Fluorescent microneedle sensor based on DNA-silver nano-cluster hydrogel as well as preparation method and application of fluorescent microneedle sensor

Abstract

The invention discloses a fluorescent microneedle sensor based on DNA-silver nanocluster hydrogel as well as a preparation method and application of the fluorescent microneedle sensor. According to the sensor, polyacrylamide hydrogel is used as a flexible matrix, and DNA-silver nanoclusters are uniformly embedded into the flexible matrix to serve as a fluorescent indicator. The preparation method comprises the following steps: mixing an acrylamide monomer, a cross-linking agent, a DNA-silver nano-cluster solution, an initiator and a catalyst, and carrying out polymerization and one-step molding in a microneedle mold under the initiation of ultraviolet light. The prepared sensor has the advantages of excellent flexibility, high stretch rate, surface adhesion and stable fluorescence property. When the fluorescent probe is used for detecting thiram, the low detection limit of 0.199 mu M and good linear response can be realized based on the specific fluorescence quenching effect of the fluorescent probe. The sensor can be used for on-site rapid screening of pesticide residues on the surfaces of foods such as fruits and seafood without complex instruments, and has a wide application prospect in the fields of food safety and environmental monitoring.

Description

Technical Field

[0001] This invention relates to the field of biosensing and food safety detection technology, specifically to a fluorescent sensor for detecting the bactericide thiram, particularly a fluorescent microneedle sensor based on DNA-silver nanocluster hydrogel, its preparation method, and its application. Background Technology

[0002] Thiram is a broad-spectrum fungicide derived from dithiocarbamates and widely used in agricultural production. However, long-term excessive use and improper disposal have led to its gradual accumulation in the environment, raising widespread concerns about ecosystems and human health. Studies have shown that thiram disrupts microbial community structure, reduces soil fertility, and is toxic to aquatic organisms, ultimately leading to ecological imbalance. Furthermore, thiram residues on fruits, vegetables, and other agricultural products can enter the human body through the food chain, causing damage to the liver, kidneys, and nervous system. For human health, thiram can also irritate the skin and mucous membranes; long-term exposure may inhibit the activity of key transcription factors such as NF-κB and HIF-1α, thereby causing reproductive and nervous system dysfunction. Therefore, effective detection of thiram residues in food is of great significance. Developing a rapid and sensitive method for detecting thiram is crucial for ensuring food safety.

[0003] Currently, conventional detection methods for thiamethoxam mainly include chromatographic methods (such as gas chromatography and high-performance liquid chromatography) and spectroscopic methods (such as surface-enhanced Raman scattering). However, these methods typically rely on large instruments, are cumbersome to operate, costly, and require professional personnel, making rapid on-site screening difficult. Although fluorescent probe-based sensing technologies have the advantages of high sensitivity and fast response, traditional solution-based fluorescent probes (such as free DNA-silver nanoclusters, DNA-AgNC) are limited. s Its poor stability, susceptibility to environmental interference, and difficulty in reuse limit its practical application.

[0004] Hydrogels are polymeric materials with a three-dimensional network structure, exhibiting good biocompatibility, tunable mechanical properties, and high water content, making them ideal matrices for immobilizing fluorescent probes. Combining fluorescent nanomaterials with hydrogels holds promise for developing stable solid-state or flexible sensors. However, in traditional solution systems, DNA-AgNC… s Susceptible to environmental factors, resulting in unstable fluorescence signals, poor anti-interference ability, and limited reusability and portability, these shortcomings severely restrict its practical application. How to integrate DNA-AgNC... s The current technical challenge is to efficiently and uniformly embed the sensor into the hydrogel while maintaining its high fluorescence performance, and at the same time endow the sensor with flexibility and adhesion to adapt to the detection needs of irregular surfaces. Summary of the Invention

[0005] In view of the problems of strong equipment dependence, complex operation, difficulty in rapid on-site screening, poor stability and limited practicality of solution fluorescent probes in the existing thiram detection methods, the present invention aims to provide a novel thiram detection scheme.

[0006] This study constructed a novel fluorescent microneedle sensor (PAAC) based on a DNA-silver nanocluster hydrogel. This was achieved by using DNA-silver nanoclusters (DNA-AgNC)... s ) Embedded polyacrylamide (PAA) m This is achieved in hydrogels.

[0007] To facilitate on-site detection, the PAAC hydrogel sensor was fabricated as a microneedle array. This microneedle structure can firmly adhere to irregular surfaces, such as seafood, making it ideal for integration into flexible sensing patches. Microscopic observation reveals that the structure consists of uniformly arranged conical microneedles with sharp tips and broad bases. The smooth surface and well-defined geometry endow the microneedles with sufficient mechanical strength and penetration capability, ensuring stable contact with the target substrate during detection.

[0008] This design ensures stable probe fixation and enhances signal response. Upon exposure to thiram, the sensor exhibits strong fluorescence quenching, allowing for highly sensitive visual detection with a detection limit of 0.199 μM. The flexibility and biocompatibility of the hydrogel enable its fabrication in portable formats, such as patches or microchips, for real-time field detection. The system also demonstrates excellent anti-interference capabilities and environmental stability when applied to curved surfaces such as fish skin. This work provides a promising method for field-deployable pesticide detection, supporting food safety and environmental monitoring.

[0009] The novel DNA-silver nanocluster hydrogel fluorescent microneedle sensor disclosed in this invention has the following advantages when used to detect thiamethoxam:

[0010] 1) A solid-state fluorescence sensor that integrates sampling and detection without the need for complex instruments is provided;

[0011] 2) Improved the stability and anti-interference ability of fluorescent probes in complex environments;

[0012] 3) It enables the sensor to adapt to irregular sample surfaces and facilitate convenient detection.

[0013] This invention is achieved through the following technical means:

[0014] In a first aspect, the present invention provides a fluorescent microneedle sensor based on a DNA-silver nanocluster hydrogel.

[0015] The sensor uses polyacrylamide hydrogel as a flexible matrix, DNA-silver nanoclusters as fluorescence sensing units, and has an array-type microneedle structure.

[0016] Preferably, the DNA-silver nanoclusters are uniformly immobilized within a three-dimensional network of polyacrylamide hydrogel.

[0017] Preferably, the DNA-silver nanoclusters emit fluorescence at 625 nm when excited by light at a wavelength of 356 nm.

[0018] Preferably, the particle size of the DNA-silver nanoclusters is 0.1-0.4 μM.

[0019] Preferably, the average particle size of the DNA-silver nanoclusters is 0.26µm.

[0020] Preferably, the sensor exhibits a specific fluorescence response to thiram, and its fluorescence intensity undergoes quantitative quenching as the concentration of thiram increases. Its detection limit for thiram is not higher than 0.199 μM, and its linear detection range is at least 0 to 2 μM.

[0021] Preferably, the sensor exhibits excellent flexibility and mechanical properties, with a maximum elongation of not less than 1000%, and has adhesion to various biological tissue surfaces (such as fish skin) underwater.

[0022] Preferably, the adhesion force of the sensor is not less than 12 kPa.

[0023] Preferably, the adhesion force of the sensor can reach 12.95 kPa.

[0024] In a second aspect, the present invention provides a method for preparing a fluorescent microneedle sensor of DNA-silver nanocluster hydrogel as described in the first aspect.

[0025] The method includes the following steps:

[0026] S1. Preparation of a solution containing DNA-silver nanoclusters;

[0027] S2. Mix the acrylamide monomer, the crosslinking agent N,N'-methylenebisacrylamide, and the solution obtained in step S1 to prepare a prepolymer solution;

[0028] S3. Add ammonium persulfate initiator and N,N,N',N'-tetramethylethylenediamine catalyst to the prepolymer solution, mix evenly, inject into a microneedle mold, and carry out polymerization reaction under ultraviolet light irradiation to obtain a composite hydrogel with microneedle structure;

[0029] S4. The obtained composite hydrogel is post-processed to obtain the microneedle sensor.

[0030] Preferably, in step S1, the DNA-silver nanoclusters are synthesized as follows: after incubating a DNA template with a concentration of 10-20 μM and silver nitrate with a concentration of 80-100 μM in a buffer solution, sodium borohydride with a final concentration of 80-100 μM is added for a reduction reaction.

[0031] Preferably, in step S2, the amount of the solution containing DNA-silver nanoclusters added is 100-500 µL.

[0032] Preferably, in step S3, the ultraviolet light wavelength is 365nm and the polymerization time is 0.5-2 hours; the mold is a polydimethylsiloxane mold with a negative microneedle array.

[0033] Preferably, in step S4, the post-treatment includes washing with deionized water or distilled water to remove unreacted monomers.

[0034] Thirdly, the present invention provides the application of the fluorescent microneedle sensor of the DNA-silver nanocluster hydrogel described in the first aspect in the detection of thiamethoxam.

[0035] Preferably, the application is for detecting thiram residues in food, agricultural products, or environmental samples.

[0036] Preferably, the detection is achieved by contacting the microneedle sensor with the analyte, causing thiram to interact with the DNA-silver nanoclusters in the sensor and triggering a change in fluorescence signal, and determining the presence or concentration of thiram by detecting this signal change.

[0037] Preferably, the fluorescence signal is detected by visual observation, observation with a portable ultraviolet lamp, or measurement using a fluorescence detection instrument.

[0038] Preferably, the sensor is particularly suitable for in-situ rapid screening of irregular surfaces such as fruit peels, vegetable leaves, and seafood shells.

[0039] Beneficial effects

[0040] Compared with the prior art, the technical solution provided by the present invention has the following significant advantages:

[0041] 1. Integration and Portability: This invention integrates a fluorescent recognition element (DNA-AgNC) s By integrating the sensor with a flexible carrier (PAM hydrogel), a solid-state, independent sensor device is constructed, eliminating the need for complex sample pretreatment and large instruments, which greatly improves the feasibility of rapid on-site detection.

[0042] 2. High sensitivity and excellent selectivity: thanks to DNA-AgNC sWith its excellent optical properties and the enrichment effect of the hydrogel matrix, the sensor exhibits high sensitivity (LOD as low as 0.199 μM) and high selectivity for thiram, and strong resistance to interference from other common pesticides.

[0043] 3. Excellent stability and practicality: The hydrogel three-dimensional network integrates DNA-AgNC s The robust embedding effectively isolates the probe from environmental interference (such as oxidation and changes in ionic strength), significantly improving its long-term storage and operational stability. Its flexibility and adhesiveness allow it to closely conform to various irregular test surfaces, enhancing sampling efficiency and detection reliability.

[0044] 4. Simple and environmentally friendly preparation: The preparation method is based on mature photopolymerization technology, with simple process, mild conditions, and good reproducibility. The materials used have good biocompatibility, avoiding the use of toxic organic dyes, which is in line with the concept of green chemistry and has the potential for large-scale production and application. Attached Figure Description

[0045] Figure 1 DNA-AgNC was demonstrated s Transmission electron microscopy (TEM) image (scale bar 500 nm).

[0046] Figure 2 DNA-AgNC was demonstrated s Particle size distribution diagram.

[0047] Figure 3 The distribution of silver in PAAC hydrogel is shown.

[0048] Figure 4 The PACC swelling ratio curve is shown.

[0049] Figure 5 Image a shows the tensile properties of hydrogels with different concentrations of silver nanoclusters; image b shows photographs of PAAC hydrogels before and after stretching; image c shows the fatigue resistance of PAAC hydrogels.

[0050] Figure 6 Figure a shows a schematic diagram of hydrogel adhesion; figure b shows the adhesion strength of PAAC hydrogel on different substrates; figure c shows photographs of PAAC hydrogel adhering to various substrates.

[0051] Figure 7 The excitation and emission wavelengths of the DNA-silver nanocluster hydrogel were demonstrated.

[0052] Figure 8 The optimized amount of DNA-silver nanoclusters was demonstrated.

[0053] Figure 9 Figure a shows DNA-AgNC at different concentrations of thiram dual concentrations.s Fluorescence spectra of the hydrogel; b shows the linear relationship between fluorescence quenching efficiency and thiram concentration; c shows the effect of reaction time on DNA-AgNC. s The effect of hydrogel fluorescence intensity.

[0054] Figure 10 a shows DNA-AgNC s The selectivity of the hydrogel for different pesticides; b demonstrates DNA-AgNC s The anti-interference properties of the hydrogel in a mixed environment; c shows microscopic images of fluorescence quenching induced by different pesticides.

[0055] Figure 11 Photographs of actual tests are shown. (a) Allergy test of PAAC on fish surface; (b) Residue test of PAAC on fish surface; (c) Images of microneedle detection and micronail characterization; (d) Detection of fish surface using PAAC microneedles; (e) Detection of leaf surface using PAAC; (f) Detection of citrus surface using PAAC. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this application clearer, the application will be further described in detail below with reference to the accompanying drawings. The described embodiments should not be regarded as limitations on this application. All other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this application.

[0057] The materials and instruments used in this experiment are shown below:

[0058] Material

[0059] Acrylamide (AA), N,N,N',N'-tetramethylethylenediamine (TEMED), N,N'-methylenebisacrylamide (BIS), and ammonium persulfate (APS) were purchased from Sigma-Aldrich. DNA template sequence 1 (SEQ ID NO: 1 5'-acccgaacctgggctaccacccttaatcccc-3') was synthesized and supplied by Shanghai Sanggong Biotechnology Co., Ltd., and purified by HPLC. Sodium borohydride (NaBH4) and silver nitrate (AgNO3) were purchased from Shanghai Guoyao Chemical Reagent Co., Ltd. Thiamethoxam and other pesticides used for selectivity testing were purchased from Shanghai Maclean's Biochemical Co., Ltd. All reagents were of analytical grade and used directly without further purification.

[0060] Experimental instruments

[0061] The instruments used in this study included: a high-speed refrigerated centrifuge (Hengnuo Instruments), an ultraviolet lamp (Qiwei), a universal testing machine (ZQ-990L, Zhiku Instruments), an LCR meter (E4980AL, Kessett), a fluorescence spectrophotometer (RF-6000, Shimadzu), a scanning electron microscope (Sigma300, Zeiss), and a vacuum freeze dryer (X0-18S, Xi'an Ou).

[0062] Example 1: Preparation method of fluorescent hydrogel (PAAC)

[0063] Operating instructions:

[0064] DNA-silver nanoclusters (DNA-AgNC) s Synthesis of the solution: 15 µM DNA template (SEQ ID NO: 15'-acccgaacctgggctaccacccttaatcccc-3') and 90 µM silver nitrate (AgNO3) were sequentially added to 20 mM, pH 6.8 sodium phosphate buffer and mixed thoroughly. The reaction mixture was incubated at room temperature in the dark for 20 minutes. Subsequently, freshly prepared sodium borohydride (NaBH4) solution was added to the mixture to a final concentration of 90 µM. After rapid vortex mixing, the reaction was continued at room temperature in the dark for 4 hours to obtain DNA-AgNC. s Solution.

[0065] Preparation of polyacrylamide hydrogel prepolymer solution: Measure 2.75 mL of 19% (w / v) acrylamide (AA) solution, and add 100, 200, 300, 400, and 500 µL of synthesized DNA-AgNC at different amounts. s Mix the solution thoroughly.

[0066] Addition of crosslinking and initiation system: Add 300µL of 0.2% (w / v) crosslinking agent N,N'-methylenebisacrylamide (BIS), 70µL of 0.2M initiator ammonium persulfate (APS), and 10µL of catalyst N,N,N',N'-tetramethylethylenediamine (TEMED) to the above mixture in sequence, and stir rapidly until homogeneous to obtain prepolymer solution.

[0067] Polymerization molding: The uniformly mixed prepolymer liquid is rapidly injected into a pre-made mold with a microneedle array structure, and then the mold is placed under a 365nm ultraviolet lamp and vertically irradiated for 1 hour to carry out photo-initiated free radical polymerization reaction.

[0068] Post-processing: After the polymerization reaction is complete, the solidified hydrogel is removed from the mold, immersed in sufficient distilled water and washed several times to thoroughly remove unreacted monomers and other impurities, finally obtaining DNA-AgNC loaded with DNA. sThe polyacrylamide fluorescent hydrogel, which is the final DNA-silver nanocluster hydrogel fluorescent microneedle sensor, is denoted as PAAC.

[0069] result:

[0070] A fluorescent microneedle sensor (PAAC) with flexible DNA-silver nanocluster hydrogel was successfully prepared.

[0071] Results Explanation:

[0072] This embodiment provides a simple and mild method for preparing fluorescent hydrogels. By using DNA-AgNC... s In-situ embedding of fluorescent probes into a polyacrylamide hydrogel network achieved solid-state immobilization of the probes. UV-initiated polymerization ensured the uniformity and controllability of the reaction, resulting in a well-structured hydrogel with both excellent mechanical and fluorescent properties, laying the foundation for subsequent sensing applications.

[0073] Example 2: Performance Characterization of Fluorescent Hydrogels

[0074] Morphology and dispersibility observation: DNA-AgNC prepared in Example 1 was used. s The solution obtained in the previous step was dropped onto a copper grid, dried, and its morphology was observed and photographed using a transmission electron microscope (TEM). Figure 1 ), statistical average particle size and distribution ( Figure 2 ).

[0075] Characterization of silver distribution: DNA-AgNC s Fluorescent hydrogels were prepared by incorporating polyacrylamide into a network. DNA-AgNC was also incorporated. s Subsequently, the distribution of silver elements was detected using scanning electron microscopy. The red dotted signals represent silver elements, and their uniform distribution indicates that the silver nanoclusters were successfully and stably embedded in the material. Figure 3 ).

[0076] Swelling performance test: A PAAC hydrogel sample with a known dry weight (W0) was immersed in excess deionized water and swollen at room temperature. Every 30 minutes, the sample was removed, the surface moisture was gently blotted dry with filter paper, and then weighed (W0). t (), until the weight no longer increases significantly. Swelling ratio (W) s The calculation formula is: W s =(W t -W0) / W0. Plot the swelling ratio as a function of time. Figure 4 ).

[0077] Mechanical property testing: PAAC hydrogel was cut into standard rectangular specimens (50mm × 10mm × 2mm) and subjected to tensile testing at a tensile rate of 100mm / min at room temperature using a universal testing machine until the specimens fractured. Cyclic tensile testing at 50% strain was performed on PAAC to determine its fatigue resistance. Figure 5 ).

[0078] Adhesion performance test: PAAC hydrogel samples were subjected to 90° peel or shear tests from the surfaces of five different substrates (fish skin, rubber, pigskin, PET, PI) at a speed of 300 mm / min. Each substrate was tested three times, and the average adhesion strength was recorded. Figure 6 ).

[0079] The results show that:

[0080] 1. TEM images show DNA-AgNC s It exhibits good dispersibility, with no obvious agglomeration, and the measured average particle size is approximately 0.26 µm. Figure 1 , Figure 2 ).

[0081] 2. SEM-EDS surface scans show that silver elements (red signal points) are uniformly distributed in the PAAC hydrogel. Figure 3 ).

[0082] 3. The swelling curve shows that the PAAC hydrogel swells rapidly in the first 4 hours and reaches swelling equilibrium after about 8 hours. Figure 4 ).

[0083] 4. Tensile tests show that when DNA-AgNC s When the addition amount is 500µL, PAAC exhibits good tensile properties, with a maximum elongation of 1140%. In addition, cyclic tensile tests were conducted on PAAC at 50% strain. During the cyclic tensile process, the hydrogel's resistance remained almost unchanged, indicating that it has excellent fatigue resistance and the ability to adapt to harsh testing environments. Figure 5 ).

[0084] 5. Adhesion tests showed that PAAC exhibited the highest shear strength on fish skin surfaces. Figure 6 ).

[0085] Results Explanation

[0086] This embodiment systematically characterized the physicochemical properties of PAAC hydrogels. The results showed that DNA-AgNC... sSuccessful and uniform embedding of the hydrogel network. The hydrogel's high water content and rapid swelling capacity facilitate analyte diffusion and enrichment. Excellent tensile strength, fatigue resistance, and broad environmental adhesion demonstrate its mechanical reliability as a flexible, wearable sensor, particularly suitable for attachment and detection on irregular biological surfaces.

[0087] Example 3: Determination of fluorescence detection sensitivity of fluorescent hydrogels

[0088] Fluorescence spectroscopy optimization: The PAAC hydrogel prepared in Example 1 was placed in the sample cell of a fluorescence spectrophotometer. The emission spectrum was scanned to determine the optimal excitation / emission wavelength pair. Figure 7 ).

[0089] DNA-AgNC s Dosage optimization: Preparation of DNA-AgNC s PAAC hydrogels were added in amounts of 100, 200, 300, 400, and 500 µL. The fluorescence intensity of each hydrogel at 625 nm was measured at the optimal excitation wavelength. Figure 8 Select DNA-AgNC s The optimal dosage is 500µL.

[0090] Sensitivity and detection limit determination: Take the optimized (DNA-AgNC) s PAAC hydrogels (500 µL each) were prepared, and different concentrations of thiram standard solutions (0-2 µM) were dropped onto their surfaces. After incubation for 2 minutes, the fluorescence intensity (F) of each hydrogel was measured using a fluorescence spectrophotometer at an excitation wavelength of 356 nm and an emission wavelength of 445 nm (commonly used wavelengths for detecting the thiram quenching effect). Figure 9 ).

[0091] result:

[0092] 1. The optimal excitation / emission wavelength pair for PAAC was determined to be 535nm / 625nm. Figure 7 ).

[0093] 2. When DNA-AgNC s The fluorescence intensity of PAAC reached its maximum value when the addition amount was 500 µL. Figure 8 ).

[0094] 3. PAAC exhibits concentration-dependent quenching of the fluorescence response to thiram. Within the concentration range of 0-2 µM, the fluorescence quenching efficiency shows a good linear relationship with the thiram concentration, R0. 2 =0.996. The limit of detection (LOD) for thiram was calculated to be 0.199 µM using the detection limit formula. The fluorescence signal remained stable during the detection time. Figure 9 ).

[0095] Results Explanation:

[0096] This embodiment demonstrates that PAAC hydrogel, as a fluorescence sensor, exhibits high sensitivity to thiram. By optimizing the probe loading, the strongest initial fluorescence signal was obtained. The established quantitative analysis method has a wide linear range, good correlation, and a detection limit lower than many existing methods, meeting the needs of trace detection. Excellent temporal stability ensures the reliability of the detection results.

[0097] Example 4: Specificity verification of fluorescent hydrogel for the detection of thiram

[0098] Single interfering agent test: Take multiple samples of the prepared PAAC hydrogel (DNA-AgNC) s Different pesticide solutions of the same concentration and content, including thiram, acetopyridine, thiamethoxam, difenoconazole, imidacloprid, glyphosate, and cypermethrin, were added dropwise to the surface of each hydrogel (500 µL). After incubation for 2 minutes, the fluorescence intensity of each hydrogel was measured. The fluorescence quenching efficiency of each pesticide relative to the blank sample was calculated.

[0099] Interference resistance test with mixed interfering substances: Prepare mixed solutions containing thiram and each of the other pesticides mentioned above. Take PAAC hydrogel, add each mixed solution dropwise, incubate using the same method, and measure the fluorescence intensity. Use the solution containing only thiram as a control to investigate the effect of coexistence of other pesticides on the detection signal.

[0100] result:

[0101] 1. Single interfering agent test results showed that only thiram caused a significant decrease in the fluorescence intensity of PAAC hydrogel (high quenching efficiency), while the fluorescence changes caused by the other six common pesticides were very small. Figure 10 (a)

[0102] 2. The anti-interference test results showed that, when thiram coexisted with any other pesticide, the fluorescence quenching response of PAAC was not significantly different from that when thiram was present alone. Figure 10 b, Figure 10 (c)

[0103] Results Explanation:

[0104] This embodiment verifies that PAAC hydrogel has high selectivity and specificity for the detection of thiamethoxam. Its fluorescent probe, DNA-AgNC... sPAAC exhibits a specific interaction mechanism with thiram (such as competitive binding) and is insensitive to the structure of other tested pesticides. Even in complex mixtures, PAAC can accurately recognize and respond to thiram, demonstrating strong anti-interference capabilities, which provides a crucial guarantee for its application in real-world samples with complex compositions.

[0105] Example 5: Application of fluorescent hydrogels in the detection of real samples

[0106] Sample pretreatment and simulated contamination:

[0107] Fish samples: Fresh fish were purchased from the market, washed with ultrapure water, and air-dried. Different concentrations of thiram standard solutions (0, 1, 10, 100 µM) were evenly sprayed onto specific areas of the fish surface to simulate pesticide residues.

[0108] Leaf and citrus samples: Fresh plant leaves and citrus fruits were collected, washed and dried, and selected areas on their surfaces were sprayed with the above-mentioned series of concentrations of thiram solution.

[0109] Microneedle sensor fabrication: The method of Example 1 was used, but the prepolymer solution was injected into a PDMS mold with a microneedle array structure, and PAAC microneedle patches were obtained after polymerization. Their morphology was observed under a microscope.

[0110] In-situ detection:

[0111] Press the prepared PAAC microneedle patch or ordinary PAAC hydrogel sheet directly onto the sample surface (fish skin, leaves, citrus peel) that has been (or has not been) treated with thiram, ensuring full contact and maintaining appropriate pressure for about 2-5 minutes.

[0112] After the reaction was complete, the hydrogel was removed, and the fluorescence changes in the area in contact with the sample were immediately observed under a 365nm UV lamp and photographed.

[0113] For fish samples, additional safety tests were conducted: PAAC hydrogel was applied to the skin of live fish for a period of time to observe whether the fish showed any allergic or stress reactions. After testing, the surface of the fish skin was examined for any fluorescent residue.

[0114] result:

[0115] 1. The prepared PAAC microneedles have a neatly arranged, sharp-tipped conical structure, which can adhere well to irregular surfaces. Figure 11 (c)

[0116] 2. Safety and residue tests showed that PAAC hydrogel was non-irritating to fish, and no obvious probe residue was found after testing. Figure 11 (a) Figure 11 (b)

[0117] 3. In-situ detection results showed that as the concentration of thiram contamination on the sample surface increased (0, 1, 10, 100 µM), the red fluorescence intensity of the PAAC hydrogel or microneedles in contact with it under UV light exhibited a significant, gradient-like decrease or even quenching. The fluorescence remained strongest at a concentration of 0 µM, and almost completely disappeared at a concentration of 100 µM. Figure 11 (d)

[0118] 4. Residues in citrus fruits and leaves can be detected with relatively clear fluorescence. Figure 11 Middle (e) Figure 11 (f)

[0119] Results Explanation:

[0120] This embodiment successfully demonstrates the application of PAAC fluorescent hydrogel and microneedle sensor in rapid and visual screening of thiram residues on the surface of real complex samples (fish, plants). The microneedle structure enhances sampling efficiency. This method requires no complex sample pretreatment, is simple to operate, allows for semi-quantitative judgment by directly observing the degree of fluorescence quenching, and is safe for biological samples. The results fully demonstrate the practicality and broad application prospects of the sensor of this invention in the field of rapid on-site detection of food safety.

Claims

1. A fluorescent microneedle sensor of DNA-silver nanocluster hydrogel, characterized in that, Comprise: a polyacrylamide hydrogel matrix; and DNA-silver nanoclusters uniformly dispersed and immobilized in the polyacrylamide hydrogel matrix; The hydrogel fluorescent microneedle sensor has an array microneedle structure.

2. The fluorescent microneedle sensor of claim 1, wherein, The polyacrylamide hydrogel is a cross-linked network structure formed by a free radical polymerization reaction, and the cross-linking agent is N,N'-methylene bisacrylamide; preferably, the particle size of the DNA-silver nanoclusters is 0.1-0.4μM.

3. The fluorescent microneedle sensor of claim 1, wherein, The sensor has a specific fluorescent response to the fungicide thiram, and the fluorescent intensity is quenched with increasing thiram concentration.

4. The fluorescent microneedle sensor of claim 3, wherein, The linear detection range of the sensor for the fungicide thiram is at least 0 to 2μM; preferably, the maximum elongation rate of the sensor is not less than 1000%.

5. A method of preparing a fluorescent microneedle sensor of DNA-silver nanoclusters hydrogel according to any one of claims 1-4, characterized in that, The method comprises the following steps: (1) preparing a DNA-silver nanocluster solution: mixing a DNA template with a silver nitrate solution in a buffer, incubating, then adding a reducing agent sodium borohydride to react, to generate a DNA-silver nanocluster solution; (2) preparing a prepolymer solution: mixing acrylamide monomer, cross-linking agent, and the DNA-silver nanocluster solution prepared in step (1) to obtain a uniform prepolymer mixture; (3) in-situ polymerization and molding: adding an initiator and a catalyst to the prepolymer mixture, mixing uniformly, then injecting into a mold with a microneedle structure, and performing a polymerization reaction under ultraviolet light irradiation to obtain a composite hydrogel with a microneedle structure; (4) post-processing: removing the obtained composite hydrogel from the mold, washing and equilibrating to obtain the microneedle sensor.

6. The method of claim 5, wherein, In step (1), the concentration of the DNA template is 10-20μM, the concentration of silver nitrate is 80-100μM, and the final concentration of the reducing agent sodium borohydride is 80-100μM; the buffer is 20mM sodium phosphate buffer, pH 6.8; the reaction is carried out at room temperature in the dark.

7. The method of claim 5, wherein, In step (2), the cross-linking agent is N,N'-methylene bisacrylamide; the addition amount of the DNA-silver nanocluster solution is 100-500µL.

8. The method of claim 5, wherein, In step (3), the initiator is ammonium persulfate, and the catalyst is N,N,N',N'-tetramethyl ethylenediamine; the wavelength of the ultraviolet light is 365nm, and the polymerization time is 0.5-2 hours.

9. The method of claim 5, wherein, In step (4), the washing is soaking and washing with deionized water or distilled water to remove unreacted monomers; the equilibration is swelling the hydrogel in the buffer to a stable state.

10. Use of the DNA-silver nanocluster hydrogel fluorescent microneedle sensor of any one of claims 1-4 in the preparation of a detection device or kit for detecting thiram.

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