A method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes

Abstract

A method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes comprises the following steps: Step 1: Determine the absorbance value A of the test solution of ethyl carbamate standard solution with different concentration gradients at 420nm; Step 2: Determine the fluorescence intensity signal ratio F at 420nm and 550nm of the test solution of ethyl carbamate standard solution with different concentration gradients under 330nm wavelength excitation 420nm / F 550nm ; Step 3: Construct a linear regression equation for 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, are substituted into the corresponding linear equations to obtain the concentration of ethyl carbamate in the sample to be tested. The dual-modal detection of the present invention combines the advantages of colorimetry and fluorescence, improving the comprehensiveness and accuracy of detection. Colorimetry provides intuitive visual information but has limited sensitivity; fluorescence, on the other hand, 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.

Description

Technical Field

[0001] The present invention belongs to the technical field of environmental and food pollution detection, and relates to a method for dual-modal detection of ethyl carbamate based on laccase-like activity nanoenzyme. 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 inhalation or the food chain, posing a potential exposure risk to public health. Long-term exposure to EC has been shown to cause serious harm to organisms. In 2007, the International Agency for Research on Cancer (IARC) officially upgraded the classification of EC from a Group 2B carcinogen to a Group 2A carcinogen, sparking widespread concern about the safety of EC in food. Several countries, including Canada, France, and Japan, have set a maximum limit of 150 μg / L for EC in distillates. Given the ubiquitous presence of EC in alcoholic beverages, rapid and effective detection methods are needed to address the problem of EC residues. Currently, the detection of EC in alcoholic beverages relies primarily on methods such as spectroscopy, high-performance liquid chromatography (HPLC), gas chromatography / mass spectrometry (GC / MS), potentiometry, and conductimetry. However, these existing technologies are often limited by complexity and high cost, hindering their widespread adoption 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 to natural enzymes, nanozymes exhibit advantages such as increased stability, ease of storage, and greater cost-effectiveness. Currently, a variety of enzyme mimics have been studied, including peroxidases, oxidases, laccases, superoxide dismutases, and phosphohydrolases. Among them, laccase, a copper-containing oxidase that uses polyphenols or polyamines as substrates, has attracted considerable attention due to its wide substrate applicability and favorable environmental profile. Compared to conventional oxidases and peroxidases, laccase does not require the production of harmful reactive oxygen species in subsequent reactions, making it safer and more environmentally friendly. Fluorescent nanozymes are nanomaterials that possess both fluorescent and catalytic activity. Currently, most developed nanozymes retain traditional catalytic properties and are used in colorimetric assays. However, a single fluorescent signal has low sensitivity and is easily affected by environmental factors, leading to inaccurate measurement results. In contrast, ratiometric fluorescence technology, which measures the ratio of two different fluorescent signals, can effectively reduce interference from external factors and improve signal sensitivity and accuracy. Here, we synthesized a dual-fluorescent laccase-like 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. This method 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 ratiometric fluorescence. The Cu-BH@MIP nanozyme in the present invention has a fluorescence signal at 420nm under 330nm excitation light; at the same time, the presence of strong laccase activity can oxidize the colorless and non-fluorescent OPD to DAP, which is dark yellow and emits yellow fluorescence at 550nm, and quench the blue fluorescence of Cu-BH@MIP at 420nm through the inner filter effect, thereby constructing a ratiometric fluorescence signal F 420nm / F 550nm When ethyl carbamate is added, EC can specifically fill the imprinted cavity of Cu-BH@MIP, blocking the catalytic substrate OPD outside the Cu-BH@MIP, thereby preventing it from further reacting with the internal catalytic center Cu-BH, resulting in the inhibition of the conversion of OPD to DAP, a decrease in the DAP colorimetric signal, and a decrease in the ratiometric fluorescence signal F 420nm / F 550nm The enhancement of the activity of nanozymes with laccase-like activity can thus be used to develop a dual-modal method for the detection of ethyl carbamate.

[0005] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: a method for dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes, comprising the following steps:

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

[0007] At room temperature, the Cu-BH@MIP nanozyme, o-phenylenediamine, and urethane standards of varying concentrations were added to a HEPES buffer solution and stirred to obtain a mixed solution, which was then transferred to a cuvette. The absorbance at 420 nm was then measured using a UV spectrophotometer and recorded as A.

[0008] Step 2: Determine the fluorescence intensity signal ratio F of the test solution of ethyl carbamate standard 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 carbamate standards with different concentration gradients were added to HEPES buffer, stirred evenly to obtain a mixed solution, and then transferred to a cuvette. The fluorescence intensity signal values ​​at 420 nm and 550 nm were then measured using a fluorescence spectrometer at an excitation wavelength of 330 nm to obtain the fluorescence intensity signal ratio F. 420nm / F 550nm ;

[0010] Step 3: Construct a 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 was measured using different concentration gradients of ethyl carbamate 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 solution 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 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 continuous 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 carboxyethylsilanol triol 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 at 20-30°C overnight; the precipitate is washed with ethanol and centrifuged to discard the supernatant; 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 the background change. 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 to patent CN202311565150.2, especially in the ability to accurately identify target molecules in complex components and generate obvious signals. The experimental results show that Cu-BH@MIP has a three-dimensional cavity with high adaptability to EC molecules, which 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 from other sources and has good selectivity and anti-interference performance.

[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 this technology is less than 3 compared with that of patent CN202410427275.7. In addition, the harmful active oxygen such as O2 generated during the reaction of the traditional method - and OH - This technology operates under mild conditions (reactions are carried out at room temperature) without generating harmful reactive oxygen species. This greatly reduces damage to the sample and ensures the safety and environmental friendliness of the experiment.

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

[0025] Figure 1 The synthesis diagram of Cu-BH@MIP and the principle diagram of EC detection in 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, and B, C, D, E, and F are the distribution of C, N, O, Cu, and Si elements in Cu-BH@MIP nanozyme, respectively.

[0027] Figure 3 Illustrations of the UV absorption patterns and corresponding solution colors 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 for the verification of the laccase activity of Cu-BH@MIP nanozyme.

[0028] Figure 4 The 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 adding 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 are shown.

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

[0034] Figure 10 The effect of alcohol concentration on the dual-modal detection system. DETAILED DESCRIPTION

[0035] The following describes the implementation of the present invention through specific embodiments. People 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 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, they have no substantive 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 efficacy and purpose 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 this invention: CuCl2·2H2O (AR) and OPD were purchased from Shanghai MacLean Biochemical Technology Co., Ltd.; 2-aminoterephthalic acid and L-histidine were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and 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; all water used in the experiments 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) CuCl2·2H2O and 2-aminoterephthalic acid are dissolved in DMF solution to obtain solution A, and histidine is dissolved in a mixture of ethanol and DMF solution to obtain solution B; then, solution A is added to solution B under continuous stirring; then, the mixture is transferred to a sealed Teflon-autoclave and heated 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; (2) Cu-BH nanozyme, ethyl carbamate and ethanol were added to a container and sonicated until dissolved; sodium carboxyethylsilanol triol and tetraethyl orthosilicate were placed in the container and stirred continuously for 25-35 minutes under dark conditions, and then ammonia water was added and stirred continuously overnight at 20-30°C; the precipitate was washed with ethanol and centrifuged to discard the supernatant; 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 55-65°C overnight to obtain Cu-BH@MIP nanozyme.

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

[0044] Common laccase active substrates 2,4-dichlorophenol (2,4-DP) and 4-aminoantipyrine (4-AP) were used as colorimetric reagents and HEPES was used as buffer. The following five groups of reactions were taken: (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, the mixture was transferred to a cuvette and the absorbance at 500 nm was measured using a UV spectrophotometer to 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 concentrations of 2,4-DP and 4-AP were 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] Steady-state kinetics experiments were performed in HEPES buffer containing Cu-BH@MIP nanozymes by varying the OPD concentration (0.0-1.0 mmol / L). The OPD concentration-reaction rate doublet plot was calculated using the following Michaelis-Menten equation:

[0048]

[0049] 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 Cu-BH@MIP to OPD is shown 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 the added Cu-BH@MIP nanozyme 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 color developer and HEPES as the buffer. The following five reaction groups 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, demonstrating the feasibility of the detection system proposed in 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 EC Optimal Conditions for Cu-BH@MIP+OPD System Detection

[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. The results are shown in Figure 2. 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 range is between 30.0°C and 50.0°C, the absorbance value of the unreacted system at 420nm increases with the increase of reaction temperature. When the temperature exceeds 50.0°C and increases to 55.0°C, the absorbance value decreases as it increases, so 50.0°C is selected as the optimal temperature of the system. Then, the reaction time was optimized, and the results are shown in Figure 3. Figure 6 C. Figure 6 As shown in D, from 0.0 to 27.0 min, the absorbance value of the reaction system at 420 nm increases with the increase of reaction time, and basically no longer changes after 27.0 min. Therefore, 27.0 min is selected as the optimal reaction time of the system.

[0056] Example 6: Detection of EC Standards with Cu-BH@MIP+OPD System

[0057] EC solutions with 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 420 nm is measured using a UV spectrophotometer. As the concentration of EC increases, the absorbance at 420 nm decreases. Therefore, with absorbance A as the ordinate and EC concentration as the abscissa, a linear equation is fitted using Origin software. 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 ECThe results show that the relative sensitivity of the two reagents is 0.16875, the correlation coefficient is 0.99835, and the detection limit is 2.11 μg / L. Furthermore, as the concentration of EC increases, the solution exhibits a color gradient (from dark yellow to light yellow). This color change can be observed with the naked eye, enabling EC detection.

[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 horizontal axis is the linear equation fitting using Origin software. Figure 8 The concentration of EC and the fluorescence intensity F are shown 420nm / F 550nm It is linear in the range of 0-600.0μg / L, and the fluorescence intensity ratio F 420nm / F 550nm The regression equation is F 420nm / F 550nm =00306C EC The results show that the relative fluorescence intensity of the solution increased from yellow to blue, with a correlation coefficient of 0.9949 and a detection limit of 0.978 μg / L. Furthermore, as the concentration of EC increased, the solution exhibited a satisfactory fluorescence gradient (from yellow to blue), and the change in reagent fluorescence could be observed with the naked eye, enabling 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, 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 minutes and then transferred to a 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 results are as follows Figure 9 A. Figure 9 As shown in C, only the absorbance value A of EC decreased significantly, and the fluorescence intensity signal ratio F 420nm / F 550nmThe results show that the 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. Figure 9 B. Figure 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 the added Cu-BH@MIP nanozyme 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. 20.0μL of EC solution with different alcohol concentrations was mixed with 100.0μg / mL Cu-BH@MIP nanozyme and 10.0mmol / 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 adjusted to 200.0μL. After complete reaction for 27.0min, the solution was transferred to a cuvette and the absorbance at 420nm was measured using a UV spectrophotometer. The results are as follows: Figure 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. Actual samples containing high concentrations of alcohol can be directly detected using this system.

[0065] Example 9: Detection of EC in actual samples using the 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 white wine as a representative of actual samples. Among them, the white wine in this work was randomly extracted from a winery in Hefei, China, without purification. Take 1mL of white 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, the reaction lasted for 27.0min and then transferred to a 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 equations, 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 of 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, the reaction lasted for 27.0min and then transferred to a 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 equations, 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 of 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, the reaction was carried out for 27.0min and then transferred to a 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 equations, 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] The results show that the reaction system can be applied to the detection of actual samples, and the recovery rate is between 98% and 103%, with a relative deviation of less than 3.

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

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

[0075] At room temperature, the Cu-BH@MIP nanozyme, o-phenylenediamine, and varying concentrations of ethyl carbamate were added to a HEPES buffer solution for reaction. The mixture was stirred thoroughly and allowed to react for 27.0 minutes before being transferred to a cuvette. The absorbance at 420 nm was measured using a UV spectrophotometer and recorded as A.

[0076] Step 2: Determine the fluorescence intensity signal ratio F of the test solution of ethyl carbamate standard 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 different concentrations of ethyl carbamate were added to a HEPES buffer for reaction. The mixture was thoroughly stirred and the reaction lasted for 27.0 minutes 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 a 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 was measured using different concentration gradients of ethyl carbamate 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 colorimetric and ratio fluorescence signals, and substitute them into the corresponding linear equations to obtain the concentration of ethyl carbamate in the sample to be tested.

[0082] A 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] A 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 of 1:1), and then slowly drip solution A into solution B while stirring continuously. The mixture is then transferred to a sealed Teflon-autoclave and heated at 120°C 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, and dried and stored to obtain Cu-BH nanozyme;

[0087] S2: Next, 0.05 g of Cu-BH, 0.2 mmol of ethyl carbamate, and 10.0 mL of ethanol were added to a 25.0 mL flask and sonicated until dissolved. 0.8 mmol of sodium carboxyethylsilanol triol and 2.4 mmol of tetraethyl orthosilicate were placed in the flask and stirred continuously for 30.0 min in the dark. Then, 200.0 μL of ammonia water was added and stirred continuously at 25.0°C overnight. The precipitate was washed with ethanol, centrifuged, and the supernatant discarded. The original EC template in the precipitate was extracted using methanol Soxhlet extraction to obtain a Cu-BH@MIP precipitate. The polymer was dried in a vacuum chamber at 60.0°C overnight to obtain the 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 dual-modal detection of ethyl carbamate in alcoholic beverages based on laccase-like activity nanozymes, characterized by: The steps include: Step 1: Determine the absorbance value A of the test solution of ethyl carbamate standard with different concentration gradients at 420 nm: At room temperature, the Cu-BH@MIP nanozyme, o-phenylenediamine, and urethane standards with varying concentrations were added to HEPES buffer and stirred to obtain a mixed solution, which was then transferred to a cuvette. The absorbance at 420 nm was then measured using a UV spectrophotometer and recorded as A. Step 2: Determine the fluorescence intensity signal ratio F of the test solution of ethyl carbamate standard 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 carbamate standards with different concentration gradients were added to HEPES buffer, stirred evenly to obtain a mixed solution, and then transferred to a cuvette. The fluorescence intensity signal values ​​at 420 nm and 550 nm were then measured using a fluorescence spectrometer at an excitation wavelength of 330 nm to obtain the fluorescence intensity signal ratio F. 420nm / F 550nm ; Step 3: Construct a linear regression equation for the standard The UV regression equation A = XC was constructed 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 was measured using different concentration gradients of ethyl carbamate 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 substituted into the corresponding linear equation to obtain the concentration of ethyl carbamate in the sample to be tested; In steps 1 and 2, the pH value of the HEPES buffer was 8.8-9.2; the concentration of o-phenylenediamine was 10.0 mmol / L; the concentration of the Cu-BH@MIP nanozyme was 100 μg / mL; the total volume of the mixed solution was 200.0 μL; the reaction temperature during stirring was 50.0°C; and the reaction time was 27.0 min. In step 3, the regression equation for UV resistance is A = -0.00201C EC + 1.6875; the linear regression equation of the ratio fluorescence signal is F 420nm / F 550nm = 0.0306C EC + 0.52762; 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 continuous 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 carboxyethylsilanol triol 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 at 20-30°C overnight; the precipitate is washed with ethanol and centrifuged to discard the supernatant; 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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