Composite hydrogel microneedle and application thereof in sulfonamide antibiotic detection

Abstract

The invention discloses a composite hydrogel microneedle and application thereof in sulfonamide antibiotic detection. The composite hydrogel microneedle is prepared from sodium alginate-acrylamide double-network hydrogel, an MXene / MoS2 heterojunction nanocomposite and silver nanoparticles generated in situ through microneedle mold forming, ionic crosslinking and a silver mirror reaction. The composite microneedle substrate can be used for high-sensitivity and high-selectivity detection of sulfonamide antibiotic residues in animal-derived food, has good reproducibility, stability and practicability, and is suitable for rapid screening of trace antibiotics in complex samples.

Description

Technical Field

[0001] This invention belongs to the field of food safety testing, specifically relating to a method for preparing a composite hydrogel microneedle surface-enhanced Raman scattering substrate and its application in the detection of sulfonamide antibiotics. Background Technology

[0002] Antibiotics are a class of organic compounds that kill or inhibit bacterial growth, and are widely used in medicine, animal husbandry, aquaculture, and other fields. Sulfonamide antibiotics (SAs), as commonly used chemically synthesized broad-spectrum antibacterial drugs, possess the p-aminobenzenesulfonamide structure and have been used clinically and in animal husbandry for over 70 years. However, sulfonamide antibiotics are stable and difficult to degrade. Overuse leads to their accumulation and residues in animal-derived foods and the environment, polluting water and soil, disrupting the balance of ecosystems, and entering the human body through the food chain, causing health problems such as allergic reactions, liver damage, and urinary system damage. They also contribute to bacterial resistance, posing a serious threat to human and animal health. Therefore, establishing rapid, sensitive, and accurate detection methods for sulfonamide antibiotics is crucial for ensuring food safety.

[0003] Currently, methods for detecting sulfonamide antibiotic residues mainly include chromatography, electrochemical detection techniques, biological methods, and spectroscopic methods. Among them, chromatographic techniques such as high-performance liquid chromatography (HPLC) have low detection limits and high separation efficiency, but the equipment is expensive, sample pretreatment is complex, and the operation is cumbersome, making it unable to meet the needs of rapid on-site detection. Electrochemical detection techniques are simple to operate and have low cost, but they are susceptible to ion interference and have complex procedures. Biological methods such as enzyme-linked immunosorbent assay (ELISA) have advantages such as high specificity and time-saving, but the sensitivity needs to be improved, and antibody preparation is difficult, making it difficult to meet the needs of rapid on-site screening.

[0004] Surface-enhanced Raman scattering (SERS) technology has shown great potential in trace substance detection due to its high sensitivity, fingerprint recognition capability, and fast response characteristics. However, traditional SERS substrates, such as flexible substrates, often suffer from problems such as poor signal uniformity. Summary of the Invention

[0005] In view of the defects or deficiencies of the prior art, the present invention provides a composite hydrogel microneedle.

[0006] Therefore, the method for preparing composite hydrogel microneedles provided by the present invention includes: Sodium alginate, MXene / MoS2 composite material, acrylamide monomer, N,N-methylenebisacrylamide, potassium persulfate and water were mixed to obtain a mother liquor; then the mother liquor was injected into a microneedle mold, and after degassing, heating and curing, the microneedle material was demolded. Next, the obtained microneedle material was immersed in a calcium ion-containing solution for cross-linking; finally, the cross-linked microneedles were successively immersed in silver ammonia solution and glucose aqueous solution, and after repeated immersion, they were dried to obtain composite hydrogel microneedles.

[0007] An optional method is to mix 10.0 mL of a 2.0 wt% sodium alginate aqueous solution, 2.0 mL of an MXene / MoS2 composite material aqueous solution, 1.5-2.0 g of acrylamide, 0.0040-0.0050 g of N,N-methylenebisacrylamide, and 0.03-0.06 g of potassium persulfate to obtain a mother liquor; the mass-volume concentration of the MXene / MoS2 composite material aqueous solution is 0.3-8 mg / mL. Alternatively, the mass-volume concentration of the MXene / MoS2 composite material aqueous solution is 3 mg / mL. The concentration of the silver ammonia solution is 0.1-0.3 M; the concentration of the glucose solution is 0.2-0.5 M. The number of cyclic impregnation cycles is 2-4.

[0008] This invention also provides the application of the above-mentioned composite hydrogel microneedles as a SERS substrate.

[0009] This invention also provides the application of the above-mentioned hydrogel microneedles in the detection of sulfonamide antibiotics in food. Further, the sulfonamide antibiotic is at least one selected from sulfadiazine, sulfamethoxypyrimidine, sulfadimethoxazole, and sulfamethoxazole. The food is meat.

[0010] Specifically, the relevant detection method of the present invention includes: (1) Concentration-Raman spectrum intensity curves of sulfonamide antibiotic standard solutions; (2) The hydrogel microneedle tip of claim 1 is inserted into the food to be tested to take a sample; the hydrogel microneedle after sampling is analyzed by laser confocal Raman spectrometer to obtain the concentration-Raman spectrum intensity curve of the food to be tested, and then the content of sulfonamide antibiotics in the food to be tested is determined according to the concentration-Raman spectrum intensity curve of the sulfonamide antibiotic standard solution.

[0011] This invention mixes sodium alginate solution with MXene / MoS2 composite material, acrylamide monomer, N,N-methylenebisacrylamide (crosslinking agent), and potassium persulfate (initiator). The mixture is injected into a microneedle mold, degassed, heated and cured, and then demolded to form microneedles. The resulting microneedles are then immersed in a calcium ion-containing solution for crosslinking. The microneedles are then sequentially immersed in silver ammonia solution and reducing agent solution, and the process is repeated multiple times to generate silver nanoparticles in situ on the surface of the microneedles, thus successfully preparing a composite hydrogel microneedle SERS substrate.

[0012] Using the composite hydrogel microneedles of this invention as a SERS substrate, and with R6G as the probe molecule under a 532 nm laser, the detection limit of this chip can reach 0.01 mg / L, demonstrating its extremely high sensitivity.

[0013] The composite hydrogel microneedle SERS substrate of this invention exhibits excellent detection performance in the detection of sulfonamide antibiotics, and can distinguish sulfadiazine, for example, by characteristic peaks (1583 cm⁻¹). -1 ), sulfadimethoxypyrimidine (1572 cm) -1 Structural analogs such as sulfonamides showed spiked recoveries of 78%-86%, making it particularly suitable for rapid on-site screening of meat matrices. When this matrix was applied to the detection of sulfonamide antibiotics, the results showed good linear responses to various antibiotics: the linear correlation coefficient (R0) for sulfadiazine was significantly higher than that for other antibiotics. 2 The values ​​for 0.995 for sulfadimethoxypyrimidine, 0.993 for sulfadimethoxypyrimidine, and 0.992 for sulfadimethoxypyrimidine indicate that the chip has high accuracy and applicability in practical applications. Attached Figure Description

[0014] Figure 1 SEM image of the composite hydrogel microneedles prepared in the example; Figure 2 shows the SERS spectrum of composite hydrogel microneedles with different concentrations of MXene / MoS2; (a) SERS spectrum of 1 mg / mL sulfadimethoxypyrimidine; (b) SERS spectrum of 1 mg / mL sulfadimethoxypyrimidine. Figure 3 shows the detection results of Example 3; (a) different concentrations of R6G at 1185 cm⁻¹ -1 (a) SERS spectrum at the location; (b) Reproducibility bar graph of R6G at a concentration of 1 mg / L in composite hydrogel microneedles; Figure 4 shows the detection results of sulfadiazine in Example 4; (a) SERS spectra of sulfadiazine at different concentrations; (b) its SERS spectrum at 1583 cm⁻¹. -1 The standard curve at that location; Figure 5 shows the detection results of sulfadimethylpyrimidine in Example 4; (a) SERS spectra of sulfadimethylpyrimidine at different concentrations; (b) its SERS spectrum at 1579 cm⁻¹. -1 The standard curve at that location; Figure 6 shows the detection results of sulfadimethoxypyrimidine in Example 4; (a) SERS spectra of sulfadimethoxypyrimidine at different concentrations; (b) its SERS spectrum at 1572 cm⁻¹. -1 The standard curve at that location; Figure 7 shows the detection results of sulfadiazine in the mutton sample in Example 5; (a) SERS spectra of sulfadiazine at different concentrations; (b) its concentration at 1583 cm⁻¹. -1 The standard curve at that location; Figure 8 shows the detection results of sulfadimethylpyrimidine in the mutton sample of Example 5; (a) SERS spectra of sulfadimethylpyrimidine at different concentrations; (b) its concentration at 1579 cm⁻¹. -1 The standard curve at that location; Figure 9 shows the detection results of sulfadimethoxypyrimidine in the mutton sample of Example 5; (a) SERS spectra of sulfadimethoxypyrimidine at different concentrations; (b) its concentration at 1572 cm⁻¹. -1 The standard curve at that location. Detailed Implementation

[0015] Unless otherwise specified, the scientific and technical terms used in this article are intended for understanding by those skilled in the art.

[0016] The synthesis method of the MXene / MoS2 composite material described in this invention can be found in the following reference: Xiao Y, Le Q, Kong Y, et al. Ultrafast and ultrastable Na-ion storage in interface engineered MoS2 / MXene nanohybrids with nanoconfinement for high-performance sodium-ioncapacitors[J]. Chemical Engineering Journal, 2025, 505159268-159268. DOI:10.1016 / J.CEJ.2025.159268.

[0017] The synthesis method of the composite hydrogel microneedle SERS substrate of this invention can be found in the following reference: Zhou Lingtong. Preparation, properties and applications of three alginate-based hydrogels [D]. Northwest A&F University, 2023. DOI:10.27409 / d.cnki.gxbnu.2023.000004.

[0018] The present invention will now be described in further detail with reference to specific embodiments. The given embodiments are merely illustrative of the invention and not intended to limit its scope. The embodiments provided below can serve as a guide for further improvements by those skilled in the art and do not constitute a limitation on the invention in any way.

[0019] Unless otherwise specified, the experimental methods used in the following examples are conventional methods, performed according to the techniques or conditions described in the literature in this field or according to the product instructions. Unless otherwise specified, the materials and reagents used in the following examples are commercially available.

[0020] Examples 1-5: This example illustrates the preparation of composite hydrogel microneedles: Preparation of MXene / MoS2 composite material: 3.2 g LiF was mixed with 40 mL 9 M HCl, and 2.0 g Ti3AlC2 powder was slowly added under magnetic stirring at 35 °C for 48 h. The resulting suspension was centrifuged at 5000 rpm for 10 min, and the black product was collected. It was washed several times with deionized water until the pH of the supernatant was about 7. The black product was freeze-dried for 24 h, and the MXene product was collected. 0.1 g MXene was dissolved in 50 mL deionized water, and 0.3 g ammonium molybdate and 0.6 g thiourea were added. The mixture was magnetically stirred for 30 min until completely dissolved. The resulting mixture was transferred to a 50 mL stainless steel autoclave (PTFE-lined) and reacted at 180 °C for 24 h. After the reaction was completed, the mixture was naturally cooled to room temperature, and the suspension was centrifuged at 8000 rpm for 10 min. The black product was collected and washed three times each with deionized water and ethanol. The black product was vacuum-dried at 60 °C for 12 h to obtain the MXene / MoS2 composite material.

[0021] Synthesis of sodium alginate-acrylamide mother liquor: 2.0 wt% sodium alginate (0.2 g) was dissolved in 10.0 mL of ultrapure water and heated and stirred at 60 °C for 30 min until completely dissolved. The solution was then cooled to room temperature. 2.0 mL of MXene / MoS2 composite material dispersion with a concentration of 0.3 mg / mL (or 0.5 mg / mL, 3 mg / mL, 5 mg / mL, 8 mg / mL) using ultrapure water as solvent was added. After stirring for 20 min to homogenize the solution, 1.7 g of acrylamide, 0.0043 g of N,N-methylenebisacrylamide (crosslinking agent), and 0.05 g of potassium persulfate (initiator) were added. The solution was stirred for 15 min until completely dissolved to obtain sodium alginate-acrylamide mother liquor.

[0022] Preparation of composite hydrogel microneedles: Sodium alginate-acrylamide stock solution was added to a dimethylsiloxane (PDMS) microneedle mold. Vacuum was applied for 15 min to remove air bubbles. Excess air bubbles were removed using a pipette, and this process was repeated twice. The mold was placed in a 37℃ oven and heated for 4 h until the microneedles were dry and formed. The microneedles were carefully demolded with tweezers to obtain the composite microneedle material. The composite microneedle material was immersed in a 2.0 wt% calcium chloride solution prepared with ultrapure water for 1 h for ionic crosslinking. After crosslinking, the composite microneedles were immersed in 1 mL of 0.2 M silver ammonia solution (prepared from silver nitrate, ammonia, and ultrapure water) for 2 min, and then immersed in 2 mL of 0.35 M glucose solution prepared with ultrapure water for 2 min. This process was repeated 3 times to obtain the corresponding composite hydrogel microneedles. The electron microscopy image of the composite hydrogel microneedles prepared from the 3 mg / mL MXene / MoS2 composite dispersion is shown below. Figure 1 As shown.

[0023] Comparative example: The difference between this comparative example and the above-described example is that 2.0 mL of MXene / MoS2 composite material dispersion was replaced with 2.0 mL of ultrapure water.

[0024] The composite hydrogel microneedles prepared in Examples 1-5 and the comparative example were subjected to SERS testing. The test conditions were: laser wavelength of 532 nm, laser power of 10 mW, integration time of 10 s, and cumulative number of times of 10. The results are shown in Figure 2. The results show that the MXene / MoS2 composite material with a concentration of 3 mg / mL is the most effective for detecting sulfonamide antibiotics.

[0025] Example 6: The SERS activity of microneedles prepared using 3 mg / mL MXene / MoS2 composite material was further investigated: the test points were selected in the central region as much as possible using a microscope, and the detection range of the 532 nm Raman spectrometer was set to 800-1200 cm⁻¹. -1 The experiment was conducted under the conditions of 10 mW laser power and 10 s acquisition time, using a single-region single-scan method without repeated signal acquisition. R6G was selected as the Raman molecular probe. Before the measurement, the composite hydrogel microneedles were taken out of the -4℃ freezer. The specific preparation process of the R6G standard solution is as follows: First, R6G powder was accurately weighed using a micro-electronic balance to prepare a stock solution with a standard concentration of 1000 mg / L. The stock solution was then diluted stepwise with ultrapure water to obtain R6G standard solutions of different concentrations (100, 10, 1, 0.1, 0.01 mg / L). The solutions were then dissolved by sonication and stored in a refrigerator at 4°C in the dark.

[0026] Sensitivity determination of composite hydrogel microneedles: The composite hydrogel microneedles were incubated in 1 mL of R6G standard solutions of different concentrations for 20 min. After being removed, the composite hydrogel microneedles were placed on a clean glass plate and dried at 37°C. After drying, parallel SERS detection was performed to determine the sensitivity of the SERS chip. The results are shown in Figure 3(a).

[0027] Uniformity determination of composite hydrogel microneedles: The composite hydrogel microneedles were incubated in 1 mg / L R6G standard solution for 20 min. Then, 16 points were randomly selected at different positions of the composite hydrogel microneedles for Raman scanning to determine the uniformity of the substrate. The results are shown in Figure 3(b).

[0028] Figure 3 shows the spectra of SERS detection using composite hydrogel microneedles in R6G standard solutions of different concentrations (100, 10, 1, 0.1, 0.01 mg / L), and demonstrates the good reproducibility of the composite hydrogel microneedles during the detection process.

[0029] Example 7: Composite hydrogel microneedles prepared using 3 mg / mL MXene / MoS2 composite material were used as a SERS substrate for the detection of three sulfonamide antibiotics. The method was as follows: Standard solutions of sulfadiazine, sulfadiazine, and sulfadimethoxypyrimidine at concentrations of 1000, 100, 10, 1, 0.1, 0.01, and 0.001 mg / L were obtained by serial dilution with ultrapure water.

[0030] Using composite hydrogel microneedles as the detection substrate, the microneedles were immersed in 1 mL of standard solutions of different concentrations and incubated for 20 min. Then, they were detected by Raman spectroscopy with an excitation wavelength of 532 nm. The laser power was 10 mW, the integration time was 10 s, and the number of times was 10. The SERS spectra obtained were used to establish standard curves for three sulfonamide antibiotics.

[0031] Figure 4-6 shows that it exhibited good linear response to different antibiotics, with sulfadiazine showing a high linear correlation coefficient (R0). 2 The value was 0.995 for sulfadimethylpyrimidine, 0.993 for sulfadimethoxypyrimidine, and 0.992 for sulfadimethoxypyrimidine.

[0032] Example 8: Composite hydrogel microneedles prepared using 3 mg / mL MXene / MoS2 composite material were used to detect three sulfonamide antibiotics in mutton samples. Specific methods included: The composite hydrogel microneedles were used as the detection substrate to detect mutton samples. The pretreatment of fresh slaughtered mutton was carried out by soaking the meat pieces in standard solutions of sulfadiazine, sulfadiazine and sulfadimethoxypyrimidine with concentrations of 20, 10, 5, 1, 0.5 and 0.1 mg / L, respectively, to prepare spiked mutton samples.

[0033] The composite hydrogel microneedles were placed on the surface of the mutton sample with the needle tip facing down. The needles were pressed into the mutton with the thumb for 5 minutes. After removal, the sample was inverted and left to stand for 5 minutes. The sample was then detected using a 532 nm excitation wavelength Raman spectrometer with a laser power of 10 mW, an integration time of 10 s, and a total of 10 measurements. The SERS spectrum obtained was used to establish standard curves for three sulfonamide antibiotics.

[0034] Figures 7-9 show that it exhibits good linear response to different antibiotics, with a linear correlation coefficient (R) of sulfadiazine. 2The values ​​for sulfadimethoxine and sulfadiazine were 0.992 and 0.995, respectively. The results were converted using a standard curve to obtain the spiked recoveries for the test concentrations, which ranged from 78% to 86%.

[0035] According to GB 31658.17—2021 "National Food Safety Standard for the Determination of Tetracycline, Sulfonamide and Quinolone Drug Residues in Animal-Derived Foods by Liquid Chromatography-Tandem Mass Spectrometry", the detection limit for sulfonamide drugs in animal-derived foods is 2 μg / kg. The detection limits of the method of this invention for sulfadiazine, sulfadimethylpyrimidine and sulfadimethoxypyrimidine are 0.07 μg / kg, 0.07 μg / kg and 0.06 μg / kg, respectively.

[0036] The present invention has been described in detail above. Those skilled in the art will recognize that the invention can be practiced in a wide range of ways with equivalent parameters, concentrations, and conditions without departing from its spirit and scope, and without requiring unnecessary experiments. While specific embodiments have been provided, it should be understood that further modifications can be made to the invention. In summary, according to the principles of the invention, this application is intended to include any changes, uses, or improvements to the invention, including changes made using conventional techniques known in the art that depart from the scope disclosed herein.

Claims

1. A composite hydrogel microneedle, characterized in that, The preparation method of the composite hydrogel microneedles includes: Sodium alginate, MXene / MoS2 composite material, acrylamide monomer, N,N-methylenebisacrylamide, potassium persulfate and water were mixed to obtain a mother liquor; then the mother liquor was injected into a microneedle mold, and after degassing, heating and curing, the microneedle material was demolded. Next, the obtained microneedle material was immersed in a calcium ion-containing solution for cross-linking; finally, the cross-linked microneedles were successively immersed in silver ammonia solution and glucose aqueous solution, and after repeated immersion, they were dried to obtain composite hydrogel microneedles.

2. The composite hydrogel microneedles according to claim 1, characterized in that, A mother liquor was prepared by mixing 10.0 mL of 2.0 wt% sodium alginate aqueous solution, 2.0 mL of MXene / MoS2 composite material aqueous solution, 1.5-2.0 g acrylamide, 0.0040-0.0050 g N,N-methylenebisacrylamide, and 0.03-0.06 g potassium persulfate; the mass-volume concentration of the MXene / MoS2 composite material aqueous solution was 0.3-8 mg / mL.

3. The composite hydrogel microneedles according to claim 1, characterized in that, The mass-volume concentration of the MXene / MoS2 composite aqueous solution is 3 mg / mL.

4. The composite hydrogel microneedles according to claim 1, characterized in that, The concentration of the silver ammonia solution is 0.1–0.3 M; the concentration of the glucose solution is 0.2–0.5 M.

5. The composite hydrogel microneedles according to claim 1, characterized in that, The immersion cycle is repeated 2 to 4 times.

6. The application of the composite hydrogel microneedles of claim 1 as a SERS substrate.

7. The application of the hydrogel microneedles of claim 1 for the detection of sulfonamide antibiotics in food.

8. The application according to claim 7, characterized in that, The sulfonamide antibiotics are at least one of sulfadiazine, sulfadiazine, sulfadimethoxypyrimidine, and sulfamethoxazole.

9. The application according to claim 5, characterized in that, The food in question is meat.

10. A method for detecting sulfonamide antibiotics in food, characterized in that the method... include: (1) Concentration-Raman spectrum intensity curves of sulfonamide antibiotic standard solutions; (2) The hydrogel microneedle tip of claim 1 is inserted into the food to be tested to take a sample; the hydrogel microneedle after sampling is analyzed by laser confocal Raman spectrometer to obtain the concentration-Raman spectrum intensity curve of the food to be tested, and then the content of sulfonamide antibiotics in the food to be tested is determined according to the concentration-Raman spectrum intensity curve of the sulfonamide antibiotic standard solution.

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