System and method for detecting corneal pathogens

Abstract

According to the system and method for detecting corneal pathogens, in the system and method, the adsorption capacity is enhanced through ionic liquid modified MXene, silk fibroin in silk fibroin composite hydrogel has good biocompatibility, pathogens on the surface of the cornea can be accurately extracted after compounding, the bacterial adsorption efficiency reaches 92.3 + / -2.7%, and the bacterial adsorption efficiency reaches 92.3 + / -2.7%. The adsorption efficiency of fungi reaches 85.4 + / -3.8%, the adsorption efficiency of viruses reaches 88.6 + / -3.5%, high adsorption efficiency is achieved, and non-invasive sampling is achieved. The combined use of the micro-fluidic chip and the LAMP technology can accurately identify the nucleic acid sequence of a target pathogen, the detected pathogen can be judged only according to the detected fluorescence signal or color change, the pathogen detection can be rapidly completed within 1 h, the detection sensitivity reaches 101-102 CFU / mL, high-sensitivity, rapid and non-invasive detection is realized, and the detection cost is reduced. The kit is suitable for early diagnosis of keratitis.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedical detection, and relates to a system and method for detecting corneal pathogens. Background Art

[0002] Keratitis is an eye disease usually caused by bacterial, viral, fungal or other microbial infections, and its main feature is the inflammation and damage of corneal tissue.

[0003] The typical symptoms of keratitis are ocular irritation symptoms, which can affect vision and cause pain, burning sensation, tearing, etc. When severe, it will develop into suppurative keratitis, presenting eyelid swelling, purulent discharge, etc. Therefore, early diagnosis of keratitis is crucial for preventing vision damage. Currently, the detection methods of keratitis usually include corneal scraping microscopy, fungal culture, etc. Corneal scraping microscopy requires scraping the patient's cornea during sampling, which will cause discomfort to the patient's eyes and may also damage corneal tissue. Fungal culture relies on laboratory culture or PCR amplification technology, and the detection cycle is as long as several hours or days, affecting the treatment of keratitis. At the same time, PCR amplification detection has the problem of missed detection of low-concentration pathogens, resulting in misdiagnosis or delayed treatment.

[0004] In recent years, microneedle technology has attracted much attention in the biomedical field due to its non-invasive sampling characteristics. However, existing microneedle materials have problems of low adsorption efficiency and insufficient biocompatibility, and cannot effectively complete the sampling and related detection of corneal pathogenic bacteria. Summary of the Invention

[0005] The purpose of the present invention is to provide a system and method for detecting corneal pathogens to solve the problems of low detection efficiency and low sensitivity of existing detection systems and methods.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions: The present application provides a system for detecting corneal pathogens, which includes composite hydrogel microneedles and a microfluidic chip. The preparation raw materials of the composite hydrogel microneedles include MXene dispersion, poly(N-isopropylacrylamide) and silk fibroin composite hydrogel. The microfluidic chip includes a sample loading channel, a LAMP reaction chamber and a fluorescence / colorimetric detection module that are connected in communication. Among them, after the composite hydrogel microneedles adsorb corneal pathogenic bacteria, they are added to the microfluidic chip through the sample loading channel.

[0007] In this application, the MXene dispersion is formed by mixing MXene, 1-Ethyl-3-methylimidazolium Tetrafluoroborate (abbreviation: [EMIM][BF4]), and 1-Butyl-3-methylimidazolium chloride (abbreviation: [BMIM]Cl). MXene is a two-dimensional inorganic compound, and its adsorption ability can be enhanced through the modification of ionic liquids [EMIM][BF4] and [BMIM]Cl. As a result, the composite hydrogel microneedles can adsorb pathogenic bacteria on the cornea and enhance the adsorption efficiency.

[0008] Poly(N-isopropylacrylamide) is mainly formed by polymerizing the monomer N-isopropylacrylamide and is used in controlled release, biochemical separation, chemical sensors, etc. In this application, the preparation method of poly(N-isopropylacrylamide) includes: S01: Dissolve the N-isopropylacrylamide monomer in deionized water to prepare an N-isopropylacrylamide monomer solution with a mass fraction of 20%. S02: Add the crosslinking agent N,N'-methylenebisacrylamide and the initiator ammonium persulfate to the N-isopropylacrylamide monomer solution. After mixing evenly, purge with nitrogen or argon to reduce the influence of oxygen in the solution on the polymerization reaction. Among them, the addition amount of N,N'-methylenebisacrylamide is 0.1 - 5% of the mass of the N-isopropylacrylamide monomer, and the addition amount of ammonium persulfate is 0.01 - 1% of the mass of the N-isopropylacrylamide monomer. S03: Polymerize the above-treated mixture at 25 - 70 °C for 3 h, and monitor the progress of the polymerization reaction by observing the viscosity change of the solution or using methods such as gel permeation chromatography. S04: When the polymerization reaction reaches the expected degree, poly(N-isopropylacrylamide) is obtained.

[0009] The silk fibroin in the silk fibroin composite hydrogel has good biocompatibility. After being compounded with the MXene dispersion and poly(N-isopropylacrylamide), it can accurately extract pathogens on the corneal surface and achieve non-invasive sampling.

[0010] More preferably, the raw materials for preparing the composite hydrogel microneedles include 10 - 20% of the MXene dispersion, 45 - 55% of poly(N-isopropylacrylamide), and 30 - 40% of the silk fibroin composite hydrogel by mass fraction. Most preferably, the raw materials for preparing the composite hydrogel microneedles include 15% of the MXene dispersion, 50% of poly(N-isopropylacrylamide), and 35% of the silk fibroin composite hydrogel by mass fraction.

[0011] In this application, the preparation method of the composite hydrogel microneedles includes: after uniformly mixing the MXene dispersion, poly(N-isopropylacrylamide) and silk fibroin composite hydrogel, injecting it into a PDMS (Polydimethylsiloxane) microneedle mold, and curing it in ultraviolet light with an intensity of 25 mW / cm 2 for 2 min, and then demolding to obtain the composite hydrogel microneedles. The tip height of the prepared composite hydrogel microneedles is 500 - 800 μm, and the bottom diameter is 200 - 300 μm.

[0012] By testing the mechanical strength, swelling ratio, cytotoxicity, etc. of the composite hydrogel microneedles respectively, it is determined that the compressive strength of the composite hydrogel microneedles is greater than 0.1 N / needle, the swelling ratio in physiological saline is greater than 150%, and the cell survival rate is greater than 80%.

[0013] The microfluidic chip includes a sample loading channel, a LAMP reaction chamber, and a fluorescence / colorimetric detection module that are connected and communicate with each other. In this application, the size of the microfluidic chip is 25 * 75 mm, the height of the sample loading channel is 100 μm, the channel width is 200 μm, the length of the main channel is 50 mm, and the length of the branch channel is 10 mm; the volume of the LAMP reaction chamber is 1 μm, and the number is 8; the shape of the LAMP reaction chamber is a diameter of 1 mm and a depth of 100 μm; the diameter of the sampling hole at the sample loading channel is 1 mm and the depth is 100 μm.

[0014] In addition, this application provides a method for detecting corneal pathogens, which includes: S01: Using the composite hydrogel microneedles to adsorb the pathogens on the corneal surface, and obtaining pathogen DNA / RNA after lysis and purification.

[0015] Press the composite hydrogel microneedles on the corneal surface at a pressure of 0.1 N / cm 2 for 10 - 15 min, so that the pathogenic bacteria on the corneal surface are adsorbed on the composite hydrogel microneedles. After the pressing is completed, take out the microneedles and wash them 3 times with a sterile PBS (phosphate buffered saline) buffer solution to remove the unadsorbed pathogens, and obtain the composite hydrogel microneedles with corneal pathogen cells on the surface. The adsorption rate of the composite hydrogel microneedles to corneal pathogen cells is greater than 90%.

[0016] The composite hydrogel microneedles adsorbed with corneal pathogens are immersed in a lysis buffer and incubated at 37 - 55 °C for 30 min. During the incubation process, vortex oscillation is performed 3 times, 10 s each time. After the incubation, centrifugation is carried out at 12,000 rpm for 10 min to obtain the supernatant. The supernatant is eluted and purified using a Qiagen DNeasy or RNeasy kit to ensure that the A260 / A280 ratio is 1.8 - 2.0, and the purified pathogen DNA / RNA is obtained. Among them, the lysis buffer can be a buffer formed by 0.5% SDS (English name: Sodium dodecyl sulfate; Chinese name: Sodium dodecyl sulfate), 50 μg / mL proteinase K, and Tris-HCl with a pH of 8.0 and a concentration of 10 mM, or it can be a buffer with a pH of 7.5 formed by 1% Triton X-100 and 1 mM EDTA, or it can also be a buffer formed by 0.5% SDS and 50 μg / mL lysozyme, which can be determined according to the actual pathogens to be detected.

[0017] S02: The purified pathogen DNA / RNA is added to the LAMP reaction chamber of the microfluidic chip through the sample loading channel and subjected to constant-temperature LAMP amplification at 60 - 65 °C for 30 - 40 min to obtain the amplification product.

[0018] The purified pathogen DNA / RNA is automatically aspirated into the LAMP reaction chamber of the microfluidic chip through the sample loading channel with capillary force. The LAMP reaction chamber includes 0.5 - 0.8 μM pre-loaded primers, 1×Bst DNA polymerase, 0.2 - 1.4 mM dNTPs, and a dye. Among them, according to the different corneal pathogens, the specific types of pre-loaded primers are different, and the selection of the dye is different, which needs to be determined according to actual needs. After the pathogen DNA / RNA is added to the LAMP reaction chamber, it is subjected to constant-temperature LAMP amplification at 60 - 65 °C for 30 - 40 min to obtain the amplification product.

[0019] S03: After the amplification product is detected by fluorescence and / or colorimetry, the detection result is obtained.

[0020] After the amplification product is detected by fluorescence and / or colorimetry, the result is analyzed in real time through the fluorescence signal or color change to obtain the detection result.

[0021] The present invention has the following beneficial effects: (1) MXene modified by ionic liquids [EMIM][BF4] and [BMIM]Cl can enhance its adsorption capacity, enabling the composite hydrogel microneedles to adsorb pathogenic bacteria on the cornea, enhancing the adsorption efficiency, with the bacterial adsorption efficiency reaching 92.3±2.7%, the fungal adsorption efficiency reaching 85.4±3.8%, and the viral adsorption efficiency reaching 88.6±3.5%, achieving high adsorption efficiency, and the standard deviation <4%, with strong data repeatability.

[0022] (2) The silk fibroin in the silk fibroin composite hydrogel has good biocompatibility. After being compounded with the MXene dispersion and poly(N-isopropylacrylamide), it can accurately extract pathogens on the corneal surface and achieve non-invasive sampling.

[0023] (3) The combined use of a microfluidic chip and LAMP (loop-mediated isothermal amplification) technology can accurately identify the nucleic acid sequence of the target pathogen. It is only necessary to judge the detected pathogen based on the detected fluorescence signal or color change. At the same time, it can quickly complete the detection of the pathogen within 1h, and the detection sensitivity reaches 10^1-10^2 CFU / mL, achieving high-sensitivity, rapid and non-invasive detection, and is suitable for the early diagnosis of keratitis. Detailed implementation mode

[0024] The technical solutions of the present invention will be further explained and illustrated below through specific examples.

[0025] Example 1 The embodiment of the present application provides a system for detecting corneal pathogens, which includes composite hydrogel microneedles with a tip height of 600μm and a bottom diameter of 250μm and a microfluidic chip. Among them, the preparation raw materials of the composite hydrogel microneedles include 15% MXene dispersion, 50% poly(N-isopropylacrylamide), and 35% silk fibroin composite hydrogel by mass fraction.

[0026] The present application also provides a method for detecting corneal pathogens, which uses Staphylococcus aureus with a concentration of 10^6 CFU / mL as the detection target, specifically including: S101: Suspend the Staphylococcus aureus ATCC 25923 standard strain in PBS buffer with a pH value of 7.4, and adjust the concentration of Staphylococcus aureus to 10^6 CFU / mL. Uniformly coat Staphylococcus aureus on the surface of a 1.5% agarose simulated corneal tissue, and let it stand for 30 min to form a simulated cornea. The composite hydrogel microneedles with a tip height of 600μm and a bottom diameter of 250μm are used at 0.1N / cm 2Press on the cornea surface with pressure for 10 min, take out the microneedles, and wash them 3 times with sterile PBS buffer to remove the unadsorbed pathogens, obtaining the composite hydrogel microneedles with corneal pathogen bodies on the surface.

[0027] Immerse the composite hydrogel microneedles adsorbed with corneal pathogens into the lysis buffer composed of 0.5% SDS, 50 μg / mL proteinase K, and Tris-HCl with a pH of 8.0 and a concentration of 10 mM, incubate at 37 °C for 30 min. During the incubation, vortex 3 times, 10 s each time. After the incubation, centrifuge at 12,000 rpm for 10 min, and elute and purify the supernatant using the Qiagen DNeasy Blood & Tissue Kit, with an elution volume of 50 μL. After the elution, measure the DNA concentration to be 50.2 ± 3.1 ng / μL using NanoDrop, and ensure that the A260 / A280 ratio is 1.89 ± 0.05, obtaining the purified pathogen DNA / RNA.

[0028] S102: Automatically aspirate 5 μL of the purified pathogen DNA into the LAMP reaction chamber through the sample loading channel, and perform isothermal LAMP amplification at 65 °C for 30 min to obtain the amplification product. Among them, the LAMP reaction chamber includes a pre-loaded primer for the nuc gene at 0.8 μM, 1× Bst DNA polymerase, 0.2 mM dNTPs, and 0.2× SYBR Green I dye.

[0029] Through three independent experiments by the plate counting method, it is known that the average adsorption efficiency of the composite hydrogel microneedles is 92.3 ± 2.7%. In the fluorescence amplification curve, the sample Ct value of the pathogen DNA is 22.5 ± 1.3 (n = 3), and the sample with sterile PBS as the negative control has no signal. The gradient dilution test shows that the lower limit of detection of this system for Staphylococcus aureus is 10^2 CFU / mL (Ct value < 30).

[0030] Example 2 The embodiment of the present application provides a system for detecting corneal pathogens, which includes a composite hydrogel microneedle with a tip height of 500 μm and a bottom diameter of 200 μm and a microfluidic chip. Among them, the preparation raw materials of the composite hydrogel microneedle include 10% MXene dispersion, 50% poly(N-isopropylacrylamide), and 40% silk fibroin composite hydrogel by mass fraction.

[0031] The present application also provides a method for detecting corneal pathogens, which uses herpes simplex virus-1 (English name: herpes simplex virus-1; abbreviation: HSV-1) with a concentration of 10^5 CFU / mL as the detection target, and specifically includes: S201: Inoculate the HSV-1 ATCC VR-733 standard strain in the Vero cell culture supernatant, centrifuge and concentrate it to 10^5 PFU / mL, drop it onto the surface of a 1.5% agarose simulated corneal tissue, and let it stand for 30 min to form a simulated cornea. Press the composite hydrogel microneedle with a tip height of 500 μm and a bottom diameter of 200 μm on the corneal surface at a pressure of 0.1 N / cm 2 for 15 min, remove the microneedle, wash it 3 times with a sterile PBS buffer to remove the unadsorbed pathogens, and obtain a composite hydrogel microneedle with corneal pathogen cells on its surface.

[0032] Immerse the composite hydrogel microneedle adsorbed with corneal pathogens in a lysis buffer with a pH of 7.5 composed of 1% Triton X-100 and 1 mM EDTA, incubate it at 55 °C for 20 min. During the incubation process, vortex it 3 times, 10 s each time. After the incubation, centrifuge it at a speed of 12,000 rpm for 10 min, and elute and purify the supernatant using the Qiagen RNeasy Mini Kit, with an elution volume of 30 μL. After the elution, measure the RNA concentration to be 28.4 ± 2.8 ng / μL using NanoDrop, and ensure that the A260 / A280 ratio is 1.91 ± 0.03 to obtain the purified pathogen RNA.

[0033] S202: Automatically aspirate the purified pathogen RNA into the LAMP reaction chamber through the sample loading channel, and perform isothermal LAMP amplification at 63 °C for 40 min to obtain an amplification product. Among them, the LAMP reaction chamber includes a pre-loaded primer for the HSV UL30 gene at 0.5 μM, 1×Bst 2.0 WarmStart DNA polymerase, 0.2 mM dNTPs, and 1×SYTO-82 dye.

[0034] Through three independent experiments by the plate counting method, it is known that the average adsorption efficiency of the composite hydrogel microneedle is 88.6 ± 3.5%. In the fluorescence amplification curve, the sample Ct value of the pathogen RNA is 24.1 ± 1.8 (n = 3), and the sample with sterile PBS as the negative control has no signal. There is no cross-reaction with cytomegalovirus (CMV) and adenovirus (AdV), showing specificity.

[0035] Example 3 An embodiment of the present application provides a system for detecting corneal pathogens, which includes a composite hydrogel microneedle with a tip height of 800 μm and a bottom diameter of 300 μm, and a microfluidic chip. Among them, the raw materials for preparing the composite hydrogel microneedle include 20% MXene dispersion, 45% poly(N-isopropylacrylamide), and 35% silk fibroin composite hydrogel by mass fraction.

[0036] The present application also provides a method for detecting corneal pathogens, which uses Candida albicans with a concentration of 10^4 CFU / mL as the detection target, and specifically includes: S301: Uniformly coat the suspension of Candida albicans ATCC 90028 standard strain with a concentration of 10^4 CFU / mL on the surface of a 1.5% agarose simulated corneal tissue, and let it stand for 30 min to form a simulated cornea. Press the composite hydrogel microneedle with a tip height of 800 μm and a bottom diameter of 300 μm on the corneal surface at a pressure of 0.1 N / cm 2 for 10 min, take out the microneedle, and wash it 3 times with sterile PBS buffer to remove the unadsorbed pathogens, obtaining a composite hydrogel microneedle with corneal pathogen cells carried on its surface.

[0037] Add 0.5 mm glass beads and a lysis buffer composed of 0.5% SDS and 50 μg / mL lysozyme to the composite hydrogel microneedle adsorbed with corneal pathogens, and incubate at 37 °C for 30 min. During the incubation process, vortex and oscillate at a frequency of 30 s / time for 5 minutes to break the fungal cell wall. After the incubation, centrifuge at 12,000 rpm for 10 min, and use the DNeasy Plant Mini Kit to elute and purify the supernatant, with an elution volume of 50 μL. After the elution, use NanoDrop to measure the DNA concentration to be 35.7 ± 2.5 ng / μL, and ensure that the A260 / A280 ratio is 1.87 ± 0.04 to obtain purified pathogen DNA.

[0038] S302: Automatically suck the purified pathogen DNA into the LAMP reaction chamber through the sample loading channel, and perform isothermal LAMP amplification at 65 °C for 35 min to obtain an amplification product. Among them, the LAMP reaction chamber includes a pre-loaded primer designed for the fungal ITS1 region at 0.5 μM, 1× Bst DNA polymerase, 0.2 mM dNTPs, and a dye.

[0039] S303: Through three independent experiments using the plate counting method, it was found that the average adsorption efficiency of the composite hydrogel microneedles was 85.4 ± 3.8%. The sample reaction solution of the pathogen DNA changed from purple to blue, while the color of the sample with sterile PBS as the negative control did not change. The gradient dilution test showed that the lower limit of detection of this system for Candida albicans was 10^2 CFU / mL, and the color change was significant.

[0040] Example 4 The embodiment of the present application provides a system for detecting corneal pathogens, which includes composite hydrogel microneedles with a tip height of 600 μm and a bottom diameter of 250 μm, and a microfluidic chip. Among them, the preparation raw materials of the composite hydrogel microneedles include 15% MXene dispersion, 55% poly(N-isopropylacrylamide), and 30% silk fibroin composite hydrogel by mass fraction.

[0041] The present application also provides a method for detecting corneal pathogens, which uses Staphylococcus aureus, Herpes simplex virus, and Candida albicans as the detection targets simultaneously. Specifically, it includes: S401: Suspend the Staphylococcus aureus ATCC 25923 standard strain in PBS buffer with a pH value of 7.4, and adjust the concentration of Staphylococcus aureus to 10^6 CFU / mL. Uniformly coat Staphylococcus aureus on the surface of 1.5% agarose simulated corneal tissue. Inoculate the HSV-1 ATCC VR-733 standard strain in the Vero cell culture supernatant, centrifuge and concentrate it to 10^5 PFU / mL, and drop it onto the surface of 1.5% agarose simulated corneal tissue. Uniformly coat the suspension of Candida albicans ATCC 90028 standard strain on the surface of this 1.5% agarose simulated corneal tissue, and let it stand for 60 min to form a simulated cornea. Press the composite hydrogel microneedles with a tip height of 600 μm and a bottom diameter of 250 μm on the corneal surface at a pressure of 0.1 N / cm 2 for 10 min, take out the microneedles, and wash them 3 times with sterile PBS buffer to remove the unadsorbed pathogens, obtaining composite hydrogel microneedles with corneal pathogen bodies on the surface.

[0042] The composite hydrogel microneedles adsorbed with corneal pathogens were immersed in a lysis buffer composed of 0.5 mm glass beads, 0.5% SDS, 50 μg / mL proteinase K, 10 mM Tris-HCl pH 8.0, 1% Triton X-100, and 1 mM EDTA, and incubated at 37 °C for 30 min. During the incubation, vortex oscillation was performed 3 times, 10 s each time. After incubation, centrifugation was carried out at 12,000 rpm for 10 min, and the supernatant was eluted and purified using the Qiagen DNeasy Blood&Tissue Kit, with an elution volume of 130 μL.

[0043] S402: 15 μL of the purified pathogen DNA / RNA was automatically aspirated into the LAMP reaction chamber through the sample loading channel and subjected to isothermal LAMP amplification at 65 °C for 30 min to obtain the amplification product. Among them, the LAMP reaction chamber included 1×Bst DNA polymerase buffer, 1.4 mM dNTPs, 0.8 μM primer targeting the nuc gene, 0.8 μM primer targeting the ITS1 region, 0.5 μM primer targeting the HSV UL30 gene, 0.2×SYBR Green I dye, 1×SYTO-82 dye, and the 0.2×SYBR Green I dye was buried in three reaction zones respectively.

[0044] S403: During the isothermal LAMP amplification process, the Ct value of the fluorescence signal was monitored in real time and Ct < 25, and the color change of the indicator hydroxynaphthol blue (abbreviation: HNB) was observed. Through three independent experiments by the plate counting method, the average adsorption efficiency of the composite hydrogel microneedles was 90.5 ± 3.2%. In the fluorescence amplification curve, the Ct value of the positive sample of Staphylococcus aureus was 22.8 ± 1.5 (n = 3), and there was no signal in the sample with sterile PBS as the negative control; the Ct value of the positive sample of herpes simplex virus was 24.3 ± 1.7 (n = 3), and there was no signal in the sample with no template as the negative control; the positive sample of Candida albicans changed from purple to blue, and the color of the sample with sterile PBS as the negative control did not change. Regarding the sensitivity, the detection limit of Staphylococcus aureus was 10^2 CFU / mL (Ct < 30), the detection limit of herpes simplex virus was 10^1 PFU / mL (Ct < 30), and the detection limit of Candida albicans was 10^1 CFU / mL (significant color change).

[0045] As can be seen from the detection and analysis of the above embodiments, the composite hydrogel microneedles can effectively adsorb a variety of corneal pathogens, and the adsorption rate is relatively high, reaching more than 85%. The LAMP technology can simultaneously detect Staphylococcus aureus, Herpes simplex virus and Candida albicans, with good specificity, and the detection sensitivity reaches 10^1 - 10^2 CFU / mL, which is suitable for the rapid and non-invasive detection of corneal pathogens.

[0046] The above are only the preferred embodiments of the present application and are not intended to limit the present application. For those skilled in the art, various changes and modifications can be made to the present application. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included within the protection scope of the present application.

Claims

1. A system for detecting corneal pathogens, characterized in that: It comprises a composite hydrogel microneedle and a microfluidic chip. The microfluidic chip comprises a connected sample loading channel, a LAMP reaction chamber and a fluorescence / colorimetric detection module. The composite hydrogel microneedle adsorbs corneal pathogens and then adds them into the microfluidic chip through the sample loading channel.

2. The system for detecting corneal pathogens according to claim 1, characterized in that: The raw materials for preparing the composite hydrogel microneedle include MXene dispersion, poly (N-isopropylacrylamide) and silk fibroin composite hydrogel.

3. The system for detecting corneal pathogens according to claim 1, characterized in that: The raw materials for preparing the composite hydrogel microneedle include 10-20% of MXene dispersion, 45-55% of poly (N-isopropylacrylamide) and 30-40% of silk fibroin composite hydrogel according to mass fraction.

4. The system for detecting corneal pathogens according to claim 1, characterized in that: The composite hydrogel microneedle has a needle tip height of 500-800 μm and a bottom diameter of 200-300 μm.

5. The system for detecting corneal pathogens according to claim 1, characterized in that: The preparation method of the composite hydrogel microneedle comprises: mixing MXene dispersion, poly (N-isopropylacrylamide) and silk fibroin composite hydrogel evenly, injecting into a PDMS microneedle mold, and irradiating under ultraviolet light intensity of 25mW / cm 2 The resulting product was cured under ultraviolet light for 2 min and demolded to obtain composite hydrogel microneedles.

6. The system for detecting corneal pathogens according to claim 1, characterized in that: The MXene dispersion includes MXene, 1-ethyl-3-methylimidazolium tetrafluoroborate and 1-butyl-3-methylimidazolium chloride.

7. A method for detecting corneal pathogens, characterized in that: Using the system for detecting corneal pathogens according to any one of claims 1 to 6, the method comprises: Composite hydrogel microneedles are used to absorb pathogens on the corneal surface, and pathogen DNA / RNA is obtained after lysis and purification; The purified pathogen DNA / RNA is added into the LAMP reaction chamber of the microfluidic chip through the sample loading channel, and amplified by constant temperature LAMP at 60-65° C. for 30-40 minutes to obtain an amplified product; The amplification product is detected by fluorescence and / or colorimetry to obtain a detection result.

8. The method for detecting corneal pathogens according to claim 7, characterized in that: The composite hydrogel microneedle is used to adsorb pathogens on the corneal surface, including: applying the composite hydrogel microneedle at 0.1N / cm 2 The microneedles were removed and washed with sterile PBS buffer to obtain composite hydrogel microneedles carrying corneal pathogens on the surface.

9. The method for detecting corneal pathogens according to claim 7, characterized in that: The LAMP reaction chamber includes 0.5 μM preloaded primer, 1×Bst DNA polymerase, 0.2 mM dNTPs, and dye.

10. The method for detecting corneal pathogens according to claim 7, characterized in that: The cleavage and purification include: The composite hydrogel microneedles adsorbing corneal pathogens were immersed in lysis buffer and incubated at 37-55°C for 30 min. During the incubation process, the microneedles were vortexed 3 times for 10 s each time. After the incubation, centrifuge and use the kit to elute and purify the supernatant to obtain purified pathogen DNA / RNA.