Method for detecting ethyl carbamate in liquor based on laccase-like active nano-enzyme bimodal

Abstract

The invention discloses a method for detecting ethyl carbamate in liquor based on laccase-like active nano-enzyme bimodal. The method comprises the following steps: step 1, determining absorbance values A of to-be-detected solutions of ethyl carbamate standard substances with different concentration gradients at 420nm; 2, measuring the fluorescence intensity signal ratio F420nm / F550nm of the to-be-measured solutions of the ethyl carbamate standard substances with different concentration gradients at 420nm and 550nm under the excitation of the wavelength of 330nm; 3, constructing a linear regression equation of the standard substance; and 4, taking a sample to be detected, repeating the steps 1 and 2 to obtain the absorbance value of the sample to be detected and the fluorescence intensity signal ratio F420nm / F550nm of the sample to be detected, and respectively substituting the absorbance value and the fluorescence intensity signal ratio F420nm / F550nm into the corresponding linear equations to obtain the concentration of ethyl carbamate in the sample to be detected. According to the bimodal detection, the advantages of a colorimetric method and a fluorescence method are combined, and the comprehensiveness and accuracy of detection are improved. The colorimetric method provides visual information, but the sensitivity is limited; and the fluorescence method has higher sensitivity and selectivity. By combining the two methods, the bimodal detection can more reliably identify and quantify the target substance in the actual sample, and has higher accuracy compared with the single detection.

Description

Technical Field

[0001] The invention belongs to the technical field of environmental and food pollution detection, and relates to a method for detecting ethyl carbamate based on a laccase-like active nanoenzyme dual-modality. Background Art

[0002] Ethyl carbamate (EC) is a byproduct produced and accumulated during the fermentation and storage of alcoholic beverages. It can enter organisms through gas inhalation or the food chain, posing a potential exposure risk to public health. It has been proven that long-term exposure to ethyl carbamate can cause serious harm to organisms. In 2007, the International Agency for Research on Cancer (IARC) officially raised EC from a Class 2B carcinogen to a Class 2A carcinogen, which has aroused widespread concern about the safety of EC in food. Some countries such as Canada, France and Japan have set the maximum limit of EC in distilled liquid at 150 μg / L. Since EC is widely present in alcoholic beverages, in order to solve the problem of ethyl carbamate residues, it is necessary to develop rapid and effective detection methods. At present, the detection of ethyl carbamate in alcoholic beverages mainly relies on spectroscopy, high performance liquid chromatography (HPLC), gas chromatography / mass spectrometry (GC / MS), potentiometric determination and conductivity determination. However, these existing technologies are often limited by complexity and high cost, making them difficult to popularize in practical applications. Therefore, a new detection method is urgently needed to quickly and effectively monitor ethyl carbamate and prevent health risks. This new method should be highly sensitive, accurate, rapid, and cost-effective, aiming to protect public health and promote environmental sustainability.

[0003] Compared with natural enzymes, nanozymes show advantages such as higher stability, easy storage and more cost-effectiveness. At present, many types of mimetic enzymes have been studied, including peroxidases, oxidases, laccases, superoxide dismutases and phosphohydrolases. Among them, laccase, as a copper-containing oxidase with polyphenols or polyamines as substrates, has attracted much attention due to its wide substrate applicability and good environmental properties. Compared with conventional oxidases and peroxidases, laccases do not need to produce harmful reactive oxygen in subsequent reactions, making them safer and more environmentally friendly. Fluorescent nanozymes are nanomaterials that have both fluorescent and catalytic activity. At present, most of the developed nanozymes still maintain traditional catalytic properties and are used for colorimetric analysis. However, the sensitivity of a single fluorescent signal is low and is easily affected by environmental factors, resulting in inaccurate measurement results. In contrast, by using ratio fluorescence technology, by measuring the ratio of two different fluorescent signals, the interference of external factors can be effectively reduced, and the sensitivity and accuracy of the signal can be improved. Herein, we synthesized a dual-fluorescent laccase-active nanozyme Cu-BH@MIP as a functional nanomaterial and combined it with the chromogenic substrate o-phenylenediamine (OPD) to create a ratiometric fluorescence-driven method for ethyl carbamate detection, which is simple to operate, requires readily available materials and low cost, and does not require expensive antibodies, showing great potential for the quantitative detection of ethyl carbamate. Summary of the invention

[0004] The purpose of the present invention is to provide an efficient and accurate method for detecting ethyl carbamate based on ratio fluorescence drive. The Cu-BH@MIP nanozyme in the present invention has a fluorescence signal at 420nm under 330nm excitation light; at the same time, there is a strong laccase activity that can oxidize the colorless and non-fluorescent OPD into a dark yellow DAP that emits yellow fluorescence at 550nm, and quench the blue fluorescence of Cu-BH@MIP at 420nm through the inner filter effect, thereby constructing a ratio fluorescence signal F 420nm / F 550nm When ethyl carbamate was added, EC could specifically fill the imprinted cavity of Cu-BH@MIP, blocking the catalytic substrate OPD outside Cu-BH@MIP, thus preventing it from further reacting with the internal catalytic center Cu-BH, resulting in the inhibition of the conversion of OPD to DAP, the decrease of DAP colorimetric signal, and the decrease of ratio fluorescence signal F 420nm / F 550nm Therefore, a dual-modal method for detecting ethyl carbamate based on laccase-like active nanozymes can be developed.

[0005] To achieve the above-mentioned purpose and other related purposes, the technical solution provided by the present invention is: a method for detecting ethyl carbamate in wine based on laccase-like activity nanozyme dual modality, comprising the following steps:

[0006] Step 1: Determine the absorbance value A of the urethane standard solution with different concentration gradients at 420nm:

[0007] At room temperature, add Cu-BH@MIP nanozyme, o-phenylenediamine and ethyl carbamate standards with different concentration gradients into HEPES buffer, stir evenly to obtain a mixed solution, and then transfer it to a cuvette; then use an ultraviolet spectrophotometer to measure the absorbance value at 420nm, which is recorded as A;

[0008] Step 2: Determine the fluorescence intensity signal ratio F of the urethane standard solution with different concentration gradients at 420nm and 550nm under 330nm wavelength excitation 420nm / F 550nm ;

[0009] At room temperature, Cu-BH@MIP nanozyme, o-phenylenediamine and ethyl formate standards with different concentration gradients were added to HEPES buffer, stirred evenly to obtain a mixed solution, and then transferred to a cuvette; then, the fluorescence intensity signal values ​​at 420nm and 550nm were measured using a fluorescence spectrometer at an excitation wavelength of 330nm to obtain the fluorescence intensity signal ratio F 420nm / F 550nm ;

[0010] Step 3: Construct the linear regression equation for the standard

[0011] The UV regression equation A=XC was constructed by using the absorbance value A value measured by the urethane standard with different concentration gradients and the concentration of the standard. EC +y, where C EC is the concentration of the standard;

[0012] The fluorescence intensity signal ratio F measured by using different concentration gradients of ethyl formate standards 420nm / F 550nm The linear regression equation F of the ratio fluorescence signal was constructed based on the concentration of the standard 420nm / F 550nm =XC EC +y, where C EC is the concentration of the standard;

[0013] Step 4: Take the sample to be tested and repeat steps 1 and 2 to obtain the absorbance value of the sample to be tested and the fluorescence intensity signal ratio F of the sample to be tested. 420nm / F 550nm , respectively, and substituted into the corresponding linear equation to obtain the concentration of ethyl carbamate in the sample to be tested.

[0014] The preferred technical scheme is: in step 1 and step 2, the pH value of the HEPES buffer is 8.8-9.2; the concentration of o-phenylenediamine is 10.0 mmol / L; the concentration of the Cu-BH@MIP nanozyme is 100 μg / mL; the total amount of the mixed solution is 200.0 μL; the reaction temperature during stirring is 50.0°C; and the reaction time is 27.0 min.

[0015] The preferred technical solution is: in step 3, the ultraviolet regression equation is A = -0.00201C EC +1.6875; the linear regression equation of the ratio fluorescence signal is F 420nm / F 550nm =00306C EC +0.52762.

[0016] The preferred technical solution is: the preparation method of the Cu-BH@MIP nanozyme comprises the following steps:

[0017] S1: Dissolve CuCl2·2H2O and 2-aminoterephthalic acid in DMF solution to obtain solution A, and dissolve histidine in a mixture of ethanol and DMF solution to obtain solution B; then add solution A to solution B under constant stirring; then transfer the mixture to a sealed Teflon-autoclave and heat at 100-140°C for 5-12 hours; after the autoclave is naturally cooled, the obtained product is centrifuged, and the precipitate is washed with alcohol and water, dried and stored to obtain Cu-BH nanozyme;

[0018] S2: Cu-BH nanozyme, ethyl carbamate and ethanol are added to a container and sonicated until dissolved; sodium carboxyethylsilanetriol and tetraethyl orthosilicate are placed in the container, stirred continuously for 25-35 minutes in the dark, then ammonia water is added and stirred continuously overnight at 20-30°C; the precipitate is washed with ethanol, centrifuged and the supernatant is discarded; the original EC template in the precipitate is extracted by methanol Soxhlet extraction to obtain Cu-BH@MIP precipitate; the polymer is dried in a vacuum chamber at 55-65°C overnight to obtain Cu-BH@MIP nanozyme.

[0019] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0020] 1. Economical and environmentally friendly synthesis: The synthesis process of Cu-BH@MIP nanozyme is relatively simple, the raw materials are easily available and the cost is low, and by selecting non-toxic materials and optimizing the process, the use of toxic bromine (Br2) as a reaction raw material is successfully avoided. Therefore, compared with patent CN202311565150.2, the related environmental and operational risks are significantly reduced.

[0021] 2. High-sensitivity ratio fluorescence: The present invention simultaneously detects two fluorescence signals of different wavelengths (such as 420nm and 550nm) and calculates their ratio. This method can effectively reduce the impact of background signals caused by interfering substances in the sample matrix. When the background interference signal changes, since the signal ratio is calculated based on two independent fluorescence signals, the ratio is relatively stable and has nothing to do with background changes. Compared with patent CN202410427275.7, the detection limit (LOD) of this method is 0.978μg / L. In comparison, the LOD of the comparative patent is 1.73μg / L. Therefore, the present invention has a higher sensitivity for detecting EC.

[0022] 3. Molecularly imprinted polymer: The molecularly imprinted nanozyme used in the present invention exhibits more outstanding performance in terms of selectivity and anti-interference compared with patent CN202311565150.2, especially in the complex components, it can accurately identify the target molecules and generate obvious signals. The experimental results show that Cu-BH@MIP has a three-dimensional cavity with high adaptability to EC molecules, so that it excludes representative substrates in fermented foods and EC structural analogs such as urea, ethyl acetate, Na + , K + , Ca 2+ Mg 2 + It is not affected by interference, and has good selectivity and anti-interference.

[0023] 4. Laccase-like activity: The laccase-like activity of the present invention performs well in actual sample detection. For example, the experimental results show that the relative deviation rate of actual sample detection of the present technology is less than 3 compared with that of patent CN202410427275.7. In addition, harmful active oxygen such as O2 - and OH - , while this technology operates under mild conditions (reaction at room temperature) without generating harmful active oxygen. This greatly reduces damage to the sample and ensures the safety and environmental protection of the experiment.

[0024] 5. Dual-modal detection: Dual-modal detection combines the advantages of colorimetry and fluorescence to improve the comprehensiveness and accuracy of detection. Colorimetry provides intuitive visual information, but has limited sensitivity; while fluorescence has higher sensitivity and selectivity. By combining the two, dual-modal detection can more reliably identify and quantify target substances in actual samples, with higher accuracy than single detection. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The synthesis diagram of Cu-BH@MIP and the schematic diagram of the EC detection of Cu-BH@MIP+OPD system.

[0026] Figure 2 Characterization diagram of Cu-BH@MIP nanozyme. A is the TEM image of Cu-BH@MIP nanozyme, B, C, D, E, and F are the distribution of C, N, O, Cu, and Si elements of Cu-BH@MIP nanozyme, respectively.

[0027] Figure 3 UV absorption graphs and corresponding solution color illustrations of different groups (1) 2,4-DP+4-AP, (2) Cu-BH+2,4-DP+4-AP, (3) Cu-BH@MIP+2,4-DP (4) Cu-BH@MIP+4-AP, (5) Cu-BH@MIP+2,4-DP+4-AP to verify the laccase activity of Cu-BH@MIP nanozyme.

[0028] Figure 4 Double reciprocal plot corresponding to the laccase catalytic kinetics of Cu-BH@MIP nanozyme.

[0029] Figure 5 To verify the feasibility of detecting EC in the Cu-BH@MIP+OPD system, the UV absorption and fluorescence intensity graphs of different groups (1) EC, (2) OPD, (3) Cu-BH@MIP, (4) Cu-BH@MIP+OPD, and (5) Cu-BH@MIP+OPD+EC as well as the corresponding solution colors and fluorescence illustrations were obtained.

[0030] Figure 6 The experimental parameters for detecting EC in the Cu-BH@MIP+OPD system, such as pH value, temperature, and reaction time before and after the addition of EC, were optimized.

[0031] Figure 7 Shown are the absorption spectra and standard curves of the reaction solutions containing different concentrations of EC.

[0032] Figure 8 The fluorescence intensity of the reaction solution containing different concentrations of EC at an excitation wavelength of 330 nm and the standard curve.

[0033] Fig. 9 This is a test of the selectivity and anti-interference of the Cu-BH@MIP+OPD detection system. Fig. 9 A, 9C 1-8 are blank, EC, urea, ethyl acetate, Na + , K + , Ca 2+ Mg 2+ ; Fig. 9 B. 1-7 in 9D are EC+blank, urea, ethyl acetate, Na + , K + , Ca 2+ Mg 2+.

[0034] Fig.10 This is the effect of alcohol concentration on the dual-modal detection system. DETAILED DESCRIPTION

[0035] The following is a description of the implementation of the present invention by means of specific embodiments. Those skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0036] See also Figure 1-10 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings in 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, so they have no substantial technical significance. Any modification of the structure, change in the proportional relationship, or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the effects and purposes that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0037] Related reagents used in the embodiments of the present invention:

[0038] The raw materials used in the present invention: CuCl2·2H2O (AR), OPD were purchased from Shanghai McLean Biochemical Technology Co., Ltd.; 2-aminoterephthalic acid, L-histidine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES) buffer was purchased from Hefei White Shark Biotechnology Co., Ltd. All chemicals were purchased directly from suppliers without further purification; the water used in the experimental process was ultrapure water.

[0039] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0040] The technical solution of the present invention is further described in detail below with reference to examples.

[0041] Example 1: Synthesis of Cu-BH@MIP nanozyme

[0042] (1) Dissolving CuCl2·2H2O and 2-aminoterephthalic acid in a DMF solution to obtain a solution A, and dissolving histidine in a mixture of ethanol and DMF solution to obtain a solution B; then adding solution A to solution B under constant stirring; then transferring the mixture to a sealed Teflon-autoclave and heating it at 100-140°C for 5-12 hours; after the autoclave is naturally cooled, the obtained product is centrifuged, and the precipitate is washed with alcohol and water, and dried and stored to obtain a Cu-BH nanozyme; (2) Cu-BH nanozyme, ethyl carbamate and ethanol are added to a container and sonicated until dissolved; sodium carboxyethylsilanetriol salt and tetraethyl orthosilicate are placed in the container, stirred continuously for 25-35 minutes under dark conditions, and then ammonia water is added, and stirred continuously overnight at 20-30°C; the precipitate is washed with ethanol, centrifuged and the supernatant is discarded; the original EC template in the precipitate is extracted by methanol Soxhlet extraction to obtain Cu-BH@MIP precipitate; the polymer is dried in a vacuum chamber at 55-65°C overnight to obtain Cu-BH@MIP nanozyme.

[0043] Example 2: Verification of laccase activity of Cu-BH@MIP nanozyme

[0044] Use common laccase active substrates 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) as colorimetric agents and HEPES as buffer. Take the following five groups of reactions: (1) 2,4-DP + 4-AP, (2) Cu-BH + 2,4-DP + 4-AP, (3) Cu-BH@MIP + 2,4-DP (4) Cu-BH@MIP + 4-AP, (5) Cu-BH@MIP + 2,4-DP + 4-AP. After sufficient reaction, transfer to a cuvette, measure the absorbance at 500nm with a UV spectrophotometer, and compare the catalytic ability of the nanozymes. See Figure 3 , only the absorbance values ​​of Cu-BH+2,4-DP+4-AP and Cu-BH@MIP+2,4-DP+4-AP combinations were relatively large, indicating that the synthesized Cu-BH@MIP nanozyme had good laccase activity.

[0045] In the above verification steps, the concentration of Cu-BH@MIP nanozyme added was 100.0 μg / mL; the concentration of 2,4-DP and 4-AP was 1.0 mmol / L; the pH of the added HEPES buffer was 8.8-9.2; the total mixed solution was 200.0 μL; the reaction temperature was 50.0 °C; and the mixing and sufficient reaction time was 27.0 min.

[0046] Example 3: Catalytic kinetics test of Cu-BH@MIP nanozyme

[0047] In the HEPES buffer containing Cu-BH@MIP nanozyme, steady-state kinetic experiments were performed by changing the concentration of OPD (0.0-1.0mmol / L). The double inverse plot of OPD concentration and reaction rate was calculated using the following Michaelis-Menten formula:

[0048]

[0049] Where V is the initial reaction rate, [S] is the substrate concentration, and K m is the Michaels constant, V max represents the maximum reaction rate. Figure 4 The K of Cu-BH@MIP to OPD is shown in m The value is 1.802 mM, indicating that the Cu-BH@MIP nanozyme prepared in the present invention has good catalytic activity.

[0050] In the above verification steps, the concentration of Cu-BH@MIP nanozyme added was 100.0 μg / mL; the pH of the added HEPES buffer was 8.8; the total mixed solution was 200.0 μL; the reaction temperature was 50.0°C; and the mixing and sufficient reaction time was 27.0 min.

[0051] Example 4: Feasibility verification of Cu-BH@MIP+OPD system for EC detection

[0052] OPD was used as the colorimetric agent and HEPES was used as the buffer. The following five groups of reactions were selected: (1) EC, (2) OPD, (3) Cu-BH@MIP, (4) Cu-BH@MIP+OPD, and (5) Cu-BH@MIP+OPD+EC. After sufficient reaction, the mixture was transferred to a cuvette and the absorbance at 420 nm was measured using a UV spectrophotometer. The fluorescence intensity signal ratio F at 420 nm and 550 nm was measured using a fluorescence spectrometer. 420nm / F 550nm , compare their sizes. See the results in Figure 5 , indicating that EC can inhibit the laccase activity and cause significant changes in absorbance and fluorescence intensity, showing the feasibility of the detection system proposed by the present invention.

[0053] In the above verification steps, the concentration of Cu-BH@MIP nanozyme added was 100.0 μg / mL; the OPD concentration was 10.0 mmol / L; the EC concentration was 500 μg / L; the pH of the added HEPES buffer was 9.0; the total mixed solution was 200.0 μL; the reaction temperature was 50.0 °C; and the mixing and sufficient reaction time was 27.0 min.

[0054] Example 5: Optimization of the optimal conditions for EC detection in the Cu-BH@MIP+OPD system

[0055] In order to optimize the analytical performance of the Cu-BH@MIP+OPD system, the experimental parameters such as pH value, temperature, and reaction time in the detection system were studied in detail. The experiment first explored the performance of the system in the pH range of 7.0-9.5, and HEPES was selected as the reaction buffer. Figure 6 As shown in A, when the pH value is in the range of 7.0 to 9.0, the absorbance value of the reaction system at 420nm increases with the increase of the pH value of the buffer system. When the pH value exceeds 9.0, the absorbance value decreases as it increases, so 9.0 is selected as the optimal pH value of the system. Next, the performance of the system in the temperature range of 30.0-55.0℃ was explored. The results are shown in Figure 6 As shown in Figure B, when the temperature is in the range of 30.0℃ to 50.0℃, the absorbance value of the unreacted system at 420nm increases with the increase of the reaction temperature. When the temperature exceeds 50.0℃ and increases to 55.0℃, the absorbance value decreases as it increases, so 50.0℃ is selected as the optimal temperature of the system. Then, the reaction time is optimized, and the results are shown in Figure 6 C. Figure 6 As shown in D, within 0.0 to 27.0 min, the absorbance value of the reaction system at 420 nm increased with the increase of reaction time, and basically did not change after 27.0 min. Therefore, 27.0 min was selected as the optimal reaction time of the system.

[0056] Example 6: Cu-BH@MIP+OPD system to detect EC standards

[0057] EC solutions of different concentrations, including 0.0, 100.0, 200.0, 3000.0, 400.0, 500.0 and 600.0 μg / L, were fully reacted with 100.0 μg / mL of Cu-BH@MIP nanozyme and 10.0 mmol / L of OPD solution in HEPES buffer (pH = 8.8-9.2) at 50.0 °C for 27.0 min.

[0058] After the reaction is completed, the complete reaction solution is transferred to a cuvette and the absorbance at 420nm is measured using a UV spectrophotometer. As the concentration of EC increases, the absorbance at 420nm decreases. Therefore, the linear equation is fitted using Origin software with absorbance A as the ordinate and EC concentration as the abscissa. Figure 7 As shown, the concentration of EC and absorbance A are linear in the range of 0-600.0 μg / L, and the absorbance regression equation is A=-0.00201C EC+1.6875, the correlation coefficient is 0.99835, and the detection limit is 2.11μg / L. In addition, as the concentration of EC increases, the solution presents a color gradient (from dark yellow to light yellow), and the change in the color of the reagent can be observed by the naked eye, thus realizing the detection of EC.

[0059] Take the above reaction solution and measure the fluorescence intensity signal at 420nm and 550nm with a fluorescence spectrophotometer under 330nm excitation light. As the concentration of EC increases, the ratio of fluorescence intensity at 420nm to 550nm increases. Therefore, the fluorescence intensity ratio F 420nm / F 550nm The vertical axis is the concentration of EC, and the linear equation is fitted using Origin software. Figure 8 The concentration of EC and the fluorescence intensity F are shown in 420nm / F 550nm The fluorescence intensity ratio F 420nm / F 550nm The regression equation is F 420nm / F 550nm =00306C EC +0.52762, the correlation coefficient is 0.9949, and the detection limit is 0.978μg / L. In addition, as the concentration of EC increases, the solution presents a satisfactory fluorescence gradient (from yellow to blue), and the change in reagent fluorescence can be observed by the naked eye, thus realizing the detection of EC.

[0060] Example 7: Selectivity and anti-interference test of Cu-BH@MIP+OPD system for EC detection

[0061] In order to evaluate the selectivity of Cu-BH@MIP+OPD system for EC, some representative contents were used as proof of concept, including blank, EC, urea, ethyl acetate, Na + , K + , Ca 2+ Mg 2+ At room temperature, the Cu-BH@MIP nanozyme and o-phenylenediamine (OPD) were reacted with the above substances in HEPES buffer respectively; after mixing evenly, the mixture was reacted for 27.0 min and then transferred to a cuvette. The absorbance value A at 420 nm and the fluorescence intensity signal ratio F at 420 nm and 550 nm under 330 nm wavelength excitation were measured respectively. 420nm / F 550nm The results are as follows Fig. 9 A. Fig. 9 As shown in C, only the absorbance value A of EC decreased significantly, and the fluorescence intensity signal ratio F 420nm / F 550nmThe detection system has good selectivity for EC. At the same time, we explored whether the coexistence of EC and these substances would affect the reaction system. The results are as follows Fig. 9 B. Fig. 9 As shown in D, the absorbance value at 420nm of each coexistence system and the fluorescence intensity signal ratio F at 420nm and 550nm under 330nm wavelength excitation 420nm / F 550nm There is no obvious change, indicating that the constructed detection system has good anti-interference ability.

[0062] In the above verification steps, the concentration of Cu-BH@MIP nanozyme added was 100.0 μg / mL; the OPD concentration was 10.0 mmol / L; the concentration of the selectivity verification substance was 500.0 μg / L; the pH of the added HEPES buffer was 8.8–9.2; the total mixed solution was 200.0 μL; the reaction temperature was 50.0 °C; and the mixing and sufficient reaction time was 27.0 min.

[0063] Example 8: Effect of alcohol concentration on the detection system of the Cu-BH@MIP+OPD system detection method.

[0064] In order to verify the effect of alcohol concentration on the dual-modal 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 100.0 μg / mL Cu-BH@MIP nanozyme and 10.0 mmol / L OPD solution in a centrifuge tube, and then HEPES buffer with a pH value of 8.8-9.2 was added and the volume was fixed to 200.0 μL. After 27.0 minutes of complete reaction, the solution was transferred to a cuvette and the absorbance at 420 nm was measured with a UV spectrophotometer. The results are shown in Figure 2. Fig.10 As shown, the absorbance values ​​of various alcohol concentrations are almost the same, indicating that alcohol concentration has almost no effect on this experiment, and actual samples containing high concentrations of alcohol can be directly detected using this system.

[0065] Example 9: Detection of EC in actual samples by Cu-BH@MIP+OPD system.

[0066] In order to verify the sensing performance of EC detection of Cu-BH@MIP+OPD system in actual wine applications, we selected liquor as a representative of actual samples. Among them, the liquor in this work was randomly extracted from a brewery in Hefei, China, without purification. Take 1mL of liquor and add it to a 5.0mL centrifuge tube and adjust it to pH = 9.0. Then mix it with 0, 250.0, and 500.0μg / L EC respectively. At room temperature, Cu-BH@MIP nanozyme and o-phenylenediamine (OPD) were reacted with the above mixed solutions in HEPES buffer respectively; after mixing evenly, react for 27.0min and move to the cuvette. The absorbance value A at 420nm and the fluorescence intensity signal ratio F at 420nm and 550nm under 330nm wavelength excitation were measured respectively. 420nm / F 550nm The obtained absorbance value A and fluorescence intensity signal ratio F 420nm / F 550nm Substituting into the corresponding linear equation, the EC results measured under the two detection modes are shown in Table 1.

[0067] In order to verify the sensing performance of EC detection of Cu-BH@MIP+OPD system in actual wine applications, we selected rice wine as a representative of actual samples. Among them, the rice wine in this work was taken from a large supermarket in Hefei, Anhui. Take 1mL of rice wine and add it to a 5.0mL centrifuge tube and adjust it to pH = 9.0. Then mix it with 0, 250.0, and 500.0μg / L EC respectively. At room temperature, Cu-BH@MIP nanozyme and o-phenylenediamine (OPD) were reacted with the above mixed solutions in HEPES buffer respectively; after mixing evenly, react for 27.0min and move to the cuvette. The absorbance value A at 420nm and the fluorescence intensity signal ratio F at 420nm and 550nm under 330nm wavelength excitation were measured respectively. 420nm / F 550nm The obtained absorbance value A and fluorescence intensity signal ratio F 420nm / F 550nm Substituting the corresponding linear equation, the EC results measured under the two detection modes are as follows Figure 1 shown.

[0068] In order to verify the sensing performance of EC detection of Cu-BH@MIP+OPD system in practical applications, we selected cooking wine as a representative of actual samples. Among them, the cooking wine in this work was taken from a large supermarket in Hefei, Anhui. Take 1mL of cooking wine and add it to a 5.0mL centrifuge tube and adjust it to pH = 9.0. Then mix it with 0, 250.0, and 500.0μg / L EC respectively. At room temperature, Cu-BH@MIP nanozyme and o-phenylenediamine (OPD) were reacted with the above mixed solutions in HEPES buffer respectively; after mixing evenly, react for 27.0min and move to the cuvette. The absorbance value A at 420nm and the fluorescence intensity signal ratio F at 420nm and 550nm under 330nm wavelength excitation were measured respectively. 420nm / F 550nm The obtained absorbance value A and fluorescence intensity signal ratio F 420nm / F 550nm Substituting into the corresponding linear equation, the EC results measured under the two detection modes are shown in Table 1.

[0069] Table 1 shows the detection of EC in actual samples by the Cu-BH@MIP+OPD system. The actual samples are white wine, yellow wine, and cooking wine.

[0070]

[0071]

[0072] It can be seen from the results that the reaction system can be applied to the detection of actual samples, and the recovery rate is between 98% and 103%, and the relative deviation is less than 3.

[0073] Example 10: A method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes. The method comprises the following steps:

[0074] Step 1: Determine the absorbance value A of the urethane standard solution with different concentration gradients at 420nm:

[0075] At room temperature, add Cu-BH@MIP nanozyme, o-phenylenediamine and ethyl carbamate of different concentration gradients into HEPES buffer for reaction. Stir thoroughly and react for 27.0 min before transferring to a cuvette. Then use a UV spectrophotometer to measure the absorbance at 420 nm, which is recorded as A.

[0076] Step 2: Determine the fluorescence intensity signal ratio F of the urethane standard solution with different concentration gradients at 420nm and 550nm under 330nm wavelength excitation 420nm / F 550nm ;

[0077] At room temperature, the Cu-BH@MIP nanozyme, o-phenylenediamine, and ethyl carbamate of different concentration gradients were added to the HEPES buffer for reaction. The mixture was stirred thoroughly and the reaction lasted for 27.0 min before being transferred to a cuvette. The fluorescence intensity signal ratio F at 420 nm and 550 nm was then measured using a fluorescence spectrometer at an excitation wavelength of 330 nm. 420nm / F 550nm ;

[0078] Step 3: Construct the linear regression equation for the standard

[0079] The UV regression equation A=XC was constructed by using the absorbance value A value measured by the urethane standard with different concentration gradients and the concentration of the standard. EC +y, where C EC is the concentration of the standard;

[0080] The fluorescence intensity signal ratio F measured by using different concentration gradients of ethyl formate standards 420nm / F 550nm The linear regression equation F of the ratio fluorescence signal was constructed based on the concentration of the standard 420nm / F 550nm =XC EC +y, where C EC is the concentration of the standard;

[0081] Step 4: Take the sample to be tested, repeat steps 1 and 2 to obtain the colorimetric and ratio fluorescence signals, substitute them into the corresponding linear equations, and obtain the concentration of ethyl carbamate in the sample to be tested.

[0082] The preferred embodiment is: in steps 1 and 2, the pH value of the HEPES buffer is 8.8-9.2; the concentration of the o-phenylenediamine solution is 10.0 mmol / L; the concentration of the Cu-BH@MIP nanozyme is 100.0 μg / mL; the total amount of the mixed solution is 200.0 μL; the reaction temperature is 50.0°C; and the reaction time is 27.0 min.

[0083] The preferred embodiment is: in steps 1 and 2, the pH value of the HEPES buffer is 8.8; the concentration of the o-phenylenediamine solution is 10.0 mmol / L; the concentration of the Cu-BH@MIP nanozyme is 100.0 μg / mL; the total amount of the mixed solution is 200.0 μL; the reaction temperature is 50.0°C; and the reaction time is 27.0 min.

[0084] The preferred embodiment is: in step 3, the linear regression equation of the UV spectrophotometer is A=-0.00201C EC +1.6875; the linear regression equation of the ratio fluorescence signal is F 420nm / F 550nm =00306CEC +0.52762.

[0085] A preferred embodiment is: the preparation method of the Cu-BH@MIP nanozyme comprises the following steps:

[0086] S1: Dissolve 0.8mmol of CuCl2·2H2O and 0.4mmol of 2-aminoterephthalic acid in 16mL of DMF solution A, then dissolve 0.4mmol of histidine in a mixture of 32mL of ethanol and DMF solution B (ratio 1:1), and then slowly drip solution A into solution B under constant stirring. Then transfer the mixture to a sealed Teflon-autoclave and heat at 120℃ for 8 hours. The autoclave is cooled naturally. The product is centrifuged at 10000rpm / min for 5min, the precipitate is washed 3 times with alcohol and water, dried and preserved to obtain Cu-BH nanozyme;

[0087] S2: Then, 0.05g Cu-BH, 0.2mmol ethyl carbamate and 10.0mL ethanol were added to a 25.0mL flask and sonicated until dissolved. 0.8mmol sodium salt of carboxyethylsilanetriol and 2.4mmol tetraethyl orthosilicate were placed in a flask, stirred continuously for 30.0min in the dark, and then 200.0μL of ammonia water was added and stirred continuously at 25.0℃ 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 Cu-BH@MIP precipitate. The polymer was dried in a vacuum chamber at 60.0℃ overnight to obtain Cu-BH@MIP nanozyme.

[0088] 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 method for detecting ethyl carbamate in alcoholic beverages based on a laccase-like active nanozyme dual modality, characterized in that: The steps include: Step 1: Determine the absorbance value A of the urethane standard solution with different concentration gradients at 420nm: At room temperature, add Cu-BH@MIP nanozyme, o-phenylenediamine and ethyl carbamate standards with different concentration gradients into HEPES buffer, stir evenly to obtain a mixed solution, and then transfer it to a cuvette; then use an ultraviolet spectrophotometer to measure the absorbance value at 420nm, which is recorded as A; Step 2: Determine the fluorescence intensity signal ratio F of the urethane standard solution with different concentration gradients at 420nm and 550nm under 330nm wavelength excitation 420nm / F 550nm ; At room temperature, Cu-BH@MIP nanozyme, o-phenylenediamine and ethyl formate standards with different concentration gradients were added to HEPES buffer, stirred evenly to obtain a mixed solution, and then transferred to a cuvette; then, the fluorescence intensity signal values ​​at 420nm and 550nm were measured using a fluorescence spectrometer at an excitation wavelength of 330nm to obtain the fluorescence intensity signal ratio F 420nm / F 550nm ; Step 3: Construct the linear regression equation for the standard The UV regression equation A=XC was constructed by using the absorbance value A value measured by the urethane standard with different concentration gradients and the concentration of the standard. EC +y, where C EC is the concentration of the standard; The fluorescence intensity signal ratio F measured by using different concentration gradients of ethyl formate standards 420nm / F 550nm The linear regression equation F of the ratio fluorescence signal was constructed based on the concentration of the standard 420nm / F 550nm =XC EC +y, where C EC is the concentration of the standard; Step 4: Take the sample to be tested and repeat steps 1 and 2 to obtain the absorbance value of the sample to be tested and the fluorescence intensity signal ratio F of the sample to be tested. 420nm / F 550nm , respectively, and substituted into the corresponding linear equation to obtain the concentration of ethyl carbamate in the sample to be tested.

2. The method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes according to claim 1, characterized in that: In step 1 and step 2, the pH value of HEPES buffer is 8.8-9.2; the concentration of o-phenylenediamine is 10.0 mmol / L; the concentration of Cu-BH@MIP nanozyme is 100 μg / mL; the total amount of the mixed solution is 200.0 μL; the reaction temperature during stirring is 50.0°C; and the reaction time is 27.0 min.

3. The method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes according to claim 1, characterized in that: In step 3, the regression equation for ultraviolet radiation is A = -0.00201C EC +1.6875; the linear regression equation of the ratio fluorescence signal is F 420nm / F 550nm =00306C EC +0.52762.

4. The method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes according to claim 1, characterized in that: The preparation method of the Cu-BH@MIP nanozyme comprises the following steps: S1: Dissolve CuCl2·2H2O and 2-aminoterephthalic acid in DMF solution to obtain solution A, and dissolve histidine in a mixture of ethanol and DMF solution to obtain solution B; then add solution A to solution B under constant stirring; then transfer the mixture to a sealed Teflon-autoclave and heat at 100-140°C for 5-12 hours; after the autoclave is naturally cooled, the obtained product is centrifuged, and the precipitate is washed with alcohol and water, dried and stored to obtain Cu-BH nanozyme; S2: Cu-BH nanozyme, ethyl carbamate and ethanol are added to a container and sonicated until dissolved; sodium carboxyethylsilanetriol and tetraethyl orthosilicate are placed in the container, stirred continuously for 25-35 minutes in the dark, then ammonia water is added and stirred continuously overnight at 20-30°C; the precipitate is washed with ethanol, centrifuged and the supernatant is discarded; the original EC template in the precipitate is extracted by methanol Soxhlet extraction to obtain Cu-BH@MIP precipitate; the polymer is dried in a vacuum chamber at 55-65°C overnight to obtain Cu-BH@MIP nanozyme.

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