Method for detecting ochratoxin a by competitive colorimetric and fluorescent dual-mode immunosensor

Abstract

The application discloses a method for detecting ochratoxin A by using a competitive colorimetric fluorescent dual-mode immunosensor, and the method uses AuAg NCs-S,P-g-C3N4 nanocomposites as a label of the competitive colorimetric fluorescent dual-mode immunosensor; the S,P-g-C3N4 has the advantages of a large specific surface area and good biocompatibility; the AuAg NCs have an aggregation luminescence effect after being combined with the S,P-g-C3N4 through Au-S bonds, the fluorescence of the AuAg NCs is enhanced, the fluorescence signal is amplified, and the sensitivity of the sensor is improved; the loading of the AuAg NCs greatly enhances the enzyme-like activity of the S,P-g-C3N4; the nanocomposites are used to catalyze TMB into blue oxTMB, so that visual detection is realized. The two signals are mutually verified, so that the sensor is more sensitive, and the method has the characteristics of simple operation, fast reaction speed, low cost, low detection limit and wide linear range.

Description

Technical Field

[0001] This invention belongs to the field of nanocomposite materials and biosensing detection technology, and particularly relates to a method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor. Background Technology

[0002] Ochratoxins are among the most toxic and widely distributed fungal toxins. Ochratoxin A (OTA), in particular, is classified as a Group 2B carcinogen by the International Agency for Research on Cancer (IARC) due to its nephrotoxicity, teratogenicity, and carcinogenicity. It is primarily found in various matrices, such as moldy food and pharmaceuticals. Due to its excellent thermal and chemical stability, it can accumulate in animals and humans through the food chain, posing a serious threat to human and animal health. Therefore, the sensitive and accurate determination of ochratoxin A is urgently needed. Currently, OTA detection mainly employs traditional methods such as liquid chromatography-mass spectrometry and thin-layer chromatography. These methods are generally limited in application due to high cost, low sensitivity, and complex sample pretreatment. Therefore, it is necessary to establish a sensitive, rapid, and selective method for OTA detection.

[0003] In recent decades, fluorescent nanoclusters (NCs) have been widely used in fields such as biolabeling, bioimaging, catalysis, and ion sensing due to their ultra-small size, low toxicity, and strong fluorescence. Gold-silver nanoclusters have similar properties to gold nanoclusters, but due to silver ion doping, they exhibit greater stability, stronger fluorescence, and certain peroxidase-like catalytic oxidation properties. They possess more attractive advantages than single-metal nanomaterials in terms of electronic, optical, and catalytic properties.

[0004] Graphitic carbon nitride (g-C3N4) is a metal-free organic polymer semiconductor and the most stable allotrope of carbon nitride. It exhibits excellent thermal stability, can be calcined in air up to 600°C, and possesses perfect acid and alkali resistance. g-C3N4 is a two-dimensional sheet composed of triazine rings and conical nitrogen bridges, possessing a suitable band gap (Eg = 2.7 eV) and good stability. However, its photocatalytic performance is limited by the high recombination rate of photocarriers and low absorption of visible light. To improve the catalytic performance of g-C3N4, methods such as designing porous structures, elemental doping, and creating heterojunctions with other semiconductor or metal nanoparticles have been developed.

[0005] The colorimetric fluorescence dual-mode immunosensor combines colorimetric and fluorescence analysis, utilizing the two signals for mutual verification. Using nanozymes as markers, it achieves the determination of antigens or antibodies through the specific reaction between antibodies and antigens. It possesses advantages such as high sensitivity, high selectivity, high specificity, and low detection limit. However, the combination of g-C3N4 with the colorimetric fluorescence dual-mode immunosensor for OTA detection has been rarely reported. Summary of the Invention

[0006] To address the shortcomings of existing technologies, the present invention aims to provide a competitive colorimetric fluorescence dual-mode immunosensor, its preparation method, and its application. This invention uses AuAg NCs-S,Pg-C3N4 nanocomposite materials as markers to prepare a competitive colorimetric fluorescence dual-mode sensor, which is then applied to the detection of ochratoxin A.

[0007] The objective of this invention is achieved through the following technical solution:

[0008] A method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor includes the following steps:

[0009] (1) First, carboxylated S,Pg-C3N4 nanosheet powder and AuAg NCs solution were mixed and stirred to react. After the reaction was completed, dialysis was performed to obtain AuAg NCs-S,Pg-C3N4 nanocomposite material. Then the solution was freeze-dried for later use.

[0010] (2) The freeze-dried AuAg NCs-S,Pg-C3N4 nanocomposite material, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution were mixed, activated at room temperature and centrifuged. The solid sample after centrifugation was washed with PBS buffer, and then the antibody was added. After incubation in the dark, it was centrifuged and washed again to obtain the antibody labeled with AuAg NCs-S,Pg-C3N4 nanocomposite material. The antibody was reconstituted with PBS buffer and stored in the dark for later use.

[0011] (3) First, dopamine solution was injected into a 96-well plate, incubated and dried until a membrane appeared. Then, the coated antigen was injected and reacted overnight at 4°C. The plate was washed three times, and then a blocking agent was added to continue the reaction to block the excess binding sites on the dopamine. The plate was washed three times. Antibodies labeled with AuAg NCs-S and Pg-C3N4 nanocomposite materials were added and competitively reacted for 0.5-2 hours. Then, different concentrations of ochratoxin A standards were added and incubated. The plate was washed with PBST and its fluorescence intensity was detected using a multi-functional microplate reader. The solution was then aspirated and HAc-NaAc buffer, TMB and H2O2 were added to the solution for colorimetric detection. The plate was reacted at 25°C-70°C for 5-17 minutes. The absorbance change and corresponding color change at 652 nm were recorded. A standard curve was plotted with the concentration of ochratoxin A as the x-axis and the fluorescence value and absorbance as the y-axis.

[0012] (4) Replace the ochratoxin A standard solution with the test sample solution for detection, and calculate the concentration of ochratoxin A in the test sample according to the standard curve.

[0013] Preferably, the AuAg NCs solution in step (1) is prepared according to the following method:

[0014] Mix glutathione solution, chloroauric acid solution and silver nitrate solution, add water, heat and stir at 85°C for 6.5 h in the dark, cool to room temperature, remove and centrifuge, then dialyze with a 3500 Da dialysis bag for 48 h. The resulting yellow solution is denoted as AuAg NCs solution.

[0015] Preferably, the molar ratio of glutathione, chloroauric acid, and silver nitrate in the glutathione solution, chloroauric acid solution, and silver nitrate solution is 15:10:1.

[0016] Preferably, the carboxylated S,Pg-C3N4 nanosheet powder in step (1) is prepared as follows:

[0017] S1: Thiourea was weighed and dissolved in water. The mixture was stirred at room temperature, and then ammonium dihydrogen phosphate was added. The mixture was ultrasonically treated to obtain a solution. The solution was evaporated by rotary evaporation and then vacuum dried. After cooling to room temperature, the solution was taken out and reacted at 550℃ for 2 hours. After the reaction was completed, the product was cooled and ground into powder. The resulting yellow powder was denoted as S,Pg-C3N4 nanoblocks.

[0018] S2: Take S,Pg-C3N4 nanoblocks, add 5M nitric acid solution, reflux at 120℃ for 8-12 h, cool the suspension to room temperature and centrifuge, then wash repeatedly with water until neutral to obtain carboxylated S,Pg-C3N4; disperse the carboxylated S,Pg-C3N4 in water, sonicate to exfoliate the mixture, then centrifuge, dry the precipitate for 6-10 h, and finally obtain carboxylated S,Pg-C3N4 nanosheet powder.

[0019] Preferably, the mass ratio of thiourea to ammonium dihydrogen phosphate in S1 is 1:0.2.

[0020] Preferably, the rotary evaporation temperature in S1 is 60°C and the time is 30 min.

[0021] Preferably, the amount of S,Pg-C3N4 nanoblocks and nitric acid solution added in S2 is 0.1 g / mL.

[0022] Preferably, the volume ratio of the carboxylated S,Pg-C3N4 nanosheet powder and AuAg NCs solution in step (1) is 5:1 to 5, and the reaction time is 2 to 10 h.

[0023] Preferably, the dialysis method in step (1) is: dialysis for 24 to 48 hours in a dialysis bag with a pressure of 3500 Da.

[0024] Preferably, the concentration ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and the N-hydroxysuccinimide solution in step (2) is 1:0 to 10.

[0025] Preferably, the mass ratio of the freeze-dried AuAg NCs-S,Pg-C3N4 nanocomposite material and 1-ethyl-(3-dimethylaminopropyl)carbodiimide in step (2) is 5:1 to 5.

[0026] Preferably, the activation time in step (2) is 1 to 24 hours.

[0027] Preferably, the temperature for incubation in step (2) is 37°C and the time is 1 to 24 hours.

[0028] Preferably, the ratio of the amount of antibody added in step (2) to the mass of the freeze-dried AuAg NCs-S,Pg-C3N4 nanocomposite material is 0.001 to 0.05:1.

[0029] Preferably, the antibody in step (2) is an ochratoxin A antibody.

[0030] Preferably, the volume ratio of the dopamine solution to the coated antigen in step (3) is 1:1.

[0031] Preferably, the coating antigen in step (3) is the antigen corresponding to ochratoxin A, which was purchased from Shenzhen Antibiotechnology Co., Ltd.

[0032] Preferably, the concentration of the coating antigen in step (3) is 2 mg / mL; the competition temperature is 4℃~55℃. Preferably, the blocking agent in step (3) is one of bovine serum albumin.

[0033] Preferably, the volume ratio of the antibody labeled with AuAg NCs-S,Pg-C3N4 nanocomposite material to the coated antigen in step (3) is 1:1 to 5.

[0034] Preferably, the concentration of the different concentrations of ochratoxin A standard in step (3) is selected from 0.001 to 10 μg / L.

[0035] Preferably, in step (3), HAc-NaAc buffer, TMB solution and H2O2 solution are added to the solution for colorimetric detection, wherein the concentration of TMB solution is 30 mM and the concentration of H2O2 solution is 30%.

[0036] Preferably, the volume ratio of HAc-NaAc buffer, TMB solution and H2O2 solution in step (3) is 10:1 to 10:1 to 10.

[0037] Preferably, the excitation wavelength of the multifunctional microplate reader in step (3) is 320 nm and the emission wavelength is 440 nm.

[0038] Compared with the prior art, the beneficial effects of the present invention include:

[0039] (1) This invention uses thiourea and ammonium dihydrogen phosphate as raw materials to obtain sulfur and phosphorus co-doped graphitic carbon nitride (S,Pg-C3N4) by high-temperature calcination; using glutathione as template and chloroauric acid and silver nitrate as metal precursors, gold and silver nanoclusters (AuAg NCs) are synthesized by high-temperature hydrothermal method; and AuAg NCs-S,Pg-C3N4 nanocomposite material is synthesized by using the gold-sulfur bond (Au-S) formed between gold and silver nanoclusters and sulfur and phosphorus co-doped graphitic carbon nitride. Based on the aggregation luminescence principle, S,Pg-C3N4 significantly enhances the fluorescence intensity of AuAg NCs, and due to the incorporation of metal, AuAg NCs greatly enhances the enzyme catalytic activity of S,Pg-C3N4.

[0040] (2) In this invention, AuAg NCs-S,Pg-C3N4 nanocomposite material is used as a marker. Due to the enhancement of the fluorescence effect of S,Pg-C3N4 on AuAg NCs, the fluorescence signal of the sensor can be amplified. Furthermore, due to the enhancement of the enzyme catalytic activity of S,Pg-C3N4 by AuAg NCs, the colorimetric signal of the sensor can be amplified. The amplification of both signals can improve the sensitivity of the sensor and reduce the detection limit of the sensor.

[0041] (3) The present invention utilizes the immune reaction of antigens and antibodies to improve the specificity of the detection method.

[0042] (4) The competitive colorimetric fluorescence dual-mode sensor prepared in this invention uses AuAg NCs-S,Pg-C3N4 nanocomposite material as a label, which is coupled with an antibody. The antigen coated on the ELISA plate competes with the added standard antigen for the antibody labeled by the nanocomposite material. The antigen content is detected by comparing the changes in fluorescence intensity and absorbance before and after the addition of the standard antigen. It has the characteristics of low detection limit, wide linear range, high sensitivity, simple operation, short response time, and high specificity. Attached Figure Description

[0043] Figure 1 The images shown are transmission electron microscope (TEM) images of AuAg NCs, S,Pg-C3N4, and AuAg NCs-S,Pg-C3N4 from Example 1, where A corresponds to S,Pg-C3N4, B corresponds to AuAg NCs, and C corresponds to AuAg NCs-S,Pg-C3N4.

[0044] Figure 2 The image shows a comparison of the fluorescence spectrum (left image) and ultraviolet spectrum (right image) of AuAg NCs in Example 1 with that of Au NCs.

[0045] Figure 3 The fluorescence spectra of AuAg NCs-S, Pg-C3N4, and AuAg NCs are shown.

[0046] Figure 4 The images show a comparison of the UV spectra of g-C3N4 (corresponding to GCN in the figure) and S,Pg-C3N4 (corresponding to SPCN in the figure).

[0047] Figure 5 XRD patterns of g-C3N4 (corresponding to GCN in the figure), S,Pg-C3N4 (corresponding to SPCN in the figure), and AuAg NCs-S,Pg-C3N4 (corresponding to AuAg NCs-SPCN in the figure).

[0048] Figure 6Infrared spectra of AuAg NCs, S,Pg-C3N4 and AuAg NCs-S,Pg-C3N4.

[0049] Figure 7 XPS image of AuAg NCs-S,Pg-C3N4 nanocomposite material.

[0050] Figure 8 The image shows the UV absorption spectrum of the enzyme-mimicking activity of the AuAg NCs-S,Pg-C3N4 (corresponding to AuAg NCs-SPCN in the figure) nanocomposite material.

[0051] Figure 9 The enzyme kinetic reaction rate curves for AuAg NCs-S,Pg-C3N4 are shown.

[0052] Figure 10 The curves are double reciprocal curves of the enzyme kinetics of AuAg NCs-S,Pg-C3N4, where A corresponds to TMB and B corresponds to H2O2.

[0053] Figure 11 This is a schematic diagram illustrating the linear relationship between the FL value and the concentration of ochratoxin A in Example 3.

[0054] Figure 12 This is a schematic diagram showing the linear relationship between absorbance value and ochratoxin A concentration in Example 3. Detailed Implementation

[0055] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention.

[0056] The coating antigen was the antigen corresponding to ochratoxin A, purchased from Shenzhen Antibiotechnology Co., Ltd., with an antigen concentration of 6.1 mg / mL and an antibody concentration of 6.05 mg / mL; it was then prepared to the required concentration upon receipt.

[0057] Example 1

[0058] A method for preparing AuAg NCs-S,Pg-C3N4 nanocomposite material, comprising the following steps:

[0059] (1) Add 138 μL of 100 mg / mL glutathione, 102 μL of 100 mg / mL chloroauric acid, and 51 μL of 100 mg / mL silver nitrate to a round-bottom flask, then add pure water to a final volume of 15 mL. Place the flask in an oil bath at 85 °C and stir magnetically for 6.5 h in the dark. After cooling to room temperature, remove the flask and centrifuge at 10,000 rpm for 15 min. Then dialyze the flask through a 3500 Da dialysis bag for 48 h. The resulting yellow solution is the AuAgNCs solution, and its transmission electron microscopy image is attached. Figure 1 The fluorescence spectrum and ultraviolet spectrum are attached. Figure 2 The infrared spectrum is attached. Figure 6 .

[0060] (2) Weigh 1g of thiourea and dissolve it in 10mL of pure water. Stir magnetically for 30min at room temperature. Add 0.2g of ammonium dihydrogen phosphate to the reactants and sonicate for 10min. Then, evaporate the water in the solution at 60℃ for 30min and dry it in a vacuum drying oven at 90℃ for 2h. After cooling to room temperature, take out the white solid and put it in a crucible. Cover the crucible and put it in a muffle furnace at 550℃ for 2h. After turning off the muffle furnace, let it cool for 1h. Take out the product and grind it into powder. The yellow powder obtained is S,Pg-C3N4 nanoblocks.

[0061] (3) Take 1 g of S,Pg-C3N4 nanobulbs in a round-bottom flask, add 10 mL of 5 M nitric acid solution, reflux at 120 °C for 8 h, cool the suspension to room temperature, centrifuge at 8000 rpm for 20 min, and then wash repeatedly with ultrapure water until neutral to obtain carboxylated S,Pg-C3N4. Then disperse the precipitate in 50 mL of ultrapure water and sonicate continuously at 600 W for 8 h to exfoliate the mixture. Then centrifuge at 8000 rpm for 20 min, place the precipitate in an oven at 60 °C and dry overnight. After removal, carboxylated S,Pg-C3N4 nanosheet powder is obtained. Its scanning electron microscope image is attached. Figure 1 The ultraviolet spectrum is attached. Figure 4 XRD pattern is attached. Figure 5 The infrared spectrum is attached. Figure 6 .

[0062] (4) 2 mg of carboxylated S,Pg-C3N4 nanosheet powder was dispersed in 5 mL of ultrapure water and sonicated for 15 min. 4 mL of AuAg NCs solution was added, and the mixture was then magnetically stirred at 37 °C for 8 h. After the reaction was complete, the mixture was dialyzed at 3500 Da for 24 h to obtain the AuAg NCs-S,Pg-C3N4 nanocomposite material. Its scanning electron microscope image is attached. Figure 1 The fluorescence spectrum is attached. Figure 3 XRD pattern is attached. Figure 5 The infrared spectrum is attached. Figure 6 XPS graph is attached. Figure 7 .

[0063] Example 2

[0064] The method for analyzing the enzyme-mimicking activity and enzyme kinetics of AuAg NCs-S,Pg-C3N4 nanocomposites is as follows:

[0065] (1) Take four 2mL centrifuge tubes. Under pH 4 conditions, add 50μL of 30mM TMB solution to tube a, 50μL of 30mM TMB solution and 20μL of 30% H2O2 solution to tube b, 50μL of 30mM TMB solution and 200μL of 1mg / mL AuAg NCs-S,Pg-C3N4 nanocomposite material to tube c, and 50μL of 30mM TMB solution, 200μL of 1mg / mL AuAg NCs-S,Pg-C3N4 nanocomposite material and 20μL of 30% H2O2 solution to tube d. After heating and reacting in a shaker at 50℃ for 13min, measure the absorbance at 652nm. The UV absorption spectrum is shown in the appendix. Figure 8 .

[0066] (2) Under pH=4 conditions, detection was performed by changing the concentration of one substrate while keeping the concentration of another substrate constant. The specific experimental procedure was as follows: First, keeping the H2O2 concentration constant, 100 μL of HAc-NaAc buffer solution and 100 μL of 1 mg / mL AuAg NCs-S,Pg-C3N4 were added to the microplate. Then, 30 μL of 0.1–0.7 mM TMB was added, followed by 20 μL of H2O2 with a fixed concentration of 30%. Then, keeping the TMB concentration constant, 100 μL of HAc-NaAc buffer solution and 100 μL of 1 mg / mL AuAg NCs-S,Pg-C3N4 were added to the microplate. Then, 30 μL of TMB with a fixed concentration of 30 mM was added, followed by 20 μL of 2–50 mM H2O2. Using the kinetic cycle mode of a multi-functional microplate reader, absorbance values ​​were measured every 1 minute at a wavelength of 652 nm at the corresponding optimal temperature, for a continuous period of 15 minutes. Kinetic parameters were calculated using Lineweaver-Burk double reciprocal curves, and the Michaelis-Ment equation is as follows: 1 / V=(1 / [S])·(K m / V max )+1 / V max The rate curve and the double reciprocal curve are attached. Figure 9 and Figure 10 .

[0067] Example 3

[0068] A method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor, comprising the following steps:

[0069] (1) Take 1 mL of AuAg NCs-S,Pg-C3N4 nanocomposite material (AuAg NCs-S,Pg-C3N4 nanocomposite material was lyophilized and prepared into a 5 mg / mL liquid), add 100 μL of EDC solution (20 mg / mL) and 100 μL of NHS solution (10 mg / mL), and stir to activate at 25 °C for 30 min. Then centrifuge at 10000 rpm for 30 min, wash 3 times with PBS, and then add 100 μL of 50 μg / mL ochratoxin A antibody (Ab) (purchased from Shenzhen Antibiotechnology Co., Ltd.) to the mixed solution. Incubate in a shaker at 37 °C in the dark for 2 h, centrifuge the resulting solution at 10000 rpm for 30 min, and then wash 3 times with PBS to obtain the antibody labeled with AuAg NCs-S,Pg-C3N4 nanocomposite material, disperse it in 2 mL of PBS and store it at 4 °C for later use.

[0070] (2) First, inject dopamine solution (1 mg / mL, 50 μL) into a 96-well plate, incubate at 37°C for half an hour, and then dry until a membrane appears. Second, inject 50 μL of coated antigen (2 mg / mL), react at 4°C overnight, wash three times, then add 50 μL of BSA and react for 2 hours to block excess binding sites on the dopamine, wash three times. Third, add 50 μL of antibody labeled with AuAgNCs-S,Pg-C3N4 nanocomposite material, incubate at 37°C for 2 hours, then add 50 μL of standard antigen (standard antigen refers to ochratoxin A standard), incubate for 2 hours, wash with PBST, and then detect its absorbance using a multi-functional microplate reader; then aspirate the solution, add HAc-NaAc buffer (100 μL, 0.2 M, pH=4), TMB (30 μL, 30 mM) and 10 μL H2O2 to the solution for colorimetric detection. After reacting at 50℃ for 13 min, the absorbance change and corresponding color change of the solution at 652 nm were recorded.

[0071] (3) A series of standard antigen solutions (50 μL) of different concentrations and antigens (50 μL, i.e., coating antigens) coated on an ELISA plate at a fixed concentration competed with a fixed concentration of nanozyme-labeled antibody (50 μL, i.e., antibody labeled with AuAg NCs-S, Pg-C3N4 nanocomposite material) for 1 h. Under optimized experimental conditions, the fluorescence signal was detected using a multi-functional ELISA reader with an excitation wavelength of 320 nm and an emission wavelength of 400 nm. Then, HAc-NaAc solution (100 μL, 0.2 M, pH = 4), TMB (30 μL, 30 mM), and H2O2 (10 μL, 30%) were added. After the reaction under optimized experimental conditions, colorimetric detection was performed, and the absorbance of the solution at 652 nm and the corresponding color change were recorded. The standard curve is shown in Appendix. Figure 11 and Figure 12 .

[0072] The antigen concentrations were prepared by increasing sequentially from top to bottom (0.001 μg / L, 0.005 μg / L, 0.01 μg / L, 0.1 μg / L, 0.5 μg / L, 1 μg / L, 5 μg / L, 10 μg / L). A standard curve was plotted with OTA concentration on the x-axis and FL and absorbance on the y-axis. The OTA concentration was determined based on the standard curve to establish the detection limit (FL is the fluorescence value after adding the standard antigen solution). In this implementation case, the linear range for OTA detection was 0.001 μg / L-10 μg / L, and the detection limits were 0.155 ng / L (fluorescence) and 0.213 ng / L (colorimetric). The linear relationship is shown in the appendix. Figure 7 and Figure 8 As shown in Table 1, the competitive colorimetric fluorescence dual-mode sensor is more sensitive than most previous methods for detecting OTA, with a wider detection range and a lower detection limit.

[0073] Table 1. Comparison of Results from Various Detection Methods

[0074]

[0075] As can be seen from Table 1, the competitive fluorescence colorimetric dual-mode immunosensor in Embodiment 3 of the present invention has a lower detection limit and a wider detection range.

[0076] Example 4

[0077] A method for detecting ochratoxin A in corn and red wine using a competitive colorimetric fluorescence dual-mode immunosensor, comprising the following steps:

[0078] (1) First, pretreatment of the test sample: ① Grind 4g of commercially available corn sample and then dissolve it in 20mL of mixed solution (V 乙酰水杨酸 V H2O V CH3COOHIn a mixture of 79:20:1, the mixture was sonicated and filtered multiple times. The mixture was then centrifuged at 10,000 rpm for 5 minutes, and the supernatant was collected. ② 3 mL of commercially available red wine was diluted to 3% using 100 mL of PBS buffer.

[0079] (2) Then, following the standard addition method, 50 μL of 1 μg / L, 50 μL of 0.1 μg / L, and 50 μL of 0.01 μg / L OTA were added to the above samples respectively for recovery tests. Three replicates were set up for each test. The fluorescence intensity and absorbance were measured according to the established competitive fluorescence colorimetric dual-mode detection method. The results were substituted into the established standard curve to calculate the OTA concentration. The feasibility of the method was evaluated by comparing the recovered concentration with the OTA spiked amount and calculating the recovery rate. The formulas for calculating the sample recovery rate and coefficient of variation are as follows:

[0080] Sample recovery rate = (Measured concentration of standard sample / Added concentration of standard sample) × 100%

[0081] Coefficient of variation (RSD) = (Standard deviation / Mean) × 100%

[0082] The recovery results are shown in Table 2. The spiked recoveries for red wine were 95.5–105.8% (fluorescence) and 96.0–109.9% (colorimetric), while the spiked recoveries for corn were 97.6–105.7% (fluorescence) and 96.1–104.9% (colorimetric). This means the fluorescence method recovery rate was between 95.5% and 105.8%, and the colorimetric method recovery rate was between 96.0% and 109.9%. The coefficients of variation (relative standard deviations) were between 1.29% and 7.77% (fluorescence) and 1.84% and 6.23% (colorimetric), respectively. Since the allowable limit for recovery rate is 80%–120% when the content of the analyte is ≤1 mg / L, these results indicate that the detection method has good recovery and low relative standard deviation, suggesting that the competitive fluorescence-colorimetric dual-mode immunosensor strategy has great potential for detecting OTA in real samples.

[0083] Table 2 Recovery rates of OTA at different concentrations in actual samples (n=3)

[0084]

[0085] Figure 1 The images shown are transmission electron microscopy (TEM) images of AuAg NCs, S,Pg-C3N4, and AuAg NCs-S,Pg-C3N4 from Example 1, where A corresponds to S,Pg-C3N4, B corresponds to AuAg NCs, and C corresponds to AuAg NCs-S,Pg-C3N4. Figure 1As can be seen from the figure, S,Pg-C3N4 is in the form of thin sheets, while AuAg NCs is an ultra-small spherical structure. Figure C shows that the spherical structure is loaded on the sheet-like S,Pg-C3N4.

[0086] Figure 2 The image shows a comparison of the fluorescence spectrum (left image) and ultraviolet spectrum (right image) of AuAg NCs from Example 1 with that of Au NCs. Figure 2 As can be seen from this, AuAg NCs have good optical properties.

[0087] Figure 3 The fluorescence spectra of AuAg NCs-S, Pg-C3N4, and AuAg NCs are shown below. Figure 3 It can be seen that the fluorescence intensity of AuAgNCs-S,Pg-C3N4 is significantly stronger than that of AuAgNCs.

[0088] Figure 4 The images show a comparison of the UV spectra of g-C3N4 (corresponding to GCN in the figure) and S,Pg-C3N4 (corresponding to SPCN in the figure). From... Figure 4 As can be seen, SPCN exhibits an absorption peak at 326 nm. Compared with GCN, SPCN shows a significant blue shift in its absorption edge. This is likely because the co-doping of sulfur and phosphorus affects the electronic structure of carbon nitride, widening its band gap and providing more active centers to enhance visible light absorption, resulting in a better optical response and thus improving the catalytic performance of carbon nitride.

[0089] The figure shows the XRD patterns of 5g-C3N4 (corresponding to GCN in the figure), S,Pg-C3N4 (corresponding to SPCN in the figure), and AuAg NCs-S,Pg-C3N4 (corresponding to AuAg NCs-SPCN in the figure). Figure 5 It can be seen that the X-ray diffraction patterns of SPCN and AuAg NCs-SPCN are similar to those of GCN, indicating that the co-doping of P and S and the loading of AuAg NCs have no significant effect on the crystal structure of graphitic carbon nitride.

[0090] Figure 6 Infrared spectra of AuAg NCs, S,Pg-C3N4, and AuAg NCs-S,Pg-C3N4. From... Figure 6 It can be seen from the data that the FT-IR spectra of SPCN and AuAg NCs-SPCN are similar, with similar values ​​at 807 cm⁻¹. -1 The same peak appears at 900–1700 cm⁻¹, which is a characteristic breathing peak of the 3-S-triazine ring unique to graphitic carbon nitride. -1The peaks appearing within this range are typical stretching bands of CN heterocycles containing C-NH-C. Furthermore, compared to SPCN, AuAg NCs-SPCN exhibits peaks in the 2900–3600 cm⁻¹ range. -1 A broader absorption peak was added within the range, which is the characteristic absorption band of NH provided by the amino group in glutathione, the ligand of AuAg NCs (v). N-H This indicates that AuAgNCs was loaded successfully.

[0091] Figure 7 XPS plot of AuAg NCs-S,Pg-C3N4 nanocomposite material, from Figure 7 The image shows six distinct peaks at 283.8, 531.2, 399, 83.4, 367.2, and 169.2 eV. These peaks represent carbon (C), oxygen (O), nitrogen (N), gold (Au), silver (Ag), and sulfur (S), with atomic percentages of 59%, 33%, 5.8%, 1.3%, 0.4%, and 0.2%, respectively. Because the doping level of phosphorus (P) is very low (less than 0.1%), the peak representing P at 133.9 eV is not prominent in the full spectrum (A). This indicates that AuAg NCs-S,Pg-C3N4 contains the aforementioned elements.

[0092] Figure 8 The image shows the UV absorption spectrum of the enzyme-mimicking activity of the AuAg NCs-S,Pg-C3N4 (corresponding to AuAg NCs-SPCN in the figure) nanocomposite material. Figure 8 It can be seen from the figure that curves (a) and (b) have no absorption peaks, while curves (c) and (d) have obvious absorption peaks at 652 nm, and the absorbance with H2O2 is much higher than that without H2O2. Observing the inset of the figure, it can be seen that TMB is colorless (a), and the color of the solution does not change after adding H2O2 (b). When AuAg NCs-S,Pg-C3N4 is added, the solution turns dark blue (d), which is much darker than the TMB+AuAg NCs-S,Pg-C3N4 system (c). This indicates that AuAg NCs-S,Pg-C3N4 has strong enzyme-like activity, and the enzyme activity is stronger in the presence of H2O2.

[0093] Figure 9 The image shows the enzyme kinetics rate curves for AuAg NCs-S,Pg-C3N4, where A corresponds to TMB and B corresponds to H2O2. From... Figure 9 It can be seen that the reaction rate increases with increasing concentration.

[0094] Figure 10The graph shows the double reciprocal kinetic curves for the enzyme kinetics of AuAg NCs-S,Pg-C3N4, where A corresponds to TMB and B corresponds to H2O2. From... Figure 10 It can be seen that the two reciprocals have a linear relationship. The Kc of AuAg NCs-S,Pg-C3N4 for the substrates TMB and H2O2 can be calculated using the formula. m The concentrations were 0.21 mM and 2.26 mM, respectively, V max The values ​​are 5.75 × 10 -8 Ms -1 and 5.95×10 -8 Ms -1 This indicates that AuAg NCs-S,Pg-C3N4 has a good affinity for TMB and H2O2 and strong catalytic ability.

[0095] Figure 11 This is a schematic diagram illustrating the linear relationship between the FL value and the concentration of ochratoxin A in Example 3. (FL is the fluorescence value after adding the standard antigen solution). From Figure 11 As can be seen from the graph, the fluorescence intensity increases with increasing antigen concentration. The equation of the standard curve is I. 440 =2698.903+676.679*lgC OTA (μg / L)(R 2 =0.995). This result indicates that the competitive fluorescence assay for detecting ochratoxin A (OTA) exhibits a wide linear range of 0.001 μg / L–10 μg / L, with a low limit of detection of 0.155 ng / L.

[0096] Figure 12 This is a schematic diagram illustrating the linear relationship between absorbance values ​​and ochratoxin A concentration in Example 3. From... Figure 12 As can be seen from the graph, the absorbance increases with increasing antigen concentration. The equation of the standard curve is shown in the figure as Abs... 652 =1.0636 + 0.3094 * lgC OTA (μg / L)(R 2 =0.997), as shown in the inset, the solution color gradually darkens, corresponding to the trend of the standard curve. This result indicates that the colorimetric analysis method for detecting ochratoxin A (OTA) exhibits a wide linear range of 0.001 μg / L–10 μg / L, with a low detection limit of 0.213 ng / L.

[0097] The specific embodiments of the present invention described above do not constitute a limitation on the scope of protection of the present invention. Any other corresponding changes and modifications made in accordance with the technical concept of the present invention should be included within the scope of protection of the claims of the present invention.

Claims

1. A method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor, characterized in that, Includes the following steps: (1) The carboxylated S,Pg-C3N4 nanosheet powder and AuAg NCs solution were mixed and stirred to react. After the reaction was completed, the mixture was dialyzed to obtain AuAg NCs-S,Pg-C3N4 nanocomposite material, which was then freeze-dried for later use. (2) The freeze-dried AuAg NCs-S,Pg-C3N4 nanocomposite material, 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and N-hydroxysuccinimide solution were mixed, activated at room temperature and centrifuged. The solid sample after centrifugation was washed with PBS buffer, antibody was added, incubated in the dark, centrifuged and washed again to obtain the antibody labeled with AuAg NCs-S,Pg-C3N4 nanocomposite material. The antibody was reconstituted with PBS buffer and stored in the dark for later use. (3) First, dopamine solution was injected into a 96-well plate, incubated, and dried until a membrane appeared. Then, the coated antigen was injected and reacted overnight at 4°C. After washing, blocking agent was added and the reaction was continued. The plate was washed three times. Antibody labeled with AuAg NCs-S and Pg-C3N4 nanocomposite was added and competitively reacted for 0.5-2 hours. Then, different concentrations of ochratoxin A standard were added and incubated. After washing with PBST, the fluorescence intensity was detected. The solution was then aspirated and HAc-NaAc buffer, TMB solution, and H2O2 solution were added to the solution for colorimetric detection. After reacting at 25°C-70°C for 5-17 minutes, the absorbance change and corresponding color change at 652 nm were recorded. A standard curve was plotted with the concentration of ochratoxin A as the x-axis and the fluorescence value and absorbance as the y-axis. (4) Replace the ochratoxin A standard with the test sample solution and calculate the concentration of ochratoxin A in the test sample according to the standard curve.

2. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to claim 1, characterized in that, In step (1), the volume ratio of the carboxylated S,Pg-C3N4 nanosheet powder to the AuAg NCs solution is 5:1 to 5, and the reaction time is 2 to 10 h. The dialysis method described in step (1) is as follows: dialysis for 24 to 48 hours under a 3500 Da dialysis bag.

3. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to claim 1, characterized in that, The concentration ratio of the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution and the N-hydroxysuccinimide solution in step (2) is 1:0 to 10; The mass ratio of the freeze-dried AuAg NCs-S,Pg-C3N4 nanocomposite material and the 1-ethyl-(3-dimethylaminopropyl)carbodiimide solution in step (2) is 5:1 to 5; The ratio of the amount of antibody added in step (2) to the mass of the freeze-dried AuAg NCs-S,Pg-C3N4 nanocomposite material is 0.001 to 0.05:

1.

4. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to any one of claims 1 to 3, characterized in that, The activation time in step (2) is 1 to 24 hours; The incubation in step (2) is carried out at a temperature of 37°C for 1 to 24 hours. In step (3), the volume ratio of the dopamine solution to the coated antigen is 1:1; The concentration of the coating antigen in step (3) is 2 mg / mL; the competition temperature is 4℃~55℃.

5. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to claim 4, characterized in that, In step (3), the volume ratio of the antibody labeled with AuAg NCs-S,Pg-C3N4 nanocomposite material to the coated antigen is 1:1 to 5. The concentrations of the ochratoxin A standards in step (3) are selected from 0.001 to 10 μg / L; In step (3), HAc-NaAc buffer, TMB solution and H2O2 solution are added to the solution for colorimetric detection. The concentration of TMB solution is 30 mM and the concentration of H2O2 solution is 30%.

6. A method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to any one of claims 1 to 3, characterized in that, The volume ratio of HAc-NaAc buffer, TMB solution and H2O2 solution in step (3) is 10:1 to 10:1 to 10.

7. A method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to any one of claims 1 to 3, characterized in that, The antibody mentioned in step (2) is an ochratoxin A antibody; The coating antigen mentioned in step (3) is the antigen corresponding to ochratoxin A; The blocking agent mentioned in step (3) is one of bovine serum albumin.

8. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to claim 1, characterized in that, The AuAg NCs solution in step (1) was prepared according to the following method: Mix glutathione solution, chloroauric acid solution and silver nitrate solution, add water, heat and stir at 85°C for 6.5 h in the dark, cool to room temperature, remove and centrifuge, then dialyze with a 3500 Da dialysis bag for 48 h. The resulting yellow solution is denoted as AuAg NCs solution.

9. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to claim 1, characterized in that, The carboxylated S,Pg-C3N4 nanosheet powder described in step (1) was prepared as follows: S1: Thiourea was weighed and dissolved in water. The mixture was stirred at room temperature, and then ammonium dihydrogen phosphate was added. The mixture was ultrasonically treated to obtain a solution. The solution was evaporated by rotary evaporation and then vacuum dried. After cooling to room temperature, the solution was taken out and reacted at 550℃ for 2 hours. After the reaction was completed, the product was cooled and ground into powder. The resulting yellow powder was denoted as S,Pg-C3N4 nanoblocks. S2: Take S,Pg-C3N4 nanoblocks, add 5M nitric acid solution, reflux at 120℃ for 8-12 h, cool the suspension to room temperature and centrifuge, then wash repeatedly with water until neutral to obtain carboxylated S,Pg-C3N4; disperse the carboxylated S,Pg-C3N4 in water, sonicate to exfoliate the mixture, then centrifuge, dry the precipitate for 6-10 h, and finally obtain carboxylated S,Pg-C3N4 nanosheet powder.

10. The method for detecting ochratoxin A using a competitive colorimetric fluorescence dual-mode immunosensor according to claim 9, characterized in that, The mass ratio of thiourea to ammonium dihydrogen phosphate in S1 is 1:0.2; S1, the rotary evaporation temperature is 60℃ and the time is 30min; S2, the amount of S,Pg-C3N4 nanoblocks and nitric acid solution added is 0.1 g / mL.

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