A trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch

Abstract

A trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch, comprising the following steps: Step 1: Determine the absorbance value A of the mixed reaction solution of ethyl carbamate at 650nm n Step 2: Under the excitation wavelength of 365nm, the fluorescence intensity value F of the mixed reaction solution of ethyl carbamate at 440nm is measured. n Step 3: Determine the temperature T of the mixed reaction solution of ethyl carbamate under 660nm laser irradiation n :Step 4: Construct a linear regression equation for the concentration of ethyl carbamate; Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the absorbance signal of the colorimetric mode, the fluorescence intensity signal of the fluorescence mode and the temperature signal T of the photothermal mode n The synthesis process of Co@MOF‑MIP nanozyme is relatively simple, the raw materials are readily available and low-cost, and it does not require complex processes and expensive equipment. It has high stability and high peroxidase activity and fluorescence properties, which meet the needs of subsequent multimodal detection of EC.

Description

Technical Field

[0001] The present invention relates to a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of a Co@MOF-MIP enzyme activity switch, and belongs to the technical field of food detection. Background Art

[0002] Ethyl carbamate (EC) is a byproduct of fermentation and is widely found in fermented foods such as fermented bean curd and soy sauce, as well as alcoholic beverages such as baijiu, wine, beer, sake, and rice wine. EC is primarily ingested through the consumption of foods containing it or through long-term exposure during the production process. Although EC is not considered a highly toxic substance, long-term exposure or excessive intake may increase the risk of certain diseases. Studies have shown that long-term exposure or excessive intake of EC may increase the risk of lung cancer, lymphoma, skin cancer, and other diseases. To prevent EC contamination of food and mitigate potential threats to public health, there is an urgent need to develop efficient detection methods that can provide real-time warnings of EC presence and contamination, as well as conduct traceability analysis to identify its source and distribution pathways. Such measures can safeguard the healthy development of the environment and food-related industries, while also protecting public health and safety.

[0003] Commonly used methods for detecting ethyl carbamate include gas chromatography, liquid chromatography, and mass spectrometry. These methods can be used to quickly and accurately detect the presence and concentration of ethyl carbamate, contributing to environmental and food safety. However, these methods may have drawbacks, such as requiring expensive equipment and specialized technicians to operate, a time-consuming detection process, and some methods requiring complex sample pretreatment steps, which can introduce errors or increase costs. Furthermore, some methods may have significant environmental impacts, such as the use of organic solvents, which may generate volatile organic compound (VOC) contamination. Therefore, further research and improvement of ethyl carbamate detection methods are needed to overcome these shortcomings and improve the accuracy, efficiency, and environmental friendliness of detection. Molecular imprinting technology is a method for the recognition and capture of specific molecules, similar to the principle of matching a lock and key. This technology uses specific molecules to form channels or grooves in a polymer matrix, effectively capturing and identifying only target molecules with a matching structure. Summary of the Invention

[0004] The present invention aims to provide an efficient and accurate trimodal detection method for ethyl carbamate using a molecularly imprinted Co@MOF-MIP nanozyme with high peroxidase activity. The Co@MOF-MIP nanozyme exhibits peroxidase activity and, due to its TPE doping, exhibits blue fluorescence at 440 nm under 365 nm excitation light. In the presence of H₂O₂, the high peroxidase activity of the Co@MOF-MIP nanozyme catalyzes the H₂O₂ to produce hydroxyl radicals, which oxidize the colorless TMB to blue ox-TMB, which exhibits a significant UV absorption signal at 650 nm. Under 660 nm laser irradiation, ox-TMB produces a typical photothermal effect, resulting in a dramatic temperature increase. Simultaneously, the generated ox-TMB quenches the blue fluorescence of the Co@MOF-MIP through an internal filter effect (IFE), reducing the fluorescence intensity at 440 nm. However, when EC is present, EC specifically binds to the molecularly imprinted sites on Co@MOF-MIP, reducing the contact area between H2O2 and Co@MOF-MIP and leading to a decrease in the enzymatic activity of Co@MOF-MIP. This blocks the formation of ox-TMB and results in an EC concentration-dependent decrease in the blue colorimetric signal, a decrease in the absorption peak at 650 nm, a decrease in the temperature rise under 660 nm laser irradiation, and an increase in the blue fluorescence color signal and the fluorescence peak intensity at 440 nm. Combining a smartphone equipped with the Color Picker App and a portable handheld thermal imager, a trimodal detection method for ethyl carbamate based on molecular imprinting to regulate the enzymatic activity switch of Co@MOF-MIP can be developed.

[0005] To achieve the above and other related objectives, the present invention provides a technical solution: a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch, comprising the following steps:

[0006] Step 1: Determine the absorbance value A of the mixed reaction solution containing different concentrations of ethyl carbamate at 650 nm n The colorimetric signal is collected using a smartphone with the Color Picker App installed, converted into RGB values, and recorded as B / R in colorimetric mode. n (C):

[0007] 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0-18.0 min, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, which was recorded as the absorbance value A. n Next, the colorimetric signal of the solution was captured using a smartphone equipped with the Color Picker App, converted into RGB values, and the B / R ratio was recorded. n (C);

[0008] Step 2: Under the excitation wavelength of 365 nm, the fluorescence intensity value F of the mixed reaction solution containing different concentrations of ethyl carbamate at 440 nm was measured. n The fluorescence signal was collected using a smartphone with the Color Picker App installed, converted into RGB values, and recorded as B / R in fluorescence mode. n (F):

[0009] 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0-18.0 min, the solution was transferred to a fluorescence cuvette and the fluorescence intensity at 440 nm was measured using a fluorescence spectrophotometer under excitation at a wavelength of 365 nm, which was recorded as the fluorescence value F n Then, under the excitation of 365nm wavelength, the fluorescence signal of the solution was captured using a smartphone installed with the Color Picker App, converted into RGB values, and recorded as B / R n (F);

[0010] Step 3: Determine the temperature T of the mixed reaction solution containing different concentrations of ethyl carbamate under 660nm laser irradiation n :

[0011] 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0-18.0 min, the solution was irradiated with a 660 nm laser and the temperature was measured using a portable photothermal imager, which was recorded as the temperature T n ;

[0012] Step 4: Construct the relationship between urethane concentration and absorbance A in colorimetric mode n , B / R n (C), F in fluorescence mode n , B / R n (F) and the temperature T in the photothermal mode n The linear regression equation is:

[0013] The absorbance signal A was obtained by testing the standard sample with a series of urethane concentrations. n and B / R n (C), construct the linear equation, A n =XC EC + y and B / R n (C) = XC EC + y, where C EC is the concentration of ethyl carbamate standard;

[0014] The fluorescence intensity signal F was obtained by testing the standard sample with a series of urethane concentrations. n and B / R n (F), construct the linear equation, F n =XC EC + y and B / R n (F) = XC EC + y, where C EC is the concentration of ethyl carbamate standard;

[0015] The temperature signal T is obtained by testing the standard sample with a series of ethyl carbamate concentrations n , construct the linear equation, T n =XC EC + y, where C EC is the concentration of ethyl carbamate standard;

[0016] Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the colorimetric mode absorbance signal An and B / R n (C) Fluorescence intensity signal F in fluorescence mode n and B / R n (F) and the temperature signal T of the photothermal mode n , A n and B / R n (C), F n and B / R n (F) and T n Substitute into the corresponding linear equation to obtain the concentration of ethyl carbamate in the sample to be tested.

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

[0018] S1: Dissolve Co(NO₃)₂·6H₂O and terephthalic acid in N,N-dimethylformamide, transfer to a high-temperature reactor, and react at 100-120°C for 10-30 hours. After cooling to room temperature, centrifuge at 10,000-14,000 rpm for 5-15 minutes, wash with DMF and ultrapure water, and then lyophilize into a powder for later use, thereby obtaining the Co@MOF nanozyme.

[0019] S2: Add Co@MOF nanozyme, tetraphenylethylene and 0.2 mmol / L ethyl carbamate to a flask, add ethanol, and sonicate until dissolved; then put carboxyethylsilanetriol sodium salt and silicon tetraacetate into the flask, stir continuously for 25-40 minutes under dark conditions, add ammonia water to the mixture, and stir continuously at 20-30℃ overnight; the precipitate is washed with ethanol, centrifuged and the supernatant is discarded; the original ethyl carbamate template in the precipitate is extracted by methanol Soxhlet extraction to obtain Co@MOF-MIP sol-gel; the polymer is dried to obtain Co@MOF-MIP nanozyme.

[0020] The preferred technical solution is: the ratio of Co(NO3)2·6H2O, terephthalic acid and N,N-dimethylformamide is: 0.15-0.4g: 0.1-0.2g: 30-60mL.

[0021] The preferred technical scheme is: in the final synthesis system, the ratio of Co@MOF, tetraphenylethylene, ethyl carbamate solution and ethanol is: 0.01-0.08 g: 0.01-0.1 g: 15-20 mg: 5-15 mL; the concentration of ethyl carbamate solution is 0.2 mmol / L; the amount of carboxyethylsilanetriol sodium salt added is 200.0 μL, and the amount of silicon tetraacetate added is 200.0 μL; the concentration of carboxyethylsilanetriol sodium salt is 0.8 mmol / L; and the concentration of silicon tetraacetate is 2.4 mmol / L.

[0022] The preferred technical solution is: in step 4, the absorbance signal A n and B / R n The regression equation for (C) is A n = -0.00261C EC + 1.58196 and B / R n (C) = -0.00386C EC + 1.89281; fluorescence intensity signal F n and B / R n The regression equation of (F) is F n = 0.00186 C EC + 0.44144 and B / R n (F) = 0.01298C EC + 2.25309; Temperature signal T n The regression equation is T n = -0.03572C EC +44.25262.

[0023] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0024] 1. The synthesis process of Co@MOF-MIP nanozyme is relatively simple, the raw materials are easily available and low-cost, and it does not require complex processes and expensive equipment. It has high stability and high peroxidase activity and fluorescence properties, which meets the needs of subsequent multimodal detection of EC.

[0025] 2. Compared with traditional EC detection methods, this method is simple and convenient to use, eliminating the need for expensive equipment and specialized technicians, complex pretreatment steps, or hazardous chemicals. Furthermore, this method is the first known trimodal EC detection method. Multimodal detection offers certain advantages. It can combine multiple detection technologies or methods, overcoming the limitations of a single detection method and improving the comprehensiveness and accuracy of detection. Multimodal detection can also verify results through cross-correlation, enhancing detection credibility. Furthermore, multimodal detection can provide more comprehensive data, enabling a deeper understanding of sample characteristics and potential changes, better meeting practical testing needs.

[0026] 3. In particular, compared with the team's previous published patent number CN117607131, the detection time of the present invention is shorter. Compared with its detection time of 35.0 min, this method only takes about 16.0 min to detect EC through three signals: colorimetry, fluorescence, and photothermal. In addition, the present invention has a larger detection range of 4.13-300.0 μmol / L compared to the previous 4.96-200.0 μg / L. At the same time, no toxic Br2 is required to treat EC during the experiment, which is more environmentally friendly, and no expensive and unstable biological enzyme acetylcholinesterase is required, which is more economical. What is more noteworthy is that the previous method required a simple pretreatment of the sample under high alcohol concentration, while the present method does not require cumbersome alcohol concentration pretreatment under high alcohol concentration, and can directly detect actual samples with higher alcohol content. Compared with the previous colorimetric and fluorescence dual-mode detection method, this three-mode method adds photothermal detection. The photothermal output can be directly read using a portable and affordable thermometer. It is user-friendly and easy to operate, and it is easy to miniaturize the device. Since temperature changes are mainly caused by the photothermal effect, the photothermal sensor has a low background and can therefore solve the color resolution limitations of colorimetry and autofluorescence.

[0027] 4. The present invention can be used to perform portable detection of EC by combining a smartphone with the Color Picker App installed and a portable handheld thermal imager without being restricted by time, location or professional operators.

[0028] 5. The present invention has been successfully applied to the specific detection of EC in actual samples. Co@MOF-MIP has a high affinity for EC through its three-dimensional cavity with high adaptability to EC molecules, excluding representative substrates in fermented foods and EC structural analogs such as urea, lactic acid, ethyl lactate, isopentanol, arginine, glutamic acid, glycine, Mg 2+ 、Zn 2+ and Cu 2+ It has good selectivity and anti-interference performance. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 Schematic diagram of a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch.

[0030] Figure 2 Characterization diagram of Co@MOF-MIP nanozyme. (A) TEM image of Co@MOF-MIP; (B) elemental spectrum of Co@MOF-MIP (gold mesh used for high-resolution transmission electron microscopy); (CF) corresponding elemental maps contained in Co@MOF-MIP.

[0031] Figure 3For enzyme activity verification (1) Co@MOF + H2O2, (2) Co@MOF + H2O2 + TMB, (3) Co@MOF-MIP + H2O2, (4) Co@MOF-MIP + H2O2 + TMB, and (5) H2O2 + TMB absorbance of different groups and the corresponding color illustrations.

[0032] Figure 4 This is the double reciprocal graph corresponding to the Co@MOF-MIP nanozyme catalytic kinetics experiment.

[0033] Figure 5 Feasibility analysis of a colorimetric and fluorescence detection method for trimodal detection of ethyl carbamate based on molecular imprinting-regulated Co@MOF-MIP enzyme activity switch, as well as the corresponding colorimetric and fluorescence color illustrations. (1) H2O2 + TMB, (2) Co@MOF-MIP, (3) Co@MOF-MIP + H2O2, (4) Co@MOF-MIP + H2O2 + TMB, (5) Co@MOF-MIP + H2O2 + TMB + EC.

[0034] Figure 6 The feasibility of a photothermal method for the three-modal detection of ethyl carbamate based on molecular imprinting to regulate the enzyme activity switch of Co@MOF-MIP and the optimization of 660 nm laser irradiation time were analyzed. (1) Co@MOF-MIP + H2O2 + TMB, (2) Co@MOF-MIP + H2O2 + TMB + 660 nm near-infrared laser irradiation (1.5 W / cm 2 ), (3) Co@MOF-MIP+ H2O2 + TMB + EC, (4) Co@MOF-MIP + H2O2 + TMB + EC + 660 nm near-infrared laser irradiation (1.5 W / cm 2 ).

[0035] Figure 7 The pH and temperature of the detection system were optimized for a trimodal detection method of ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch.

[0036] Figure 8 The time optimization of the detection system for a trimodal detection method of ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch.

[0037] Figure 9 These are pictures of the changes in the solution's color, fluorescence color, and temperature as EC concentration increases under the three modes.

[0038] Figure 10 The absorption spectra at 650 nm and the standard curve corresponding to different concentrations of ethyl carbamate in the system under colorimetric mode, as well as the B / R of the color RGB n (C) Standard curve.

[0039] Figure 11 The fluorescence intensity at 440 nm corresponding to different concentrations of ethyl carbamate in the system under fluorescence mode and the standard curve as well as the B / R of the fluorescence color RGB n (F) Standard curve.

[0040] Figure 12 These are the temperatures and standard curves corresponding to different concentrations of ethyl carbamate in the system under photothermal mode.

[0041] Figure 13 This is a selective verification of a three-modal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch. 1-12, EC, urea, lactic acid, ethyl lactate, isopentanol, arginine, glutamic acid, glycine, Mg 2+ 、Zn 2+ 、Cu 2+ and blank.

[0042] Figure 14 This is a three-modal detection method for ethyl carbamate based on molecular imprinting to regulate the enzyme activity switch of Co@MOF-MIP. 1-11 are blank, urea, lactic acid, arginine, glutamic acid, ethyl lactate, isopentanol, glycine, Mg 2+ 、Zn 2+ and Cu 2+ .

[0043] Figure 15 The effect of alcohol concentration on the three-modal detection system. DETAILED DESCRIPTION

[0044] 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.

[0045] See also Figure 1-15. 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.

[0046] The technical solution of the present invention is further described in detail below with reference to examples.

[0047] The raw materials used in this invention: tetraphenylethylene (TPE), carboxyethylsilanetriol sodium salt (CEST), tetraacetic acid (TEOS), terephthalic acid (TA), N,N-dimethylformamide (DMF), cobalt chloride hexahydrate, and 3,3',5,5'-tetramethylbenzidine (TMB) were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; acetic acid-sodium acetate buffer was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; 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.

[0048] Example 1: Preparation method of Co@MOF-MIP nanozyme

[0049] First, 0.29 g of Co(NO₃)₂·6H₂O and 0.165 g of terephthalic acid (TA) were dissolved in 40 mL of N,N-dimethylformamide (DMF) and then transferred to a high-temperature reactor for reaction at 110.0°C for 20.0 h. After cooling to room temperature, the mixture was centrifuged at 12,000 rpm for 10.0 min, washed three times with DMF and ultrapure water, and then freeze-dried to a powder for later use, yielding the Co@MOF nanozyme. Next, 0.05 g of Co@MOF, 0.05 g of tetraphenylethylene (TPE), and 0.2 mmol / L EC (17.82 mg) were added to a 25 mL flask. 10.0 mL of ethanol was added and sonicated until dissolved. Then 0.8 mmol / L of carboxyethylsilanetriol sodium salt CEST (functional monomer) and 2.4 mmol / L of tetraacetic acid silicon TEOS (cross-linker) were placed in a flask, and after continuous stirring for 30.0 min under dark conditions, 200.0 μL of ammonia water was added to the mixture and continued stirring at 25 °C overnight. The precipitate was washed with ethanol, centrifuged and the supernatant was discarded. The original EC template in the precipitate was extracted by methanol Soxhlet extraction to obtain Co@MOF-MIP sol-gel. The polymer was dried in a vacuum oven at 60 °C overnight to obtain Co@MOF-MIP nanozyme. The TEM image of the synthesized nanozyme is shown below. Figure 2 shown.

[0050] Example 2: Verification of peroxidase activity of Co@MOF-MIP nanozyme

[0051] Use H2O2 as substrate, TMB as colorimetric reagent, and HAc-NaAc as buffer. Take the following reaction groups: (1) Co@MOF + H2O2, (2) Co@MOF + H2O2 + TMB, (3) Co@MOF-MIP + H2O2, (4) Co@MOF-MIP + H2O2 +TMB, (5) H2O2 + TMB. After sufficient reaction, transfer to a cuvette and measure the absorbance at 650 nm using a UV spectrophotometer. Figure 3 As shown, only the combinations of Co@MOF + H2O2 + TMB and Co@MOF-MIP + H2O2 + TMB had larger absorbance values, indicating the high peroxidase activity of Co@MOF-MIP, and this activity originated from the Co@MOF precursor. The enzyme activity was slightly reduced during the elution process of molecular imprinting.

[0052] In the above verification steps, the concentration of Co@MOF nanozyme and Co@MOF-MIP nanozyme added was 50.0 μg / mL; the concentration of TMB solution was 1.0 mmol / L; the total volume of the mixed solution was 200.0 μL; the concentration of H2O2 solution was 200.0 μmol / L; the reaction temperature was 40°C; the reaction time was 16 min; and the pH value of the HAc-NaAc buffer was 4.

[0053] Example 3: Catalytic kinetics test of Co@MOF-MIP nanozyme

[0054] Steady-state kinetic experiments were conducted by varying the concentration of H2O2 (0.0 - 200.0 μmol / L) or TMB (0.0 - 1.0 mmol / L) while maintaining the concentration of the other substance. Co@MOF-MIP nanozyme was added, and the volume was fixed to 200.0 μL with HAc-NaAc buffer. After complete reaction, the reaction was transferred to a cuvette, and the absorbance peak at 650 nm was measured using a UV spectrophotometer. The reversed-contrast plots of H2O2 concentration and reaction rate, and TMB concentration and reaction rate, were calculated using the following Michaelis-Menten equation:

[0055]

[0056] 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@MOF-MIP to H2O2 and TMB is shown m The K values ​​were 2.91 and 0.20, which were significantly higher than those of natural horseradish peroxidase HPR. m The values ​​are comparable or even lower, indicating that the Co@MOF-MIP nanozyme prepared in the present invention has good catalytic activity.

[0057] In the above verification steps, the concentration of Co@MOF nanozyme and Co@MOF-MIP nanozyme added was 50.0 μg / mL; the concentration of TMB solution was 1.0 mmol / L; the total volume of the mixed solution was 200.0 μL; the concentration of H2O2 solution was 200.0 μmol / L; the reaction temperature was 40°C; the reaction time was 16 min; and the pH value of the HAc-NaAc buffer was 4.

[0058] Example 4: Feasibility analysis of a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch

[0059] Take the following reaction groups: (1) H2O2 + TMB, (2) Co@MOF-MIP, (3) Co@MOF-MIP + H2O2, (4) Co@MOF-MIP + H2O2 + TMB, (5) Co@MOF-MIP + H2O2 + TMB + EC. After sufficient reaction, transfer to a cuvette and measure the absorbance at 650 nm using a UV spectrophotometer. The results are as follows: Figure 5 A and Figure 5 As shown in Figure B, only the Co@MOF-MIP + H2O2 + TMB and Co@MOF-MIP + H2O2 + TMB + EC groups showed significant absorbance and color changes, but the color and absorbance intensity of the latter were both smaller than those of the former. This indicates that the peroxidase activity of Co@MOF-MIP is inhibited after the addition of EC, demonstrating the feasibility of colorimetric detection of EC.

[0060] Take the above reaction solution and measure the fluorescence intensity at 440 nm using a fluorescence spectrophotometer under 365 nm excitation light. Figure 5 C and Figure 5 As shown in Figure D, the fluorescence intensities of both the Co@MOF-MIP + H2O2 + TMB and Co@MOF-MIP + H2O2 + TMB + EC groups were inhibited. This is because the oxidation product of TMB, oxTMB, can quench the fluorescence of Co@MOF-MIP. However, the fluorescence intensity of the latter was significantly restored after the addition of EC, indicating the feasibility of fluorescence detection of EC.

[0061] The following four groups were tested: (1) Co@MOF-MIP + H2O2 + TMB, (2) Co@MOF-MIP + H2O2 + TMB + 660 nm near-infrared laser irradiation (1.5 W / cm 2 ), (3) Co@MOF-MIP + H2O2 + TMB + EC, (4) Co@MOF-MIP + H2O2 + TMB + EC + 660 nm near-infrared laser irradiation (1.5 W / cm 2 ). The result is as follows Figure 6 As shown, only groups (2) and (4) have significant temperature changes, and after adding EC, the temperature change of (4) is lower than that of (2) without EC. This is because the addition of EC reduces the peroxidase activity of Co@MOF-MIP, reduces the oxTMB product, and reduces the photothermal signal, showing the feasibility of photothermal detection of EC. At the same time, after an irradiation time of 150.0 s, the temperature increase approaches saturation. Therefore, an irradiation time of 150.0 s is selected as the optimal irradiation time for the photothermal mode.

[0062] Example 5: Optimization of the overall reaction conditions for a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch

[0063] In order to optimize the analytical performance of the trimodal system, we optimized the experimental conditions of the detection system, such as pH value, temperature and reaction time. First, we optimized the pH value of the system. Since the chromogenic substrate is TMB, we selected NaAc-HAc as the reaction buffer, which is compatible with it. The results are shown in the figure below. Figure 7 A and Figure 7 As shown in Figure B, when the pH value is in the range of 3.5 to 4.0, the absorbance of the reaction system at 650 nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 4.0, the absorbance gradually decreases as it increases, so 4.0 is selected as the optimal pH value of the system. The catalytic performance of the system was then investigated in the temperature range of 30.0 ℃ - 50.0 ℃. The results are shown in Figure 2. Figure 7 C and Figure 7 As shown in D, when the temperature range is between 30.0°C and 40.0°C, the absorbance value at 650 nm of the reaction system increases with the increase of reaction temperature. When the temperature exceeds 400°C and increases to 50.0°C, the absorbance difference gradually decreases as it increases. Therefore, 40.0°C is selected as the optimal temperature for this detection system. Finally, we optimized the reaction time of the system, and the results are shown in the figure below. Figure 8 As shown in the figure, the absorbance at 650 nm of the reaction system increases with reaction time from 0.0 to 16.0 min, remaining essentially unchanged after 16.0 min. Therefore, 16.0 min was selected as the optimal reaction time for this system. Furthermore, a comparison revealed a significant difference in absorbance between the reaction with and without EC, which gradually increased over time, further demonstrating the feasibility of this system for EC detection.

[0064] Example 6: A trimodal detection method for ethyl carbamate based on molecular imprinting to regulate the Co@MOF-MIP enzyme activity switch to detect ethyl carbamate standards

[0065] First, EC solutions were prepared at concentrations of 0.0, 1.0, 10.0, 20.0, 40.0, 80.0, 120.0, 160.0, 200.0, 250.0, 300.0, and 400.0 μmol / L, respectively. Then, 50.0 μg / mL of Co@MOF-MIP nanozyme and 1.0 mmol / L of TMB solution were added, and the volume was adjusted to 200.0 μL with NaAc-HAc at pH 4.0. The reaction was completed at 40.0°C for 16.0 min.

[0066] The amount of urethane standard solution with different concentrations added was 20.0 μL, the amount of 1.0 mg / mL Co@MOF-MIP nanozyme solution added was 10.0 μL, the amount of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution added was 10.0 μL, and the amount of 2.0 mmol / L H2O2 added was 20.0 μL.

[0067] After the reaction is completed, the complete reaction solution is transferred to a cuvette and the absorbance value A at 650nm is measured using a UV spectrophotometer. As the concentration of EC increases, the absorbance value A decreases. Therefore, with A as the ordinate and the concentration of EC as the abscissa, the linear equation is fitted using Origin software. Figure 10 A and Figure 10 As shown in B, the concentration of EC and A values ​​are linear in the range of 5.93 - 300.0 μmol / L, and the regression equation is A = -0.00261 C EC + 1.58196, the correlation coefficient was 0.99056, and the detection limit was 1.78 μmol / L. Figure 9 As shown in the figure, as the concentration of EC increases, the blue color of the solution decreases gradually. Then, a smartphone with the Color Picker App installed was used to capture the color signal of the solution and convert it into RGB values. With B / R(C) as the vertical axis and the concentration of EC as the horizontal axis, a linear equation was fitted using Origin software. Figure 10 As shown in C, the EC concentration and B / R(C) value are linear in the range of 4.67 - 300.0 μmol / L, and the regression equation is B / R(C) = -0.00386C EC The correlation coefficient was 0.99697, and the detection limit was 1.64 μmol / L.

[0068] After the reaction is completed, the complete reaction solution is transferred to a fluorescent dish and the fluorescence intensity F at 440 nm is measured using a fluorescence spectrometer under 365 nm excitation. As the concentration of EC increases, the fluorescence intensity F at 440 nm increases. Therefore, with F as the vertical axis and the concentration of EC as the horizontal axis, a linear equation is fitted using Origin software. Figure 11 A and Figure 11 As shown in Figure B, the EC concentration and F value are linear in the range of 4.37 - 300.0 μmol / L, and the regression equation is F = 0.00186 C EC +0.44144, the correlation coefficient was 0.99079, and the detection limit was 1.31 μmol / L. Figure 9As shown in the figure, as the concentration of EC increases, the solution shows a gradient of blue fluorescence color. Then, under the excitation of 365nm wavelength, the fluorescence signal of the solution is captured by a smartphone installed with ColorPicker App and converted into RGB value. With B / R(F) as the vertical axis and EC concentration as the horizontal axis, the linear equation is fitted using Origin software. Figure 11 As shown in C, the concentration of EC and B / R(F) values ​​are linear in the range of 4.13 - 300.0 μmol / L, and the regression equation is B / R(F) = 0.01298C EC The correlation coefficient was 0.99182, and the detection limit was 1.24 μmol / L.

[0069] After the reaction is completed, the reaction solution is cooled to room temperature using a metal bath or water bath, and then transferred to a centrifuge tube. 2 The reaction solution was irradiated with a 660 nm near-infrared laser for 150.0 s, and the temperature of the reaction solution was recorded using a portable handheld thermal imager. As the concentration of EC increased, the upward trend of the solution temperature T decreased. Therefore, a linear equation was fitted using Origin software, with temperature T as the ordinate and EC concentration as the abscissa. Figure 12 As shown, the concentration of EC and temperature T values ​​are linear in the range of 5.93 - 300.0 μmol / L, and the regression equation is T = -0.03572C EC + 44.25262, the correlation coefficient was 0.99324, and the detection limit was 1.78 μmol / L. Figure 9 As shown in Figure 3, as the concentration of EC increases, the solution presents a satisfactory temperature decrease gradient.

[0070] Example 7: Selectivity of a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch

[0071] In order to evaluate the selectivity of EC detection based on this trimodal system, we selected some representative substrates in fermented foods and EC structural analogs such as urea, lactic acid, ethyl lactate, isopentanol, arginine, glutamic acid, glycine, Mg 2+ 、Zn 2+ and Cu 2+ As a proof of concept, three-modal quantitative detection, colorimetric, fluorescence and photothermal, was performed. Figure 13As shown, these reagents do not cause obvious colorimetric, fluorescence and photothermal changes compared with EC. This shows that the proposed trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch has good selectivity in detecting EC. The obvious selectivity of EC can be estimated by the high affinity of the three-dimensional cavities that are highly adaptable to EC molecules to EC, which can distinguish them by different functional groups, sizes and shapes. Despite having a similar structure to EC, the analogs cannot be captured by the recognition sites on the surface of the nanomaterial. In addition, the anti-interference ability of this proposed sensing platform to the representative reagents of the above-mentioned coexisting substrates was also explored. The results are shown in Figure 14 The results show that the colorimetric, fluorescence and photothermal changes of EC triggered by each coexisting reagent are not obvious, indicating that the proposed trimodal detection method of ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch is anti-interference in the detection of EC and can be used for the detection of EC in actual samples.

[0072] Example 8: Effect of alcohol concentration on the detection system of a trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch.

[0073] In order to verify the effect of alcohol concentration on the trimodal detection system, we prepared solutions with 0-70% alcohol concentration to dissolve EC and conduct detection and verification. 20.0 μL of EC solutions with different alcohol concentrations were mixed with 50.0 μg / mL Co@MOF-MIP nanozyme, 1.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution (TMB) and 200.0 μmol / L H2O2 in a centrifuge tube. Then, HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After complete reaction for 14.0-18.0 min, the solution was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer. The results are shown as follows: Figure 15 As shown, the absorbance values ​​of various alcohol concentrations are almost the same, indicating that alcohol concentration has almost no effect on this experiment. Actual samples containing high concentrations of alcohol can be directly detected using this system.

[0074] Example 9: Detection of ethyl carbamate in actual samples using a trimodal detection method based on molecular imprinting to regulate the Co@MOF-MIP enzyme activity switch

[0075] To verify the EC detection performance of this trimodal system in real samples, we selected 187 ml of Orlan Ao Tailang red wine as the test sample. The wine, purchased at the Zhonghuancheng Shopping Center in Hefei, China, had an alcohol content of 12.5% ​​and was stored in a room-temperature cabinet. 20.0 μL of the purchased wine was mixed with 50.0 μg / mL of Co@MOF-MIP nanozyme, 1.0 mmol / L of 3,3',5,5'-tetramethylbenzidine (TMB) solution, and 200.0 μmol / L of H2O2 in a centrifuge tube. HAc-NaAc buffer with a pH of 3.8-4.2 was then added to the mixture, bringing the volume to 200.0 μL. After complete reaction for 14.0-18.0 min, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, recorded as the absorbance value (A). Next, the colorimetric signal of the solution was captured using a smartphone equipped with the Color Picker app, converted to RGB values, and the B / R(C) ratio was recorded. These results were then substituted into the corresponding linear equations. Next, the fluorescence intensity (F) of the mixed reaction solution at 440 nm was measured under 365 nm excitation. The fluorescence signal was collected using a smartphone equipped with the Color Picker app and converted to RGB values, recorded as B / R(F) in fluorescence mode. These results were then substituted into the corresponding linear equations. Finally, after irradiation with a 660 nm laser, the temperature was measured using a portable photothermal imager, recorded as T, and the results were substituted into the corresponding linear equations. The EC concentrations in the red wine obtained under the three detection modes were 15.86 μg / L, 16.47 μg / L, 16.22 μg / L, 16.37 μg / L, and 15.44 μg / L, respectively.

[0076] To verify the EC detection performance of this trimodal system in real samples, we selected Tsingtao beer as the actual test sample. The beer in this work was purchased from the Zhonghuancheng Shopping Center in Hefei, China, with an alcohol content of 4%, and stored in a room temperature cabinet. 20.0 μL of the purchased beer was mixed with 50.0 μg / mL of Co@MOF-MIP nanozyme, 1.0 mmol / L of 3,3',5,5'-tetramethylbenzidine (TMB) solution, and 200.0 μmol / L of H2O2 in a centrifuge tube. HAc-NaAc buffer with a pH of 3.8-4.2 was then added to a constant volume of 200.0 μL. After complete reaction for 14.0-18.0 min, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, recorded as the absorbance value A. Next, the colorimetric signal of the solution was captured using a smartphone equipped with the ColorPicker app, converted to RGB values, and the B / R(C) ratio was recorded. These results were then incorporated into the corresponding linear equations. Next, the fluorescence intensity (F) of the mixed reaction solution at 440 nm was measured under 365 nm excitation. The fluorescence signal was collected using a smartphone equipped with the ColorPicker app and converted to RGB values, recorded as B / R(F) in fluorescence mode. These results were then incorporated into the corresponding linear equations. Finally, after irradiation with a 660 nm laser, the temperature was measured using a portable photothermal imager, recorded as T, and the resulting values ​​were incorporated into the corresponding linear equations. The EC concentrations in beer obtained under the three detection modes were 11.41 μg / L, 10.22 μg / L, 11.86 μg / L, 11.11 μg / L, and 11.08 μg / L, respectively.

[0077] To verify the EC detection performance of this trimodal system in real samples, rice wine was selected as the test sample. The rice wine, purchased from the Zhonghuancheng Shopping Center in Hefei, China, had an alcohol content of 6% and was stored in a room temperature cabinet. 20.0 μL of the purchased rice wine was mixed with 50.0 μg / mL of Co@MOF-MIP nanozyme, 1.0 mmol / L of 3,3',5,5'-tetramethylbenzidine (TMB) solution, and 200.0 μmol / L of H2O2 in a centrifuge tube. HAc-NaAc buffer with a pH of 3.8-4.2 was then added to a constant volume of 200.0 μL. After complete reaction for 14.0-18.0 min, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, recorded as the absorbance value (A). Next, the colorimetric signal of the solution was captured using a smartphone equipped with the ColorPicker app, converted to RGB values, and the B / R(C) ratio was recorded. These results were then incorporated into the corresponding linear equations. Next, the fluorescence intensity (F) of the mixed reaction solution at 440 nm was measured under 365 nm excitation. The fluorescence signal was collected using a smartphone equipped with the ColorPicker app and converted to RGB values, recorded as B / R(F) in fluorescence mode. These results were then incorporated into the corresponding linear equations. Finally, after irradiation with a 660 nm laser, the temperature was measured using a portable photothermal imager, recorded as T, and the resulting values ​​were incorporated into the corresponding linear equations. The EC concentrations of rice wine obtained under the three detection modes were 8.20 μg / L, 8.92 μg / L, 9.44 μg / L, 9.28 μg / L, and 9.12 μg / L, respectively.

[0078] To verify the EC detection performance of this trimodal system in real samples, we selected unpurified baijiu (raw liquor) as the test sample. The baijiu in this work was obtained from a distillery in Hefei, China, with an alcohol content of approximately 40%, and stored in plastic barrels at room temperature. 20.0 μL of purchased rice wine was mixed with 50.0 μg / mL of Co@MOF-MIP nanozyme, 1.0 mmol / L of 3,3',5,5'-tetramethylbenzidine (TMB) solution, and 200.0 μmol / L of H2O2 in a centrifuge tube. HAc-NaAc buffer (pH 3.8-4.2) was then added to the mixture, bringing the volume to 200.0 μL. After complete reaction for 14.0-18.0 min, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, recorded as the absorbance value (A). Next, the colorimetric signal of the solution was captured using a smartphone equipped with the Color Picker app, converted to RGB values, and the B / R(C) ratio was recorded. The results were then substituted into the corresponding linear equations. Next, the fluorescence intensity (F) of the mixed reaction solution at 440 nm was measured under 365 nm excitation. The fluorescence signal was collected using a smartphone equipped with the Color Picker app and converted to RGB values, recorded as B / R(F) in fluorescence mode, and the results were substituted into the corresponding linear equations. Finally, after irradiation with a 660 nm laser, the temperature was measured using a portable photothermal imager, recorded as T, and the results were substituted into the corresponding linear equations. The EC concentrations of the liquor in the three detection modes were 41.53 μg / L, 40.88 μg / L, 41.96 μg / L, 39.86 μg / L, and 40.08 μg / L, respectively.

[0079] The results in the table below show that the trimodal system can be applied to the detection of actual samples, with a recovery rate between 96.55% and 103.40% and a relative deviation of less than 5%.

[0080]

[0081] Example 10: A trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch

[0082] A trimodal detection method for ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch, characterized by comprising the following steps:

[0083] Step 1: Determine the absorbance value A of the mixed reaction solution containing different concentrations of ethyl carbamate at 650 nm nThe colorimetric signal is collected using a smartphone with the Color Picker App installed, converted into RGB values, and recorded as B / R in colorimetric mode. n (C):

[0084] 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8 was added and the volume was adjusted to 200.0 μL. After reacting for 14 minutes, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, which was recorded as the absorbance value A. n Next, the colorimetric signal of the solution was captured using a smartphone equipped with the Color Picker App, converted into RGB values, and the B / R ratio was recorded. n (C);

[0085] Step 2: Under the excitation wavelength of 365 nm, the fluorescence intensity value F of the mixed reaction solution containing different concentrations of ethyl carbamate at 440 nm was measured. n The fluorescence signal was collected using a smartphone with the Color Picker App installed, converted into RGB values, and recorded as B / R in fluorescence mode. n (F):

[0086] 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution, and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0 minutes, the solution was transferred to a fluorescence cuvette and the fluorescence intensity at 440 nm was measured using a fluorescence spectrophotometer under excitation at a wavelength of 365 nm, which was recorded as the fluorescence value F n Then, under the excitation of 365nm wavelength, the fluorescence signal of the solution was captured using a smartphone installed with the ColorPicker App, converted into RGB values, and the B / R ratio was recorded. n (F);

[0087] Step 3: Determine the temperature T of the mixed reaction solution containing different concentrations of ethyl carbamate under 660nm laser irradiation n :

[0088] 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution, and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube. Then, HAc-NaAc buffer with a pH value of 3.8 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0 minutes, the solution was irradiated with a 660 nm laser and the temperature was measured using a portable photothermal imager, which was recorded as the temperature T n ;

[0089] Step 4: Construct the relationship between urethane concentration and absorbance A in colorimetric mode n , B / R n (C), F in fluorescence mode n , B / R n (F) and the temperature T in the photothermal mode n The linear regression equation is:

[0090] The absorbance signal A was obtained by testing the standard sample with a series of urethane concentrations. n and B / R n (C), construct the linear equation, A n =XC EC + y and B / R n (C) = XC EC + y, where C EC is the concentration of ethyl carbamate standard;

[0091] The fluorescence intensity signal F was obtained by testing the standard sample with a series of urethane concentrations. n and B / R n (F), construct the linear equation, F n =XC EC + y and B / R n (F) = XC EC + y, where C EC is the concentration of ethyl carbamate standard;

[0092] The temperature signal T is obtained by testing the standard sample with a series of ethyl carbamate concentrations n , construct the linear equation, T n =XC EC + y, where C EC is the concentration of ethyl carbamate standard;

[0093] Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the colorimetric mode absorbance signal A n and B / R n (C) Fluorescence intensity signal F in fluorescence mode n and B / R n (F) and the temperature signal T of the photothermal mode n , A n and B / R n (C), F n and B / R n (F) and T n Substitute into the corresponding linear equation to obtain the concentration of ethyl carbamate in the sample to be tested.

[0094] The preparation method of the Co@MOF-MIP nanozyme comprises the following steps:

[0095] S1: Co(NO₃)₂·6H₂O and terephthalic acid were dissolved in N,N-dimethylformamide and then transferred to a high-temperature reactor for reaction at 100°C for 10 hours. After cooling to room temperature, the mixture was centrifuged at 10,000 rpm for 5 minutes, washed with DMF and ultrapure water, and then freeze-dried into a powder for later use, yielding the Co@MOF nanozyme.

[0096] S2: Co@MOF nanozyme, tetraphenylethylene and 0.2 mmol / L ethyl carbamate were added to a flask, ethanol was added, and the mixture was ultrasonically dissolved. Carboxyethylsilanetriol sodium salt and silicon tetraacetate were then placed in the flask. After continuous stirring for 25 minutes in the dark, ammonia water was added to the mixture and continuous stirring was continued at 20°C overnight. The precipitate was washed with ethanol, centrifuged and the supernatant was discarded. The original ethyl carbamate template in the precipitate was extracted by methanol Soxhlet extraction to obtain Co@MOF-MIP sol-gel. The polymer was dried to obtain Co@MOF-MIP nanozyme.

[0097] A preferred embodiment is as follows: the ratio of Co(NO3)2·6H2O, terephthalic acid and N,N-dimethylformamide is: 0.15g:0.1g:30mL.

[0098] A preferred embodiment is as follows: in the final synthesis system, the ratio of Co@MOF, tetraphenylethylene, urethane solution and ethanol is: 0.01g:0.01g:15mg:5mL; the concentration of urethane solution is 0.2 mmol / L; the amount of carboxyethylsilanetriol sodium salt added is 200.0 μL, and the amount of silicon tetraacetate added is 200.0 μL; the concentration of carboxyethylsilanetriol sodium salt is 0.8 mmol / L; and the concentration of silicon tetraacetate is 2.4 mmol / L.

[0099] The preferred embodiment is: in step 4, the absorbance signal A n and B / R n The regression equation for (C) is A n = -0.00261C EC + 1.58196 and B / R n (C) = -0.00386C EC + 1.89281; fluorescence intensity signal F n and B / R n The regression equation of (F) is F n = 0.00186 C EC + 0.44144 and B / R n (F) = 0.01298C EC + 2.25309; Temperature signal T n The regression equation is T n = -0.03572C EC +44.25262.

[0100] 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 ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch, characterized by: The following steps are involved: Step 1: Determine the absorbance value A of the mixed reaction solution containing different concentrations of ethyl carbamate at 650 nm n The colorimetric signal is collected using a smartphone with the Color Picker App installed, converted into RGB values, and recorded as B / R in colorimetric mode. n (C): 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0-18.0 min, the mixture was transferred to a cuvette and the absorbance at 650 nm was measured using a UV spectrophotometer, which was recorded as the absorbance value A. n ; Then, use a smartphone installed with the Color Picker App to capture the colorimetric signal of the solution, convert it into RGB values, and record the B / R n (C); Step 2: Under the excitation wavelength of 365nm, measure the fluorescence intensity value F of the mixed reaction solution containing different concentrations of ethyl carbamate at 440nm n The fluorescence signal was collected using a smartphone with the Color Picker App installed, converted into RGB values, and recorded as B / R in fluorescence mode. n (F): 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0-18.0 min, the solution was transferred to a fluorescence cuvette and the fluorescence intensity at 440 nm was measured using a fluorescence spectrophotometer under excitation at a wavelength of 365 nm, which was recorded as the fluorescence value F n Then, under the excitation of 365nm wavelength, the fluorescence signal of the solution was captured using a smartphone installed with the Color Picker App, converted into RGB values, and recorded as B / R n (F); Step 3: Determine the temperature T of the mixed reaction solution containing different concentrations of ethyl carbamate under 660nm laser irradiation n : 20.0 μL of urethane standard solution of different concentrations, 10.0 μL of 1.0 mg / mL Co@MOF-MIP nanozyme solution, 10.0 μL of 20.0 mmol / L 3,3',5,5'-tetramethylbenzidine solution and 20.0 μL of 2.0 mmol / L H2O2 were mixed in a centrifuge tube, and then HAc-NaAc buffer with a pH value of 3.8-4.2 was added and the volume was adjusted to 200.0 μL. After reacting for 14.0-18.0 min, the solution was irradiated with a 660 nm laser and the temperature was measured using a portable photothermal imager, which was recorded as the temperature T n ; Step 4: Construct the relationship between urethane concentration and absorbance A in colorimetric mode n , B / R n (C), F in fluorescence mode n , B / R n (F) and the temperature T in the photothermal mode n The linear regression equation is: The absorbance signal A was obtained by testing the standard sample with a series of urethane concentrations. n and B / R n (C), construct the linear equation, A n =XC EC + y and B / R n (C) = XC EC + y, where C EC is the concentration of ethyl carbamate standard; The fluorescence intensity signal F was obtained by testing the standard sample with a series of urethane concentrations. n and B / R n (F), construct the linear equation, F n =XC EC + y and B / R n (F) = XC EC + y, where C EC is the concentration of ethyl carbamate standard; The temperature signal T is obtained by testing the standard sample with a series of ethyl carbamate concentrations n , construct the linear equation, T n =XC EC +y, where C EC is the concentration of ethyl carbamate standard; Step 5: Take the sample to be tested and repeat steps 1, 2 and 3 to obtain the colorimetric mode absorbance signal A n and B / R n (C) Fluorescence intensity signal F in fluorescence mode n and B / R n (F) and the temperature signal T of the photothermal mode n , A n and B / R n (C), F n and B / R n (F) and T n Substitute into the corresponding linear equation to obtain the concentration of ethyl carbamate in the sample to be tested; The preparation method of the Co@MOF-MIP nanozyme comprises the following steps: S1: Dissolve Co(NO3)2·6H2O and terephthalic acid in N,N-dimethylformamide, then transfer to a high-temperature reactor and react at 100-120°C for 10-30 hours; after cooling to room temperature, centrifuge at 10,000-14,000 rpm for 5-15 minutes, wash with DMF and ultrapure water, and then freeze-dry into a powder for use to obtain Co@MOF nanozyme; S2: Add Co@MOF nanozyme, tetraphenylethylene and 0.2 mmol / L ethyl carbamate to a flask, add ethanol, and sonicate until dissolved; then put carboxyethylsilanetriol sodium salt and silicon tetraacetate into the flask, stir continuously for 25-40 minutes under dark conditions, add ammonia water to the mixture, and stir continuously at 20-30℃ overnight; the precipitate is washed with ethanol, centrifuged and the supernatant is discarded; the original ethyl carbamate template in the precipitate is extracted by methanol Soxhlet extraction to obtain Co@MOF-MIP sol-gel; the polymer is dried to obtain Co@MOF-MIP nanozyme.

2. The method for trimodal detection of ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch according to claim 1, characterized in that: The ratio of Co(NO3)2·6H2O, terephthalic acid and N,N-dimethylformamide is: 0.15-0.4g: 0.1-0.2g: 30-60mL.

3. The method for trimodal detection of ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch according to claim 1, characterized in that: In the final synthesis system, the ratio of Co@MOF, tetraphenylethylene, ethyl carbamate solution and ethanol is: 0.01-0.08 g: 0.01-0.1 g: 15-20 mg: 5-15 mL; the concentration of ethyl carbamate solution is 0.2 mmol / L; the amount of carboxyethylsilanetriol sodium salt added is 200.0 μL, and the amount of silicon tetraacetate added is 200.0 μL; the concentration of carboxyethylsilanetriol sodium salt is 0.8 mmol / L; and the concentration of silicon tetraacetate is 2.4 mmol / L.

4. The method for trimodal detection of ethyl carbamate based on molecular imprinting regulation of Co@MOF-MIP enzyme activity switch according to claim 1, characterized in that: In step 4, the absorbance signal A n and B / R n The regression equation for (C) is A n = -0.00261C EC + 1.58196 and B / R n (C) = -0.00386C EC + 1.89281; fluorescence intensity signal F n and B / R n The regression equation of (F) is F n = 0.00186 C EC + 0.44144 and B / R n (F) = 0.01298C EC + 2.25309; Temperature signal T n The regression equation is T n = -0.03572C EC +44.25262.

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