A trimodal detection method for clenbuterol based on Co@C3N4 nanozyme

Abstract

A trimodal detection method for clenbuterol based on Co@C3N4 nanozymes comprises the following steps: Step 1: measuring the fluorescence intensity F1 at 460 nm of a reaction solution containing a clenbuterol standard at varying concentrations under 365 nm excitation; Step 2: measuring the absorbance A1 at 650 nm of a reaction solution containing a clenbuterol standard at varying concentrations; Step 3: measuring the temperature T1 of a reaction solution containing a clenbuterol standard at varying concentrations under laser irradiation; and Step 4: constructing a linear regression equation for the fluorescence F, absorbance A, and temperature T of the standard. This method has been successfully applied to the detection of Clenbuterol in real samples, demonstrating excellent sensitivity and selectivity. Furthermore, the trimodal detection method demonstrates greater adaptability in practical applications, enabling it to meet diverse testing requirements based on different instruments.

Description

Technical Field

[0001] The present invention relates to a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme, belonging to the technical field of environmental and food pollution detection. Background Art

[0002] Clenbuterol (CLB), a member of the β2-adrenergic agonist family, has been widely used in livestock farming as a growth promoter and feed additive, as it reduces fat accumulation and promotes lipolysis. However, it can accumulate in the body through the food chain, causing various health problems such as fatigue, dizziness, palpitations, metabolic disorders, and muscle tremors. Currently, CLB has been banned as a livestock growth promoter in many countries, but there are still many cases of illegal use of CLB as a feed additive for farm animals. In this context, the development of a sensitive and specific method to detect CLB residues in commercially available meat products is of great significance for ensuring food safety and human health.

[0003] Common CLB detection methods include gas chromatography-mass spectrometry, liquid chromatography, polymer molecular imprinting, enzyme-linked immunosorbent assay (ELISA), electrochemical methods, and colorimetric sensing. However, these detection methods often have some disadvantages, including expensive instruments, complex and time-consuming pretreatment procedures, and sophisticated technical operators, which seriously hinder their widespread application. Summary of the Invention

[0004] The purpose of the present invention is to provide an efficient and accurate trimodal detection method for clenbuterol based on Co@C3N4 nanozyme. The Co@C3N4 nanozyme in the present invention has peroxidase activity and has blue fluorescence characteristics under 365nm excitation light. When H2O2 is present, it can catalyze H2O2 to produce hydroxyl radicals, thereby oxidizing colorless TMB to blue ox-TMB; under 660nm laser irradiation, ox-TMB will produce a typical photothermal effect and the temperature will increase; at the same time, the generated ox-TMB will quench the blue fluorescence of Co@C3N4 through the fluorescence resonance energy transfer effect (FRET). However, when CLB is present, it will competitively react with H2O2, thereby reducing the hydroxyl radicals, thereby blocking the formation of ox-TMB, showing a CLB concentration-dependent colorimetric signal decrease, temperature decrease and fluorescence signal enhancement, thereby developing a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme, comprising the following steps:

[0006] Step 1: Determine the fluorescence intensity F1 of the reaction solution containing different concentrations of clenbuterol standard at 460nm under 365nm wavelength excitation

[0007] The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was allowed to proceed for 30-40 minutes before the tube was transferred to a cuvette. The fluorescence intensity at 460 nm was measured using a fluorescence spectrometer under 365 nm excitation, and recorded as the fluorescence value F1.

[0008] Step 2: Determine the absorbance value A1 of the reaction solution containing different concentrations of clenbuterol standard at 650nm

[0009] The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was carried out for 30-40 minutes and then the solution was transferred to a cuvette. The absorbance at 650 nm was measured using a UV spectrophotometer and recorded as absorbance A1.

[0010] Step 3: Determine the temperature T1 of the reaction solution containing different concentrations of clenbuterol standard under 660nm laser irradiation:

[0011] The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was carried out for 30-40 minutes. After irradiation with 660 nm laser, the temperature value was measured using a portable photothermal imager and recorded as temperature T1.

[0012] Step 4: Construct a linear regression equation for the fluorescence F, absorbance A, and temperature T of the standard

[0013] The fluorescence intensity signal F was obtained by testing the clenbuterol standard with a series of concentrations, and a linear equation was constructed: F = XC CLB +Y, where C CLB is the concentration of the standard;

[0014] The absorbance signal A was obtained by testing a series of concentrations of clenbuterol standard substances, and a linear equation was constructed: A = XC CLB +Y, where C CLB is the concentration of the standard;

[0015] The temperature signal T is obtained by testing a series of concentrations of clenbuterol standard substances, and a linear equation is constructed: T = XC CLB +Y, where C CLB is the concentration of the standard;

[0016] Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the fluorescence intensity signal F n , absorbance signal A n and temperature signal T n , F n 、A n and T n Substitute into the corresponding linear equation to obtain the concentration of clenbuterol in the test sample.

[0017] The preferred technical solution is: the preparation method of the Co@C3N4 nanozyme comprises the following steps:

[0018] S1: 5.0 g of melamine was calcined at 600.0°C for 2.0 h in a muffle furnace; after cooling to room temperature, the obtained C3N4 powder was ultrasonically treated in deionized water for 12.0 h, and then freeze-dried to obtain C3N4;

[0019] S2: 10.0 mg of C3N4 obtained in step S1 was mixed with 1.0 mg of CoCl·6H2O and dissolved in 15.0 mL of DI. After stirring for 1.0 h, the mixture was transferred to a high-temperature reactor and reacted at 130.0°C for 3.0 h. After the reaction, the mixture was washed with ethanol and centrifuged in a centrifuge at 12000 r for 3.0 min, repeated three times. The precipitate was collected and dried in a constant temperature vacuum oven at 60.0°C overnight to obtain Co@C3N4 nanozyme.

[0020] The preferred technical solution is: the concentration of the Co@C3N4 nanozyme solution is 50.0 μg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 1.0 mmol / L; the reaction temperature is 35.0°C; the reaction time after mixing is 33.0 min; in steps 1, 2 and 3, 20.0 μL of 20.0 μg / mL Co@C3N4 nanozyme solution, 20.0 μL of clenbuterol standard solutions of different concentrations, 20.0 μL of 1.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution, and 20.0 μL of 2.0 mmol / L H2O2 are added to a centrifuge tube; then, HAc-NaAc buffer with a pH of 4.0 is added to the volume to 200.0 μL, mixed evenly and reacted for 30-40 minutes, and then the measurement is performed.

[0021] The preferred technical solution is: in step 4, the regression equation of the fluorescence signal F is F = 1.00071XC CLB+194.7896; the regression equation of absorbance signal A is A=-0.00792C CLB +1.78149; the regression equation of temperature signal T is T=-0.12087C CLB +45.11938.

[0022] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0023] 1. The synthesis method of Co@C3N4 nanozyme utilizes a wide range of raw materials, is environmentally friendly, safe, and low-cost, making it economical and affordable. It also possesses inherent fluorescent properties, which meets the needs of subsequent detection.

[0024] 2. Compared with traditional CLB detection methods, the present invention does not require expensive instruments, complex and time-consuming preprocessing procedures, and sophisticated technical operators. It is simple and convenient to operate. At the same time, the three modalities provide built-in cross-reference correction, which improves the reliability of the detection results.

[0025] 3. The detection time of the present invention is relatively short. It only takes about 33.0 minutes to observe the color change of the reagent with the naked eye to achieve CLB detection.

[0026] 4. The thermal response of the present invention has a low background signal and is not limited by time, location or professional operators. It can be conveniently measured by a portable thermometer.

[0027] 5. This invention has been successfully applied to the detection of CLB in real samples, demonstrating excellent sensitivity and selectivity. Furthermore, the trimodal detection method demonstrates greater adaptability in practical applications, enabling it to meet diverse testing requirements based on different instruments. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 Schematic diagram of a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme.

[0029] Figure 2 Transmission electron microscopy image of Co@C3N4 nanozyme.

[0030] Figure 3 The absorbance of different groups of (1) Co@C3N4+H2O2+TMB, (2) Co@C3N4+TMB, (3) Co@C3N4+H2O2 (4) H2O2+TMB in enzyme activity verification and the corresponding color illustrations.

[0031] Figure 4 Verification of the catalytic kinetics of Co@C3N4 nanozyme and its double reciprocal plot.

[0032] Figure 5The feasibility of detecting CLB in the Co-C3N4+TMB+H2O2 system was verified by the UV absorption and fluorescence intensity graphs of different groups (1) Co-C3N4+TMB+H2O2, (2) Co-C3N4+TMB+H2O2+CLB, (3) Co-C3N4+TMB, (4) Co-C3N4+H2O2, (5) TMB+H2O2, and (6) TMB.

[0033] Figure 6 The feasibility analysis of the photothermal method for a trimodal detection method of clenbuterol based on Co@C3N4 nanozyme and the optimization of 660nm laser irradiation time were carried out.

[0034] Figure 7 Optimization of time, pH, and temperature for a trimodal detection method of clenbuterol based on Co@C3N4 nanozyme.

[0035] Figure 8 Shown are the fluorescence spectra and standard curves of CLB at different concentrations.

[0036] Figure 9 Figure 4 shows the absorption spectra and standard curves of CLB at different concentrations.

[0037] Figure 10 This is the standard curve of the photothermal signal of different concentrations of CLB.

[0038] Figure 11 This proves the selectivity of the trimodal detection CLB method. DETAILED DESCRIPTION

[0039] The following describes the implementation of the present invention through specific examples. People skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in these examples.

[0040] See also Figure 1-11 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, any modification of the structure, change in the proportional relationship or adjustment of the size does not have any technical significance. The following examples are provided for a better understanding of the present invention, but are not intended to limit the present invention. Unless otherwise specified, the experimental methods in the following examples are all conventional methods. At the same time, the experimental materials used in the following examples, unless otherwise specified, were purchased from conventional biochemical reagent stores.

[0041] The technical solution of the present invention is further described in detail below with reference to examples.

[0042] Related reagents used in the embodiments of the present invention:

[0043] The raw materials used in this invention: melamine (98.0%), cobalt chloride hexahydrate (99.0%), and TMB (3,3',5,5'-tetramethylbenzidine, 99.5%) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; acetic acid-sodium acetate buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; and H2O2 (hydrogen peroxide solution) was purchased from Sinopharm Group Co., Ltd. All chemicals were purchased directly from suppliers without further purification; all water used in the experiments was ultrapure water.

[0044] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0045] Example 1: A trimodal detection method for clenbuterol based on Co@C3N4 nanozyme

[0046] The preparation method of Co@C3N4 nanozyme includes the following technical steps.

[0047] (1) Synthesis of Co@C3N4 nanozyme

[0048] First, 5.0g of melamine was calcined at 600.0℃ in a muffle furnace for 2.0h. After cooling to room temperature, the obtained C3N4 powder was ultrasonically treated in DI for 12.0h and further freeze-dried. Then 10.0mg of C3N4 was mixed with 1.0mg of CoCl·6H O and dissolved in 15.0mL of DI and stirred with a magnetic stirrer for 1.0h. It was then transferred to a high-temperature reactor and reacted at 130.0℃ for 3.0h. After the reaction, it was washed with ethanol and centrifuged in a 12000r centrifuge for 3.0min, and repeated 3 times. The precipitate was collected and kept in a constant temperature vacuum drying oven at 60.0℃ overnight to obtain Co@C3N4 nanozyme. The TEM image of the synthesized nanozyme is shown below. Figure 2 shown.

[0049] Example 2: Verification of Co@C3N4 nanozyme activity

[0050] Use H2O2 as substrate, TMB as color developer, and HAc-NaAc as buffer. Take the following reaction groups: (1) Co@C3N4+H2O2+TMB, (2) Co@C3N4+TMB, (3) Co@C3N4+H2O2(4) H2O2+TMB. After sufficient reaction, transfer to a cuvette and measure the absorbance at 650nm using a UV spectrophotometer. Figure 3 , only the Co@C3N4+H2O2+TMB combination has a larger absorbance value, indicating the high peroxidase activity of the Co@C3N4 nanozyme prepared in the present invention.

[0051] In the above verification steps, the concentration of the added Co@C3N4 nanozyme was 50.0 μg / mL; the concentration of the TMB solution was 1.0 mmol / L; the total amount of the mixed solution was 200.0 μL; the reaction temperature was 35.0°C; the reaction time was 33.0 min; and the pH value of the HAc-NaAc buffer was 4.0.

[0052] Example 3: Catalytic kinetics test of Co@C3N4 nanozyme

[0053] In HAc-NaAc buffer containing Co@C3N4 nanozymes, steady-state kinetic experiments were performed by varying the concentration of H2O2 (0.0-200.0 μmol / L) or TMB (0.0-2.0 mmol / L) while keeping the concentration of the other substance fixed. The reverse-reaction plots of H2O2 concentration and TMB concentration versus reaction rate were calculated using the following Michaelis-Menten equation:

[0054]

[0055] Where V represents the initial reaction rate, [S] is the substrate concentration, and K m is the Michaels constant, V max Indicates the maximum reaction rate. Figure 4 The K of Co@C3N4 to H2O2 and TMB is shown m The K values ​​were 0.03065 and 0.2227, which were significantly higher than those of natural horseradish peroxidase HPR. m The values ​​are about 120 times and 2.0 times lower, indicating that the Co@C3N4 nanozyme prepared in the present invention has good catalytic activity.

[0056] In the above verification steps, the concentration of the added Co@C3N4 nanozyme was 50.0 μg / mL; the concentration of the TMB solution was 1.0 mmol / L; the total amount of the mixed solution was 200.0 μL; the reaction temperature was 35.0°C; the reaction time was 33.0 min; and the pH value of the HAc-NaAc buffer was 4.0.

[0057] Example 4: Feasibility analysis of a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme

[0058] Take the following reaction groups: (1) Co@C3N4+H2O2+TMB, (2) Co@C3N4+H2O2+TMB+CLB (3) Co@C3N4+TMB, (4) Co@C3N4+H2O2 (5) H2O2+TMB (6) TMB. After sufficient reaction, transfer to a cuvette and measure the absorbance at 650nm using a UV spectrophotometer. The results are as follows Figure 5 A and Figure 5 As shown in Figure B, only Co@C3N4+H2O2+TMB had obvious absorbance and color changes. After adding CLB, that is, the Co@C3N4+H2O2+TMB+CLB group, the absorbance and color were inhibited, showing the feasibility of colorimetric detection of CLB.

[0059] Take the above reaction solution and measure the fluorescence intensity at 440nm using a fluorescence photometer under 365nm excitation light. Figure 5 C and Figure 5 As shown in Figure D, the fluorescence intensity of Co@C3N4+H2O2+TMB was suppressed, and the fluorescence of the Co@C3N4+H2O2+TMB+CLB group was restored after adding CLB, demonstrating the feasibility of fluorescence detection of CLB.

[0060] The following four groups were tested: (1) Co@C3N4+H2O2+TMB, (2) Co@C3N4+H2O2+TMB+660nm near-infrared laser irradiation (1.5W / cm 2 ), (3) Co@C3N4+H2O2+TMB+CLB (4) Co@C3N4+H2O2+TMB+CLB+660nm near-infrared laser irradiation (1.5W / cm 2 ). The result is as follows Figure 6 As shown, only groups (2) and (4) showed significant temperature changes. Furthermore, the temperature change in group (4) after adding CLB was lower than that in group (2) without CLB, demonstrating the feasibility of photothermal detection of CLB. At the same time, the temperature increase approached saturation at an irradiation time of 240.0s. Therefore, an irradiation time of 240.0s was selected as the optimal detection condition for the photothermal method.

[0061] Example 5: Optimization of the overall reaction conditions of a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme

[0062] In order to optimize the analytical performance of the three-modal system, we optimized the reaction time, pH value, temperature and other experimental conditions of the detection system. The experiment first optimized the reaction time, and the results are as follows: Figure 7 As shown in A, the difference in absorbance at 650nm between the reaction system without CLB and the reaction system with CLB increased with the increase of reaction time from 0.0 to 33.0min, and basically did not change after 33.0min. Therefore, 33.0min was selected as the optimal reaction time of the system. Then, the performance of the system in the pH range of 3.5-5.5 was explored, and NaAc-HAc was selected as the reaction buffer. The results are shown in Figure 1. Figure 7As shown in Figure B, when the pH value is in the range of 3.5 to 4.0, the difference in absorbance at 650nm between the reaction system without CLB and the reaction system with CLB increases with the increase in the pH value of the buffer system. When the pH value exceeds 4.0, the difference in absorbance gradually decreases as it increases, so 4.0 is selected as the optimal pH value for the system. Next, the performance of the system in the temperature range of 25.0℃-45.0℃ was explored. The results are shown in Figure 3. Figure 7 As shown in Figure 3, when the temperature range is 25.0°C to 35.0°C, the difference in absorbance at 650nm between the reaction system without CLB and the reaction system with CLB increases with increasing reaction temperature. When the temperature exceeds 35.0°C and increases to 45.0°C, the difference in absorbance gradually decreases as it increases, so 35.0°C is selected as the optimal temperature for the system.

[0063] Example 6: A trimodal detection of clenbuterol standard based on Co@C3N4 nanozyme

[0064] First, different concentrations of CLB (including 0.0, 5.0, 10.0, 15.0, 20.0, 40.0, 50.0, and 76.0 μmol / L) were prepared. Then, 20.0 μg / mL of Co@C3N4 nanozyme and 1.0 mmol / L of TMB solution were fully reacted with different concentrations of CLB in HAc-NaAc buffer (pH = 3.8-4.2) at 32.0-37.0°C for 33.0 min.

[0065] To a centrifuge tube, add 20.0 μL of 20.0 μg / mL Co@C3N4 nanozyme solution, 20.0 μL of clenbuterol standard solution of different concentrations, 20.0 μL of 1.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution, and 20.0 μL of 2.0 mmol / L H2O2; then add HAc-NaAc buffer with a pH of 4.0 to make the volume 200.0 μL, mix well, react for 30-40 minutes, and then perform the measurement.

[0066] After the reaction is completed, the complete reaction solution is obtained and the fluorescence intensity value F at 460nm is measured using a fluorescence spectrometer under 365nm excitation. As the CLB increases, the fluorescence intensity value F increases. Therefore, with F as the vertical axis and the concentration of CLB as the horizontal axis, the linear equation is fitted using Origin software. Figure 8 As shown, the concentration of CLB and F value are linear in the range of 0.0-76.0 μmol / L, and the regression equation is F = 1.00071C CLB+194.7896, the correlation coefficient was 0.99672, and the detection limit was 0.2858 mol / L. In addition, as the concentration of CLB increased, the solution showed a satisfactory fluorescence gradient.

[0067] After the reaction is completed, the complete reaction solution is obtained and the absorbance value A at 650nm is measured using a UV spectrophotometer. As the CLB increases, the absorbance value A increases. Therefore, with A as the ordinate and the concentration of CLB as the abscissa, the linear equation is fitted using Origin software. Figure 9 As shown, the concentration of CLB and A value are linear in the range of 0.0-76.0 μmol / L, and the regression equation is A=-0.00792C CLB +1.78149, the correlation coefficient was 0.98716, and the detection limit was 0.946 μmol / L. In addition, as the concentration of CLB increased, the solution showed a satisfactory color gradient.

[0068] After the reaction is completed, the complete reaction solution is obtained and cooled to room temperature at a power of 1.5 W / cm 2 The reaction solution was irradiated with a 660nm near-infrared laser for 240.0s and the temperature of the reaction solution was recorded using a thermal imager. As the CLB concentration decreased, the maximum temperature reached about 45.2°C. Therefore, with T as the ordinate and the CLB concentration as the abscissa, the linear equation was fitted using Origin software. Figure 10 As shown, the concentration of CLB and T value are linear in the range of 0.0-76.0 μmol / L, and the regression equation is T = -0.12087C Hx +45.11938, the correlation coefficient was 0.99313, and the detection limit was 0.273 μmol / L. In addition, as the concentration of CLB increased, the solution showed a satisfactory temperature gradient.

[0069] Example 7: Selectivity of a trimodal detection method for clenbuterol based on Co@C3N4 nanozyme

[0070] In order to evaluate the selectivity of the trimodal sensor based on Co@C3N4+H2O2+TMB system to CLB, some representative contents were used as proof of concept, including Mg 2+ , K + , Ca 2+ 、Na +, alanine, glucose, arginine, methionine, glutamic acid, aspartic acid, serine, histidine, threonine, lysine, leucine and clenbuterol. At 33°C, 3,3',5,5'-tetramethylbenzidine solution, Co@C3N4 nanozyme, and H2O2 were reacted with the above substances in HAc-NaAc buffer respectively; after mixing evenly, the reaction was carried out for 30-35 minutes and then transferred to a cuvette. The absorbance value A at 650nm, the fluorescence intensity value F at 440nm under 365nm wavelength excitation and the temperature T under 660nm laser irradiation were measured respectively. The results are shown in the figure. Figure 11 A. Figure 11 B and Figure 11 As shown in Figure C, only the absorbance value A of clenbuterol decreased significantly at temperature T, while the fluorescence intensity values ​​F and C increased significantly, indicating that the detection system has good selectivity for CLB.

[0071] In the above verification steps, the concentration of the added Co@C3N4 nanozyme was 50.0 μmol / L; the TMB concentration was 1.0 mmol / L; the H2O2 concentration was 200.0 μmol / L; the concentration of the selective verification substance was 76.0 μmol / L; the pH of the added HAc-NaAc buffer was 3.8-4.2; the total mixed solution was 200.0 μL; the reaction temperature was 35.0°C; and the mixing and sufficient reaction time was 33.0 min.

[0072] Example 8: Detection of Clenbuterol in Actual Samples Using a Trimodal Detection Method Based on Co@C3N4 Nanozyme

[0073] To verify the performance of the Co@C3N4+H2O2+TMB system for CLB detection in practical crop applications, we selected pork as a representative sample. In this work, pork was purchased from a supermarket in Anhui Province, individually vacuum-packed and with a relatively recent production date. 10.0 g of pork was minced and mixed with 20.0 mL of 0.1 mol / L hydrochloric acid in a 50.0 mL stoppered glass tube. After shaking and stirring, 20.0 mL of anhydrous ethanol was added, and ultrasonic extraction was performed for 30 minutes. The mixture was then centrifuged at 4000 rpm for 10 minutes, and the supernatant was collected into a separatory funnel. The residue was extracted again with anhydrous ethanol, and the two extracts were combined. 2 mol / L sodium hydroxide was added dropwise to the supernatant in the separatory funnel to adjust the pH to 12, followed by the addition of diethyl ether. Ether extraction was repeated three times to obtain an extract. The extract was then dehydrated by adding 1-2 g of anhydrous sodium sulfate and placed in an evaporating dish in a water bath. Finally, the resulting material was dissolved in anhydrous ethanol to form the pork extract. The solution was then mixed with 1.0, 20.0, and 40.0 ppm of clenbuterol, respectively. At room temperature, the 3,3',5,5'-tetramethylbenzidine solution, Co@C3N4 nanozyme, and H2O2 were reacted with the above mixed solutions in HAc-NaAc buffer. After mixing evenly, the mixture reacted for 33.0 minutes before being transferred to a cuvette. The absorbance A at 650 nm, the fluorescence intensity F at 440 nm under 365 nm excitation, and the temperature T under 660 nm laser irradiation were measured. The absorbance A, fluorescence intensity F, and temperature T were substituted into the corresponding linear equation.

[0074] To verify the sensing performance of the Co@C3N4+H2O2+TMB system for CLB detection in practical crop applications, we selected animal feed as a representative sample. In this work, the feed was purchased fresh, bulk, and dried from a vegetable market in Anhui Province. 5.0 g of animal feed was placed in a 100 mL Erlenmeyer flask, and 50.0 mL of 0.1 mol mol / L HCl / methanol (80:20 v / v) extraction solvent was added. After shaking, the mixture was extracted in an ultrasonic bath for 15 minutes. Manual shaking was performed every 5 minutes. After ultrasonic extraction, the mixture was centrifuged at 4000 rpm for 10.0 minutes. Then, 10.0 mL of the supernatant was placed in a 150 mL separatory funnel, and sodium hydroxide was added dropwise to adjust the pH to 11-12. Extraction was then repeated twice with 30.0 mL and 25.0 mL of diethyl ether, respectively. The ether layer was then passed through anhydrous sodium sulfate. Finally, the extract was diluted to 50 mL with diethyl ether. The final extract (25.0 mL) was added to a 50 mL beaker and evaporated to dryness in a water bath at <50°C. Finally, the obtained substance was dissolved in anhydrous ethanol as an animal feed extract. It was then mixed with 1.0, 20.0, and 40.0 ppm of clenbuterol, respectively. At room temperature, 3,3',5,5'-tetramethylbenzidine solution, Co@C3N4 nanozyme, and H2O2 were reacted with the above mixed solution in HAc-NaAc buffer; after mixing evenly, the reaction was continued for 33.0 minutes and transferred to a cuvette. The absorbance value A at 650 nm, the fluorescence intensity value F at 440 nm under 365 nm wavelength excitation, and the temperature T under 660 nm laser irradiation were measured respectively. The obtained absorbance value A, fluorescence intensity value F, and temperature T were substituted into the corresponding linear equations. The clenbuterol results measured under the three detection modes are shown in the following table.

[0075]

[0076] The results show that the trimodal system can be applied to the detection of actual samples, with a recovery rate between 95% and 105% and a relative deviation of less than 5%.

[0077] Example 9: A trimodal detection method for clenbuterol based on Co@C3N4 nanozyme,

[0078] A trimodal detection method for clenbuterol based on Co@C3N4 nanozyme comprises the following steps:

[0079] Step 1: Determine the fluorescence intensity F1 of the reaction solution containing different concentrations of clenbuterol standard at 460nm under 365nm wavelength excitation

[0080] The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was allowed to proceed for 30 minutes before the tube was transferred to a cuvette. The fluorescence intensity at 460 nm was measured using a fluorescence spectrometer under 365 nm excitation, and recorded as the fluorescence value F1.

[0081] Step 2: Determine the absorbance value A1 of the reaction solution containing different concentrations of clenbuterol standard at 650nm

[0082] The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was carried out for 30 minutes and then the solution was transferred to a cuvette. The absorbance at 650 nm was measured using a UV spectrophotometer and recorded as absorbance A1.

[0083] Step 3: Determine the temperature T1 of the reaction solution containing different concentrations of clenbuterol standard under 660nm laser irradiation:

[0084] The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was carried out for 30 minutes. After irradiation with 660 nm laser, the temperature value was measured using a portable photothermal imager and recorded as temperature T1.

[0085] Step 4: Construct a linear regression equation for the fluorescence F, absorbance A, and temperature T of the standard

[0086] The fluorescence intensity signal F was obtained by testing the clenbuterol standard with a series of concentrations, and a linear equation was constructed: F = XC CLB +Y, where C CLB is the concentration of the standard;

[0087] The absorbance signal A was obtained by testing a series of concentrations of clenbuterol standard substances, and a linear equation was constructed: A = XC CLB +Y, where C CLB is the concentration of the standard;

[0088] The temperature signal T is obtained by testing a series of concentrations of clenbuterol standard substances, and a linear equation is constructed: T = XC CLB +Y, where C CLB is the concentration of the standard;

[0089] Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the fluorescence intensity signal F n , absorbance signal A n and temperature signal T n , F n 、A n and T n Substitute into the corresponding linear equation to obtain the concentration of clenbuterol in the test sample.

[0090] A preferred embodiment is: the preparation method of the Co@C3N4 nanozyme comprises the following steps:

[0091] S1: 5.0 g of melamine was calcined at 600.0°C for 2.0 h in a muffle furnace; after cooling to room temperature, the obtained C3N4 powder was ultrasonically treated in deionized water for 12.0 h, and then freeze-dried to obtain C3N4;

[0092] S2: 10.0 mg of C3N4 obtained in step S1 was mixed with 1.0 mg of CoCl·6H2O and dissolved in 15.0 mL of DI. After stirring for 1.0 h, the mixture was transferred to a high-temperature reactor and reacted at 130.0°C for 3.0 h. After the reaction, the mixture was washed with ethanol and centrifuged in a centrifuge at 12000 r for 3.0 min, repeated three times. The precipitate was collected and dried in a constant temperature vacuum oven at 60.0°C overnight to obtain Co@C3N4 nanozyme.

[0093] The preferred embodiment is: the concentration of the Co@C3N4 nanozyme solution is 50.0 μg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 1.0 mmol / L; in steps 1, 2 and 3, 20.0 μL of 20.0 μg / mL Co@C3N4 nanozyme solution, 20.0 μL of clenbuterol standard solution of different concentrations, 20.0 μL of 1.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution, and 20.0 μL of 2.0 mmol / L H2O2 are added to a centrifuge tube; then, HAc-NaAc buffer with a pH of 4.0 is added to the volume to 200.0 μL, mixed evenly and reacted for 30-40 minutes, and then the measurement is performed.

[0094] The preferred embodiment is: in step 4, the regression equation of the fluorescence signal F is F=1.00071XC CLB +194.7896; the regression equation of absorbance signal A is A=-0.00792C CLB +1.78149; the regression equation of temperature signal T is T=-0.12087C CLB +45.11938.

[0095] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. A trimodal detection method for clenbuterol based on Co@C3N4 nanozyme, characterized by: The following steps are involved: Step 1: Determine the fluorescence intensity F1 of the reaction solution containing different concentrations of clenbuterol standard at 460nm under 365nm wavelength excitation The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was allowed to proceed for 30-40 minutes before the tube was transferred to a cuvette. The fluorescence intensity at 460 nm was measured using a fluorescence spectrometer under 365 nm excitation, and recorded as the fluorescence value F1. Step 2: Determine the absorbance value A1 of the reaction solution containing different concentrations of clenbuterol standard at 650nm The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was carried out for 30-40 minutes and then the solution was transferred to a cuvette. The absorbance at 650 nm was measured using a UV spectrophotometer and recorded as absorbance A1. Step 3: Determine the temperature T1 of the reaction solution containing different concentrations of clenbuterol standard under 660nm laser irradiation: The Co@C3N4 nanozyme solution was placed in a centrifuge tube, and different concentrations of clenbuterol standard solution, 3,3',5,5'-tetramethylbenzidine solution, and H2O2 were added. Then, HAc-NaAc buffer was added and the volume was adjusted to 200.0 μL. After mixing evenly, the reaction was carried out for 30-40 minutes. After irradiation with 660 nm laser, the temperature value was measured using a portable photothermal imager and recorded as temperature T1. Step 4: Construct a linear regression equation for the fluorescence F, absorbance A, and temperature T of the standard The fluorescence intensity signal F was obtained by testing the clenbuterol standard with a series of concentrations, and a linear equation was constructed: F = XC CLB +Y, where C CLB is the concentration of the standard; The absorbance signal A was obtained by testing a series of concentrations of clenbuterol standard substances, and a linear equation was constructed: A = XC CLB +Y, where C CLB is the concentration of the standard; The temperature signal T is obtained by testing a series of concentrations of clenbuterol standard substances, and a linear equation is constructed: T = XC CLB +Y, where C CLB is the concentration of the standard; Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the fluorescence intensity signal F n , absorbance signal A n and temperature signal T n , F n 、A n and T n Substitute into the corresponding linear equation to obtain the concentration of clenbuterol in the test sample.

2. The trimodal detection method for clenbuterol based on Co@C3N4 nanozyme according to claim 1, characterized in that: The preparation method of the Co@C3N4 nanozyme comprises the following steps: S1: 5.0 g of melamine was calcined at 600.0°C for 2.0 h in a muffle furnace; after cooling to room temperature, the obtained C3N4 powder was ultrasonically treated in deionized water for 12.0 h, and then freeze-dried to obtain C3N4; S2: 10.0 mg of C3N4 obtained in step S1 and 1.0 mg of CoCl·6H2O were mixed and dissolved in 15.0 mL of DI. After stirring for 1.0 h, the mixture was transferred to a high-temperature reactor and reacted at 130.0°C for 3.0 h. After the reaction, the mixture was washed with ethanol and centrifuged in a centrifuge at 12000 r for 3.0 min. This was repeated three times. The precipitate was collected and placed in a constant temperature vacuum drying oven at 60.0°C overnight to obtain Co@C3N4 nanozyme.

3. The trimodal detection method for clenbuterol based on Co@C3N4 nanozyme according to claim 1, characterized in that: The concentration of the Co@C3N4 nanozyme solution is 20.0 μg / mL; the concentration of the 3,3',5,5'-tetramethylbenzidine solution is 1.0 mmol / L; the reaction temperature is 35.0°C; the reaction time after mixing evenly is 33.0 min; in steps 1, 2 and 3, 20.0 μL of 20.0 μg / mL Co@C3N4 nanozyme solution, 20.0 μL of clenbuterol standard solutions of different concentrations, 20.0 μL of 1.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution, and 20.0 μL of 2.0 mmol / L H2O2 are added to a centrifuge tube; then HAc-NaAc buffer with a pH of 4.0 is added to the volume to 200.0 μL, mixed evenly and reacted for 30-40 minutes, and then the measurement is performed.

4. The trimodal detection method for clenbuterol based on Co@C3N4 nanozyme according to claim 1, characterized in that: In step 4, the regression equation of the fluorescence signal F is F = 1.00071XC CLB +194.7896; the regression equation of absorbance signal A is A=-0.00792C CLB +1.78149; the regression equation of temperature signal T is T=-0.12087C CLB +45.11938.

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