A rapid detection method and enzyme tablet for ethyl carbamate based on aptamer-mediated regulation of V6O13 activity.

Abstract

This invention discloses a rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity. The method involves preparing a test sample A1 with a known target ethyl carbamate concentration; preparing a blank control sample A2; processing A1 and A2 using ImageJ to obtain regional grayscale values; plotting a standard curve of the change in target ethyl carbamate concentration versus grayscale value based on the grayscale value difference; establishing a regression equation based on the standard curve to determine the relationship between target ethyl carbamate concentration and grayscale value; preparing a test liquor solution C with an unknown target ethyl carbamate concentration; calculating the grayscale value difference between C and A2 using the regional grayscale value of C; and substituting this difference into the linear regression equation to obtain the target ethyl carbamate concentration in C. This invention offers advantages such as simple operation, high accuracy of detection results, high sensitivity and specificity for ethyl carbamate detection, short detection time, and visual identification, making it widely applicable for on-site testing.

Description

Technical Field

[0001] This invention relates to a rapid and convenient detection method for ethyl carbamate, particularly a rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity and an enzyme tablet. Background Technology

[0002] Ethyl carbamate (EC) is a Group 2A carcinogen, commonly found as a residue in fermented foods and spirits. Excessive exposure to EC can severely damage the skin and immune system. It can also be absorbed through the intestines and bloodstream, increasing the incidence of liver, lung, and skin malignancies. Therefore, the amount of EC residue in spirits is a major concern. Some countries, such as Canada, the United States, Japan, and Brazil, have set a maximum limit of 150 μg / L for EC in distilled spirits. The presence of interfering substances in spirits makes EC detection challenging, necessitating the development of new EC detection technologies. Currently, existing detection methods both domestically and internationally mainly rely on large instruments such as GC-MS, isotope dilution gas chromatography-mass spectrometry (ID GC-MS), and capillary electrophoresis. While these instruments offer high sensitivity and accurate results, they require complex pretreatment, specialized technicians, and expensive equipment, hindering rapid on-site detection of EC.

[0003] Aptamers are single-stranded DNA or RNA oligonucleotide sequences obtained through ligand phylogenetic analysis (SELEX) using exponential enrichment. They offer advantages such as high stability, safety, specificity, and non-toxicity. Our team recently screened for the nucleic acid aptamer (EC1) of endothelial cells (EC) using Capture-SELEX technology. Through molecular docking, the EC1 sequence was truncated into the more affinity EC1-34 aptamer, and a rapid EC detection test strip based on aptamer recognition was constructed and successfully applied to the rapid detection of EC in baijiu (Chinese liquor). However, the above method suffers from problems such as uneven AuNP color development, susceptibility to environmental influences, and interference in the detection results. Rapid detection of EC remains challenging, and there is an urgent need to find new sensing signal materials to construct rapid EC detection methods and new products. Summary of the Invention

[0004] The purpose of this invention is to provide a rapid detection method and enzyme tablet for ethyl carbamate based on aptamer-regulated V6O13 activity. This method utilizes V6O13... 13 Nanoribbons were used as recognition tools, nucleic acid aptamers as inhibitors of catalytic activity, and 3,3',5,5'-tetramethylbenzidine (TMB) was used as a substrate, along with V6O 13The -aptamer recognition probe is immobilized on a paper disc, and the entire detection process uses the paper disc as a carrier, requiring no grafting or modification. Furthermore, it provides a nanozyme disc that is simple to operate, highly sensitive, fast in detection, low in cost, and not limited by detection equipment, enabling rapid detection and on-site monitoring of ethyl carbamate.

[0005] The technical solution of this invention: A rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, which utilizes nucleic acid aptamers to regulate V6O13 activity. 13 The peroxidase activity of nanoribbons, combined with the specific recognition function of nucleic acid aptamers, enables rapid colorimetric detection of ethyl carbamate, specifically including the following steps:

[0006] (1) Plot a standard curve of the change in ethyl carbamate concentration versus gray value;

[0007] (2) Based on the standard curve of the change in ethyl carbamate concentration and gray value, establish a regression equation for the relationship between the change in ethyl carbamate concentration and gray value.

[0008] (3) Prepare a test solution with an unknown concentration of ethyl carbamate to obtain product C;

[0009] (4) Take product C and process it with software to obtain the gray value of the region. Calculate the difference in gray value between product C and the blank control group. Substitute the difference in gray value into the linear regression equation to obtain the concentration of ethyl carbamate in product C.

[0010] In the aforementioned rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, the specific method for step (1) of plotting the standard curve of ethyl carbamate concentration versus grayscale value change is as follows:

[0011] A1: Take multiple graduated centrifuge tubes, and add a certain amount of V6O of known concentration to each centrifuge tube. 13 Nanoribbons and nucleic acid aptamers were mixed and incubated to prepare V6O. 13 -aptamer recognition probes were used, and then the recognition probes were fixed on paper to prepare ethyl carbamate colorimetric nanozyme tablets. Then, ethyl carbamate standard solution of known concentration was added to the enzyme tablets. After reacting for a certain time, NaAc buffer was added, and finally H2O2 and substrate TMB were added to prepare the test group, which is A1 product.

[0012] A2: Take the already fixed V6O 13 The enzyme tablets for -aptamer recognition probes can be replaced with ultrapure water to replace the ethyl carbamate standard solution of known concentration. The blank control group, A2 product, can be prepared according to method A1.

[0013] A3: Take A1 and A2 products and process them separately using software to obtain the grayscale values ​​of the regions;

[0014] A4: Based on the gray values, plot the difference in gray values ​​between different concentrations of A1 and A2 products as the ordinate and the ethyl carbamate concentration as the abscissa to obtain a standard curve of target concentration versus gray value change.

[0015] In the aforementioned rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, in step (2), when the concentration C of ethyl carbamate is 20 μg / L ≤ C ≤ 200 μg / L, the regression equation for the relationship between its concentration and the change in gray value is: y = 0.003C - 0.077.

[0016] y represents the grayscale difference = grayscale value of the test sample containing the target ethyl carbamate - grayscale value of the blank control group.

[0017] In the aforementioned rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, the specific method for preparing the test solution C with an unknown concentration of ethyl carbamate in step (3) is as follows: replace the target ethyl carbamate standard solution with the actual sample solution of known concentration, and the test solution with an unknown target ethyl carbamate concentration can be prepared according to method A1, which is product C.

[0018] In the aforementioned rapid detection method for urethane based on aptamer-mediated regulation of V6O13 activity, the V6O 13 The concentration of the nanoribbon solution was 0.04 mg / mL; the concentration of the NaAc buffer was 20 mM, pH 4.0; the concentration of the H2O2 solution was 0.5 M; the concentration of the TMB solution was 35 mM; and the paper type of the enzyme sheet was CH-27.

[0019] In the aforementioned rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, the nucleic acid aptamer has a concentration of 6 μM and its sequence is: 5′-ACCGACCGTGCTGGACTCTGGGGGCACGGGAGGT-3′.

[0020] In the aforementioned rapid detection method for urethane based on aptamer-mediated regulation of V6O13 activity, the dosage ratio of each item used in the preparation of product A1, A2, or C is as follows: V6O 13 The amount of nanoribbon used is 15 μL, and the amount of nucleic acid aptamer used is 7 μL; V6O 13 The volume of the -aptamer recognition probe is 5 μL; the volumes of ethyl carbamate standard solution, ultrapure water, actual sample solution, and NaAc buffer are 10 μL; the volume of H2O2 solution is 12 μL; and the volume of TMB solution is 5 μL.

[0021] Based on aptamer-regulated V6O 13Active ethyl carbamate rapid detection enzyme tablets include paper discs on which V6O is immobilized. 13 -aptamer identification probe, V6O 13 -aptamer recognition probe consists of a certain amount of V6O at a known concentration 13 It is formed by mixing nanoribbons and nucleic acid aptamers and then incubating them.

[0022] The aforementioned aptamer-based regulation of V6O 13 In the active ethyl carbamate rapid detection enzyme tablet, the paper includes a base plate, an absorbent pad, and a sample pad, which are sequentially fixed to the base plate.

[0023] The aforementioned aptamer-based regulation of V6O 13 In the active ethyl carbamate rapid detection enzyme tablets, the paper discs are pre-treated with a processing solution and dried, and then treated with paraffin oil for hydrophobicity to form a hydrophobic ring. V6O is then added dropwise into this hydrophobic ring. 13 -aptamer recognition probes, after drying, yield nanozyme tablets.

[0024] The beneficial effects of this invention: Compared with the prior art, vanadium tridecyloxane (V6O) 13 Nanoribbons possess inherent peroxidase-mimicking activity and, due to their excellent physical, chemical, and electronic properties, are widely used in hydrogen storage, catalysis, and high-energy battery electrode materials. Considering the material's good catalytic activity, stability, and environmental tolerance, V6O... 13 Nanoribbons have also been used in bioanalysis and food safety testing in recent years.

[0025] The inventors of this application discovered that nucleic acid aptamer sequences can inhibit V6O 13 By combining the peroxidase activity of nanoribbons with the specific recognition function of nucleic acid aptamers (EC1-34), a novel rapid detection method was constructed. Subsequently, V6O... 13 A composite probe prepared from nanoribbons and EC1-34 was immobilized on a paper substrate, successfully developing an EC colorimetric nanozyme tablet. Furthermore, the inventors utilized this mechanism to develop a rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity. This rapid detection technology and enzyme tablet offer advantages such as simple operation, short color development time, low cost, and good selectivity, and the detection results can be interpreted visually.

[0026] The detection principle of this method is as follows: This application utilizes the EC1-34 aptamer sequence to regulate V6O 13 Peroxidase activity of nanoribbons, and regulation of V6O using this sequence. 13The peroxidase activity of nanoribbons was utilized to construct a rapid detection colorimetric enzyme tablet for the detection of ethyl carbamate. The aptamer sequence EC1-34 used in this method has two applications: as an inhibitor to regulate V6O 13 The catalytic activity of nanoribbons was observed, and they served as recognition elements for the binding of urethane. The EC1-34 aptamer sequence inhibited V6O via adsorption. 13 Nanoribbons catalyze the production of ·OH from H₂O₂, which reduces their affinity for 3,3',5,5'-tetramethylbenzidine (TMB), resulting in a decrease in the blue product (ox TMB) and causing the rapid detection enzyme tablets to appear light blue or even colorless. Once EC is added, EC preferentially binds to EC1-34, leading to V6O… 13 The nanoribbons have exposed active sites, catalyzing the production of ·OH from H₂O₂, generating a significant amount of ox TMB, resulting in a deep blue color on the rapid detection enzyme tablets. Furthermore, the color change of the constructed colorimetric nanozyme tablets is positively correlated with the EC concentration, thus this method can be used for the detection of ethyl carbamate. The average recovery rate of the prepared nanozyme tablets is within 95.5%-101%, with a relative standard deviation (RSD) ranging from 1.98%-3.56%, and its detection performance is similar to that of high-performance liquid chromatography (HPLC). This method can specifically detect ethyl carbamate despite interference from aspartic acid (Asp), proline (Pro), tyrosine (Tyr), glycine (Gly), glutamic acid (Glu), methyl carbamate (MC), formamide (Prop), carbaryl (CBR), carbofuran (CBF), and carbendazim (CBZ). The proposed rapid detection method and enzyme tablets are low-cost, simple to operate, and can be extended to detect other targets of interest by replacing the appropriate aptamers. Attached Figure Description

[0027] Appendix Figure 1 A schematic diagram illustrating the feasibility of detecting the target ethyl carbamate.

[0028] Appendix Figure 2 A schematic diagram for detecting the target ethyl carbamate;

[0029] Appendix Figure 3 The relationship between different concentrations of urethane and the regional grayscale difference (ΔG / G0) is shown.

[0030] Appendix Figure 4 A schematic diagram illustrating the effects of other interfering substances on the detection of urethane;

[0031] Appendix Figure 5 This is a schematic diagram illustrating the detection of target heavy metals in actual samples.

[0032] Appendix Figure 6 This is a schematic diagram of rapid detection nanozyme tablets.

[0033] Appendix Figure 1 and attached Figure 2 The principle speculation and feasibility study of this invention show that the method of this invention can be used for the detection of ethyl carbamate.

[0034] Appendix Figure 1 The following are the possible combinations: 1-Vanadium tridecyl oxide + hydrogen peroxide + substrate (TMB); 2-Vanadium tridecyl oxide + aptamer (EC1-34) + hydrogen peroxide + substrate (TMB); 3-Vanadium tridecyl oxide + aptamer (EC1-34) + ethyl carbamate (50 μg / L) + hydrogen peroxide + substrate (TMB); 4-Vanadium tridecyl oxide + aptamer (EC1-34) + ethyl carbamate (100 μg / L) + hydrogen peroxide + substrate (TMB); 5-Vanadium tridecyl oxide + aptamer (EC1-34) + ethyl carbamate (200 μg / L) + hydrogen peroxide + substrate (TMB); 6-Vanadium tridecyl oxide + ethyl carbamate (200 μg / L) + hydrogen peroxide + substrate (TMB).

[0035] Appendix Figure 3 The invention demonstrates that the linear range for detecting ethyl carbamate is 20-200 μg / L, with a detection limit of 20 μg / L, indicating that the invention has the characteristics of good stability and high sensitivity.

[0036] Appendix Figure 4 As can be seen, the method established in this invention can specifically detect ethyl carbamate, and other competing targets have almost no interference with the detection of ethyl carbamate.

[0037] Appendix Figure 5 As shown, the method of this invention was used to determine the recovery rate of light-aroma, sauce-aroma, and strong-aroma baijiu purchased from local supermarkets. 0 μg / L, 50 μg / L, and 100 μg / L of ethyl carbamate were added to the samples, respectively, and the recovery rate was 95.5%-101%, proving the reliability of this method. Detailed Implementation

[0038] The present invention will be further described below with reference to the accompanying drawings and embodiments, but this should not be construed as limiting the present invention.

[0039] Example 1 of the present invention: A rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, which regulates V6O13 activity through nucleic acid aptamers. 13 The peroxidase activity of nanoribbons, combined with the specific recognition function of nucleic acid aptamers, enables rapid colorimetric detection of ethyl carbamate, including the following steps:

[0040] (1) Plot a standard curve of the change in target ethyl carbamate concentration versus gray value. The standard curve is shown in the attached figure. Figure 3 As shown, it includes the following steps:

[0041] A1: Prepare a test solution with a known target concentration of ethyl carbamate to obtain product A1. Specifically, take multiple graduated centrifuge tubes and add 15 μL of 0.04 mg / mL V6O to each tube. 13 After mixing with 7 μL of 6 μM nucleic acid aptamer, the mixture was incubated at 37 °C for 20 min to prepare V6O. 13 -aptamer recognition probe. Then, 5 μL of the recognition probe was immobilized on a paper disc to prepare a urethane colorimetric nanozyme tablet. Next, 10 μL of urethane standard solution of known concentration was added to the enzyme tablet, and after reacting for 5 min, 10 μL of 20 mM (pH 4.0) NaAc buffer was added. Finally, 12 μL of 0.5 M H2O2 solution and 5 μL of 35 mM TMB solution were added to prepare the test group, which is sample A1.

[0042] A2: Secure V6O 13 The enzyme tablets for -aptamer recognition probes can be replaced with ultrapure water to replace the ethyl carbamate standard solution of known concentration. The blank control group, A2 product, can be prepared according to method A1.

[0043] A3: Take A1 and A2 products and process them using ImageJ software to obtain the grayscale values ​​of the regions;

[0044] A4: Based on the gray values, plot the difference in gray values ​​between different concentrations of A1 and A2 products as the ordinate and the ethyl carbamate concentration as the abscissa to obtain a standard curve of target concentration versus gray value change.

[0045] (2): A regression equation was established based on the standard curve of the target ethyl carbamate concentration and the change in gray value to determine the relationship between the target ethyl carbamate concentration and the change in gray value. When the ethyl carbamate concentration C is 20 μg / L ≤ C ≤ 200 μg / L, the regression equation for the relationship between its concentration and the change in gray value is: y = 0.003C - 0.077.

[0046] y is the gray value difference (ΔA) = gray value of the test sample containing the target ethyl carbamate - gray value of sample A2.

[0047] (3): Prepare a test solution (light-aroma baijiu) with an unknown target ethyl carbamate concentration to obtain product C. The specific preparation method is as follows: replace the target ethyl carbamate standard solution of known concentration with the actual sample solution, and prepare the test solution with an unknown target ethyl carbamate concentration according to method A1, which is product C.

[0048] (4): Take 10 μL of product C and add it to the nanozyme sheet. Use ImageJ to process the gray value of the area and calculate the difference between the gray values ​​of product C and product A2. Substitute the difference between the gray values ​​into the linear regression equation to obtain the concentration of the target ethyl carbamate in product C.

[0049] Example 2 of the present invention: A rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, which regulates V6O13 activity through nucleic acid aptamers. 13 The peroxidase activity of nanoribbons, combined with the specific recognition function of nucleic acid aptamers, enables rapid colorimetric detection of ethyl carbamate, including the following steps:

[0050] (1) Plot a standard curve of the change in target ethyl carbamate concentration versus gray value. The standard curve is shown in the attached figure. Figure 3 As shown, it includes the following steps:

[0051] A1: Prepare a test solution with a known target concentration of ethyl carbamate to obtain product A1. Specifically, take multiple graduated centrifuge tubes and add 15 μL of 0.04 mg / mL V6O to each tube. 13 After mixing with 7 μL of 6 μM nucleic acid aptamer, the mixture was incubated at 37 °C for 20 min to prepare V6O. 13 -aptamer recognition probe. Then, 5 μL of the recognition probe was immobilized on a paper disc to prepare a urethane colorimetric nanozyme tablet. Next, 10 μL of urethane standard solution of known concentration was added to the enzyme tablet, and after reacting for 5 min, 10 μL of 20 mM (pH 4.0) NaAc buffer was added. Finally, 12 μL of 0.5 M H2O2 solution and 5 μL of 35 mM TMB solution were added to prepare the test group, which is sample A1.

[0052] A2: Secure V6O 13 The enzyme tablets for -aptamer recognition probes can be replaced with ultrapure water to replace the ethyl carbamate standard solution of known concentration. The blank control group, A2 product, can be prepared according to method A1.

[0053] A3: Take A1 and A2 products and process them using ImageJ software to obtain the grayscale values ​​of the regions;

[0054] A4: Based on the gray values, plot the difference in gray values ​​between different concentrations of A1 and A2 products as the ordinate and the ethyl carbamate concentration as the abscissa to obtain a standard curve of target concentration versus gray value change.

[0055] (2): A regression equation was established based on the standard curve of the target ethyl carbamate concentration and the change in gray value to determine the relationship between the target ethyl carbamate concentration and the change in gray value. When the ethyl carbamate concentration C is 20 μg / L ≤ C ≤ 200 μg / L, the regression equation for the relationship between its concentration and the change in gray value is: y = 0.003C - 0.077.

[0056] y is the gray value difference (ΔA) = gray value of the test sample containing the target ethyl carbamate - gray value of sample A2.

[0057] (3): Prepare a test solution (sauce-flavored liquor) with an unknown target ethyl carbamate concentration to obtain product C. The specific preparation method is as follows: replace the target ethyl carbamate standard solution with the actual sample solution of known concentration, and prepare the test solution with an unknown target ethyl carbamate concentration according to method A1, which is product C.

[0058] (4): Take 10 μL of product C and add it to the nanozyme sheet. Use ImageJ to process the gray value of the area and calculate the difference between the gray values ​​of product C and product A2. Substitute the difference between the gray values ​​into the linear regression equation to obtain the concentration of the target ethyl carbamate in product C.

[0059] Example 3 of the present invention: A rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity, which regulates V6O13 activity through nucleic acid aptamers. 13 The peroxidase activity of nanoribbons, combined with the specific recognition function of nucleic acid aptamers, enables rapid colorimetric detection of ethyl carbamate, including the following steps:

[0060] (1) Plot a standard curve of the change in target ethyl carbamate concentration versus gray value. The standard curve is shown in the attached figure. Figure 3 As shown, it includes the following steps:

[0061] A1: Prepare a test solution with a known target concentration of ethyl carbamate to obtain product A1. Specifically, take multiple graduated centrifuge tubes and add 15 μL of 0.04 mg / mL V6O to each tube. 13 After mixing with 7 μL of 6 μM nucleic acid aptamer, the mixture was incubated at 37 °C for 20 min to prepare V6O. 13 -aptamer recognition probe. Then, 5 μL of the recognition probe was immobilized on a paper disc to prepare a urethane colorimetric nanozyme tablet. Next, 10 μL of urethane standard solution of known concentration was added to the enzyme tablet, and after reacting for 5 min, 10 μL of 20 mM (pH 4.0) NaAc buffer was added. Finally, 12 μL of 0.5 M H2O2 solution and 5 μL of 35 mM TMB solution were added to prepare the test group, which is sample A1.

[0062] A2: Secure V6O 13 The enzyme tablets for -aptamer recognition probes can be replaced with ultrapure water to replace the ethyl carbamate standard solution of known concentration. The blank control group, A2 product, can be prepared according to method A1.

[0063] A3: Take A1 and A2 products and process them using ImageJ software to obtain the grayscale values ​​of the regions;

[0064] A4: Based on the gray values, plot the difference in gray values ​​between different concentrations of A1 and A2 products as the ordinate and the ethyl carbamate concentration as the abscissa to obtain a standard curve of target concentration versus gray value change.

[0065] (2): A regression equation was established based on the standard curve of the target ethyl carbamate concentration and the change in gray value to determine the relationship between the target ethyl carbamate concentration and the change in gray value. When the ethyl carbamate concentration C is 20 μg / L ≤ C ≤ 200 μg / L, the regression equation for the relationship between its concentration and the change in gray value is: y = 0.003C - 0.077.

[0066] y is the gray value difference (ΔA) = gray value of the test sample containing the target ethyl carbamate - gray value of sample A2.

[0067] (3): Prepare a test solution (strong-aroma baijiu) with an unknown target ethyl carbamate concentration to obtain product C. The specific preparation method is as follows: replace the target ethyl carbamate standard solution of known concentration with the actual sample solution, and prepare the test solution with an unknown target ethyl carbamate concentration according to method A1, which is product C.

[0068] (4): Take 10 μL of product C and add it to the nanozyme sheet. Use ImageJ to process the gray value of the area and calculate the difference between the gray values ​​of product C and product A2. Substitute the difference between the gray values ​​into the linear regression equation to obtain the concentration of the target ethyl carbamate in product C.

[0069] In the above embodiments, V6O is used 13 The preparation method of the nanoribbon solution is as follows: Prepare the materials according to the following ratio: Dissolve 0.2275g of V₂O₅ powder in 18mL of distilled water, then place it under a magnetic stirrer, and successively add 1mL of H₂O₂ and 1mL of anhydrous ethanol. After the solution is mixed, transfer it to a 60mL polytetrafluoroethylene-lined reactor and react at 250℃ for 48h. Then filter the precipitate, wash it three times with anhydrous ethanol and distilled water respectively, and dry it under vacuum at 75℃ for 12h to obtain the powder. Weigh a certain amount of V₆O₅... 13 The nanoribbon powder is dissolved in ultrapure water and stored at room temperature for later use.

[0070] In the above embodiments, the specific method for obtaining the regional grayscale value in steps A3 and (4) is as follows: the sample to be tested is dropped onto the nanozyme sheet, and the grayscale values ​​of the negative control well (G0) and the sample well (G) are obtained by processing with Image J and Origin 9.0 software. Then, the colorimetric value ΔG / G0 is calculated to obtain its grayscale value. ΔG=G0-G.

[0071] In the preparation process of product A1, product A2, or product C in the above embodiments: V6O is added to a 1.5 mL centrifuge tube. 13 The solution and EC1-34 aptamer were thoroughly mixed and incubated at 37°C for 20 min for later use, completing the preparation of the recognition probe. The recognition probe complex was then immobilized on a paper disc that had been pre-treated with a processing solution, dried, and treated with paraffin oil for hydrophobicity to form a hydrophobic ring. 5 μL of V6O was then dropped into this hydrophobic ring. 13 The -aptamer recognition probe was dried at 37°C for 40 min to obtain nanozyme tablets. Then, ethyl carbamate standard solution, ultrapure water or actual sample solution were added dropwise, and the reaction was allowed to proceed for 5 min. After that, NaAc buffer was added, and finally, H2O2 and TMB solution were added dropwise in sequence.

[0072] In the above embodiments, the EC1-34 nucleic acid aptamer was purchased from Sangon Biotech (Shanghai) Co., Ltd., and its sequence is: 5′-ACCGACCGTGCTGGACTCTGGGGGCACGGGAGGT-3′. Before use, it was purified by high-performance liquid chromatography, centrifuged at 10,000 rpm for 10 min, and then dissolved in ultrapure water. All aptamers were denatured at 95℃ for 5 min before use, and then cooled to room temperature (25-30℃) for subsequent experiments.

[0073] The aptamer-based V6O modulation used in the above embodiments 13 Active ethyl carbamate rapid detection enzyme tablets include paper discs on which V6O is immobilized. 13 -aptamer identification probe, V6O 13 -aptamer recognition probe consists of a certain amount of V6O at a known concentration 13 It is formed by mixing nanoribbons and nucleic acid aptamers and then incubating them.

[0074] The paper sheet includes a base plate, an absorbent pad, and a sample pad, which are fixed to the base plate in sequence. The base plate, absorbent pad, and sample pad are cut to a uniform standard (1.3cm × 1.3cm).

[0075] The paper sheet was pre-treated with a processing solution and dried, and then treated with paraffin oil for hydrophobicity to form a hydrophobic ring. V6O was then dropped into this hydrophobic ring. 13-aptamer recognition probes, after drying, yield nanozyme tablets.

[0076] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any way. Any simple modifications, material changes, equivalent variations, or alterations made to the above embodiments based on the technical essence of the present invention without departing from the technical solution of the present invention shall still fall within the scope of the technical solution of the present invention.

[0077] Sequence list information:

[0078] DTD Version: V1_3

[0079] Filename: ethyl carbamate aptamer.xml

[0080] Software Name: WIPO Sequence

[0081] Software version: 2.3.0

[0082] Generation Date: 2023-09-19

[0083] Basic Information:

[0084] Current application / applicant file name: 12520000429203011T

[0085] Applicant's Name or Title: Guizhou University

[0086] Applicant's name or title / language:zh

[0087] Applicant's Name or Title / Latin Name: Guizhou University

[0088] Invention Title: Rapid Detection Method for Ethyl Carbamate Based on Aptamer-Regulated V6O13 Activity and Enzyme Tablet (zh)

[0089] Total sequence count: 1

[0090] sequence:

[0091] Serial Number (ID): 1

[0092] Length: 34

[0093] Molecular type: DNA

[0094] Feature location / qualifier:

[0095] -source,1.34

[0096] >mol_type, other DNA

[0097] >organism, synthetic construct

[0098] Residue:

[0099] accgaccgtg ctggactctg ggggcacggg aggt 34

[0100] END

Claims

1. A rapid detection method for ethyl carbamate based on aptamer-mediated regulation of V6O13 activity, characterized in that: Regulating V6O via nucleic acid aptamers 13 The peroxidase activity of nanoribbons, combined with the specific recognition function of nucleic acid aptamers, enables rapid colorimetric detection of ethyl carbamate, specifically including the following steps: (1) Plot a standard curve of the change in ethyl carbamate concentration versus gray value; (2) Based on the standard curve of the change in ethyl carbamate concentration and gray value, establish a regression equation for the relationship between the change in ethyl carbamate concentration and gray value; (3) Prepare a test solution with an unknown concentration of ethyl carbamate to obtain product C; (4) Take product C and process it with software to obtain the gray value of the region. Calculate the difference in gray value between product C and the blank control group. Substitute the difference in gray value into the linear regression equation to obtain the concentration of ethyl carbamate in product C. The specific method for plotting the standard curve of ethyl carbamate concentration versus grayscale value change in step (1) is as follows: A1: Take multiple graduated centrifuge tubes, and add a certain amount of V6O of known concentration to each centrifuge tube. 13 Nanoribbons and nucleic acid aptamers were mixed and incubated to prepare V6O. 13 -aptamer recognition probes were used, and then the recognition probes were fixed on paper to prepare ethyl carbamate colorimetric nanozyme tablets. Then, ethyl carbamate standard solution of known concentration was added to the enzyme tablets. After reacting for a certain time, NaAc buffer was added, and finally H2O2 and substrate TMB were added to prepare the test group, which is A1 product. A2: Take the already fixed V6O 13 The enzyme tablets for -aptamer recognition probes can be replaced with ultrapure water to replace the ethyl carbamate standard solution of known concentration. The blank control group, A2 product, can be prepared according to method A1. A3: Take A1 and A2 products and process them separately using software to obtain the grayscale values ​​of the regions; A4: Based on the gray values, plot the difference in gray values ​​between different concentrations of A1 and A2 products as the ordinate and the ethyl carbamate concentration as the abscissa to obtain a standard curve of target concentration versus gray value change.

2. The rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity according to claim 1, characterized in that: In step (2), when the concentration of ethyl carbamate C is 20 μg / L≤C≤200 μg / L, the regression equation for the relationship between its concentration and the change in gray value is: y=0.003C-0.077; y represents the grayscale difference = grayscale value of the test sample containing the target ethyl carbamate - grayscale value of the blank control group.

3. The rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity according to claim 1, characterized in that: In step (3), the specific method for preparing the test solution C with an unknown concentration of ethyl carbamate is as follows: replace the target ethyl carbamate standard solution with the actual sample solution of known concentration, and the test solution with an unknown target ethyl carbamate concentration can be prepared according to method A1, which is product C.

4. The rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity according to claim 1, characterized in that: The V6O 13 The concentration of the nanoribbon solution was 0.04 mg / mL; the concentration of the NaAc buffer was 20 mM, pH 4.0; the concentration of the H2O2 solution was 0.5 M; the concentration of the TMB solution was 35 mM; and the paper type of the enzyme sheet was CH-27.

5. The rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity according to claim 1, characterized in that: The nucleic acid aptamer has a concentration of 6 µM and its sequence is: 5′-ACCGACCGTGCTGGACTCTGGGGGCACGGGAGGT-3′.

6. The rapid detection method for ethyl carbamate based on aptamer-regulated V6O13 activity according to claim 1, characterized in that: In the preparation process of the aforementioned A1, A2, or C products, the ratio of each item used is: V6O 13 The amount of nanoribbon used is 15 μL, and the amount of nucleic acid aptamer used is 7 μL; V6O 13 The volume of the -aptamer recognition probe is 5 μL; the volumes of ethyl carbamate standard solution, ultrapure water, actual sample solution, and NaAc buffer are 10 μL; the volume of H2O2 solution is 12 μL; and the volume of TMB solution is 5 μL.

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