the sample to be tested into the corresponding area without the need for precise fluid manipulation.

[0111] (3) The LAMP reaction was carried out by incubating at 65℃ for 20 min. After the reaction was completed, the fluorescence image of the hydrogel microfluidic chip array was photographed and recorded under fluorescence imaging conditions.

[0112] Result interpretation method: By observing which area on the multiplex detection chip shows fluorescent spots, the types and quantities of nucleic acids in the sample can be determined, enabling rapid and simultaneous quantitative detection of multiple nucleic acids. In the fluorescence image, the number of fluorescent spots represents the quantity of target nucleic acids in the sample, and the location of the fluorescent spots can identify the type of bacteria.

[0113] Results analysis: Prefabricated, ready-to-use multi-hydrogel microfluidic chip arrays, such as Figure 11 As shown in a, the detection results are as follows: Figure 11 As shown in bd, when the sample contains only a single pathogen, fluorescent spots appear only in the corresponding area, while the remaining areas remain negative. Figure 11 As shown in e and f, when the genomic DNA of three target bacteria is present in the sample solution, the number of fluorescent spots corresponds to the number of pathogens in the sample, while the area where the spots are located represents the type of pathogen.

[0114] These results demonstrate that the hydrogel microfluidic chip array achieves effective partitioning, with each region accurately representing its corresponding target, and enables digital absolute quantitative detection.

[0115] Example 11 This embodiment conducts a spiked recovery experiment on actual food samples and compares it with a commercial digital detection platform. Details are as follows: (1) Six different real samples commonly found in the market, namely peach, apple, grape, pear, coconut and white wine, were inoculated with Staphylococcus aureus. The fruits were made into fresh juice and then inoculated with Staphylococcus aureus.

[0116] (2) Take 1 mL of real sample containing Staphylococcus aureus, heat it at 95℃ for 10 min, and then cool it in an ice box at -20℃ for 5 min to obtain freshly squeezed juice and alcoholic beverages containing Staphylococcus aureus DNA.

[0117] (3) The hydrogel microfluidic chip prepared in Example 7 was used to perform quantitative detection with the commercial digital nucleic acid detection chip (QuantStudio 3D Digital 20K Chip), and the recovery rates of the two were compared.

[0118] The results are as follows Figure 12 As shown in Figure a, the detection performance of this method is comparable to that of commercial platforms. Furthermore, the reliability of the method was validated in a wider range of fruit and vegetable samples, including pears, grapes, apples, peaches, coconut juice, and white wine, such as... Figure 12 As shown in c, countable fluorescent spots were successfully obtained in all six spiked samples, with recoveries ranging from 82% to 106%.

[0119] The above results demonstrate that the self-absorbing hydrogel microfluidic chip array exhibits good accuracy and robustness in detecting pathogens in complex real food samples, showing excellent potential for practical applications.

[0120] The above description is merely a specific embodiment of the present invention, intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly, and should not be construed as limiting the scope of protection of the present invention. All equivalent modifications or substitutions made based on the essence of the present invention should be covered within the scope of protection of the present invention.

Claims

1. A digital nucleic acid detection method based on a hydrogel self-absorbing microfluidic chip, characterized in that, Includes the following steps: (1) The hydrogel monomer is mixed with a nucleic acid amplification reagent containing primers and fluorescent dyes for specific detection target nucleic acids to obtain a mixture, and the mixture is gelled to form a hydrogel microfluidic chip with a thickness of 150~250 μm and an average pore size of 20~90 nm inside the gel. (2) Drop the sample solution to be tested onto the surface of the hydrogel microfluidic chip, seal it and let it stand for 8-12 min; then place it under nucleic acid amplification conditions to carry out nucleic acid amplification reaction. (3) After the amplification is completed, fluorescence imaging is performed on the hydrogel microfluidic chip, and the fluorescence spots are counted for quantitative analysis.

2. The digital nucleic acid detection method as described in claim 1, characterized in that, The hydrogel monomer is poly(ethylene glycol) diacrylate; the molecular weight of poly(ethylene glycol) diacrylate is 700 MW, and the final volume percentage concentration of poly(ethylene glycol) diacrylate in the mixture is 5~8%.

3. The digital nucleic acid detection method as described in claim 2, characterized in that, The mixture contains photoinitiator 1173, and the mixture is heated at an intensity of 20~25 mW / cm. 2 Irradiate with ultraviolet light at a wavelength of 365 nm for 2-6 minutes to solidify into a gel.

4. The digital nucleic acid detection method as described in claim 1, characterized in that, The hydrogel monomers are polyethylene glycol-acrylate and polyethylene glycol-mercapto. The molecular weight of polyethylene glycol-acrylate is 5000~10000 MW, and the molecular weight of polyethylene glycol-mercapto is 2000~3400 MW. The molar ratio of the two is 1:2, and the concentration of polyethylene glycol-acrylate in the mixed liquid system is 3~9 mM.

5. The digital nucleic acid detection method as described in claim 1, characterized in that, The thickness of the hydrogel microfluidic chip is 200 μm.

6. The digital nucleic acid detection method as described in claim 1, characterized in that, The nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, a recombinase polymerase amplification reaction, or a rolling circle amplification reaction.

7. The digital nucleic acid detection method as described in claim 6, characterized in that, When the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, the mixture comprises: 1× isothermal amplification buffer, 2~6 mM MgSO4, 1~1.8 mM dNTPs, and 120~360 U Bst 2.0 DNA polymerase, 0.5–2× fluorescent dye, 5–8% (v / v) poly(ethylene glycol) diacrylate, 0.03–0.05% (v / v) photoinitiator 1173 and a 1× LAMP primer mixture, wherein the 1× LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM F3 and B3, and 0.8 μM LF and LB.

8. The digital nucleic acid detection method as described in claim 6, characterized in that, When the nucleic acid amplification reaction is a loop-mediated isothermal amplification reaction, the mixture comprises: 1× isothermal amplification buffer, 2~6 mM MgSO4, 1~1.8 mM dNTPs, and 120~360 U / mL. Bst 2.0 DNA polymerase, 0.5–2× fluorescent dye, 3–9 mM polyethylene glycol-acrylate, 6–18 mM polyethylene glycol-thiol, and a 1× LAMP primer mixture, wherein the 1× LAMP primer mixture consists of 1.6 μM FIB and BIP, 0.2 μM F3 and B3, and 0.4 μM LF and LB; when the target analyte is RNA, 200–400 U / mL of reverse transcriptase is added to the reaction system.

9. The digital nucleic acid detection method as described in claim 6, characterized in that, The conditions for the ring-mediated isothermal amplification reaction are heating at 63~68℃ for 10~30 min.

10. The digital nucleic acid detection method as described in claim 1, characterized in that, In step (1), an array of several hydrogel microfluidic chips is prepared on the same substrate. Different hydrogel microfluidic chips contain specific primers for different targets, serving as reaction chambers for multiplex digital nucleic acid detection.

Images