Rapid electrochemical detection method for vomitoxin based on enzymatic electropolymerization signal
By using an enzyme-catalyzed electropolymerization signal method, this method utilizes an enzyme-labeled vomitoxin and a molecularly imprinted polymer-modified electrode for electrochemical detection, solving the problems of long detection time and dependence on biological antibodies in existing technologies, and achieving rapid, simple and highly sensitive detection of vomitoxin.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- HUIZHOU DINGSHUO IND CO LTD
- Filing Date
- 2026-02-28
- Publication Date
- 2026-05-08
AI Technical Summary
In existing technologies, methods for detecting vomitoxin suffer from problems such as expensive instruments, cumbersome operation, and long processing time. Furthermore, immunoassay methods, which rely on biological antibodies, have drawbacks such as long preparation cycles, high costs, and easy inactivation, making it difficult to meet the application needs of rapid screening and complex food matrices.
The method employs an enzyme-catalyzed electropolymerization signal, which involves mixing enzyme-labeled vomitoxin with the sample solution to form a competitive reaction system. The system is then incubated using a working electrode modified with a vomitoxin molecularly imprinted polymer. After cleaning, the electrode is transferred to a detection solution containing electropolymerized monomers and enzyme-labeled substrate to form a polymer film. Quantitative detection is then performed based on changes in the electrochemical signal.
It achieves high sensitivity, good stability, simple operation, and rapid detection of vomitoxin, and does not rely on biological antibodies, thus meeting the needs of rapid detection.
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Abstract
Description
Technical Field
[0001] This application relates to the field of detection technology, and in particular to a rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals. Background Technology
[0002] Vomitoxin (DON), also known as deoxynivalenol, is a common mycotoxin found in grains and their products, posing a serious threat to food safety and human health. Currently, the main methods for detecting vomitoxin include high-performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and enzyme-linked immunosorbent assay (ELISA).
[0003] While chromatography offers high accuracy, it suffers from drawbacks such as expensive equipment, complex pretreatment, cumbersome operation, and long processing times, making it difficult to meet the needs of rapid on-site screening. Immunoassay, although relatively simple to operate, relies heavily on biological antibodies. However, biological antibodies have inherent limitations, including long preparation cycles, high costs, significant batch-to-batch variability, stringent storage conditions (requiring cold chain transportation), and susceptibility to inactivation in non-aqueous phases or organic solvents, restricting their widespread application in complex food matrices. Summary of the Invention
[0004] This application provides a rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals, in order to solve the technical problem of immunoassay relying on biological antibodies.
[0005] In a first aspect, this application provides a rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals, characterized in that it includes: S1, the sample solution to be tested is mixed with enzyme-labeled vomitoxin of a predetermined concentration to form a competitive reaction system, wherein the enzyme in the enzyme-labeled vomitoxin can catalyze the enzyme-labeled substrate to produce an active substance that initiates the electropolymerization of monomers; S2, the working electrode is placed in the competitive reaction system for incubation, so that the free vomitoxin in the sample solution to be tested competes with the enzyme-labeled vomitoxin to bind to the specific recognition site on the working electrode, thereby obtaining the target working electrode, wherein the working electrode is modified with a vomitoxin molecularly imprinted polymer. S3, after removing the unbound material from the target working electrode, place it in a detection solution containing electropolymerized monomers and enzyme-labeled substrate, so that the enzyme-labeled vomitoxin on the target working electrode catalyzes the enzyme-labeled substrate to produce the active substance, and the active substance initiates the in-situ electropolymerization of the electropolymerized monomers on the surface of the target working electrode to form a polymer film; S4. Electrochemical methods are used to measure the changes in electrochemical signals of the polymer film before and after its formation, and based on the changes in electrochemical signals, the concentration of vomitoxin in the sample solution to be tested is quantitatively detected.
[0006] In some of these embodiments, the sample solution to be tested in S1 is prepared based on the following steps: The grain sample to be tested was crushed into sample powder; Add methanol-water solution or ethyl acetate to the sample powder, vortex to mix, and then perform extraction by shaking for 3 min to 5 min, followed by centrifugation at 2000 rpm to 4000 rpm for 1 min to 3 min. Collect the supernatant to obtain the sample solution to be tested.
[0007] In some of the embodiments, the enzyme-labeled vomitoxin in S1 is glucose oxidase-labeled vomitoxin, the enzyme-labeled substrate is glucose, and the active substance is hydrogen peroxide.
[0008] In some of these embodiments, the predetermined concentration of enzyme-labeled vomitoxin in S1 is 0.1 μg / mL to 5 μg / mL.
[0009] In some of these embodiments, the electropolymerization monomer in S3 is o-phenylenediamine, a phenylenediamine derivative, or pyrrole.
[0010] In some embodiments, the working electrode in S2 is prepared based on the following steps: On the surface of a bare electrode or an electrode modified with nanomaterials, a molecularly imprinted polymer film is formed by electropolymerization using vomitoxin as a template molecule and functional monomers. The template molecules are eluted to remove them, forming a cavity that has a specific ability to recognize vomitoxin, thus obtaining the working electrode.
[0011] In some of these embodiments, the functional monomer is one or more of arginine, acrylamide, o-phenylenediamine, dopamine, and pyrrole, and the nanomaterial is one or more of carboxylated carbon nanotubes, graphene oxide, and gold nanoparticles.
[0012] In some embodiments, the incubation time in S2 is 5 min to 20 min; and the polymer film formation time in S3 is 1 min to 10 min.
[0013] In some embodiments, the electrochemical method in S4 is square wave voltammetry, differential pulse voltammetry, or AC impedance spectroscopy; the change in the electrochemical signal is a decrease in peak current or an increase in electron transport impedance.
[0014] In some embodiments, the quantitative detection of the free vomitoxin concentration in the test sample solution based on the electrochemical signal change in S4 includes: Based on a preset correspondence between changes in electrochemical signals and the amount of enzyme-labeled vomitoxin, the amount of enzyme-labeled vomitoxin corresponding to the changes in electrochemical signals is determined, wherein the amount of enzyme-labeled vomitoxin is the amount of enzyme-labeled vomitoxin that binds to specific recognition sites on the working electrode. Based on the linear relationship between the amount of enzyme-labeled vomitoxin and the concentration of free vomitoxin, the concentration of free vomitoxin in the test sample solution is determined, wherein the expression for the linear relationship is: ; The concentration of free vomitoxin in the sample solution to be tested. The concentration of enzyme-labeled vomitoxin in the competitive reaction system. This represents the total number of specific recognition sites on the working electrode. The amount of enzyme-labeled vomitoxin that binds to the specific recognition site on the working electrode.
[0015] Compared with the prior art, this application has the following beneficial effects: Free vomitoxin in the sample to be tested is mixed with a predetermined concentration of glucose oxidase-labeled vomitoxin to form a competitive reaction system. When a working electrode modified with a vomitoxin molecularly imprinted polymer is incubated in this system, the free toxin and the enzyme-labeled toxin compete for binding to specific recognition sites on the electrode surface. After cleaning, the electrode is transferred to a detection solution containing electropolymerized monomers and enzyme-labeled substrates. The enzyme bound to the electrode catalyzes the enzyme-labeled substrates to generate active substances, which trigger in-situ electropolymerization of the electropolymerized monomers on the electrode surface to form an insulating polymer film. This polymer film hinders electron transfer, resulting in changes in the electrochemical signal.
[0016] Since the total number of specific recognition sites on the working electrode is constant, the ratio of the number of free vomitoxins bound to these sites to the number of enzyme-labeled vomitoxins is almost equal to the ratio of the free vomitoxin concentration to the enzyme-labeled vomitoxin concentration (specifically, a predetermined concentration). Therefore, the higher the free vomitoxin concentration, the less enzyme-labeled vomitoxin binds to the sites, resulting in a thinner polymer film and a smaller signal drop. Thus, quantitative detection of free vomitoxins can be achieved by measuring the change in the electrochemical signal. This method cleverly combines the high selectivity of molecular imprinting, the signal amplification of enzyme catalysis, and the signal conversion advantages of in-situ electropolymerization. It does not rely on biological antibodies and has significant advantages such as high sensitivity, good stability, simple operation, and rapid detection.
[0017] Meanwhile, the preparation process of the sample solution and working electrode in this application is simple and efficient, requiring no special steps; the enzyme-labeled substrate, reactive enzyme, electropolymerized monomer and functional monomer are all readily available substances, and there are no problems such as difficult storage and easy inactivation; quantitative analysis can be performed through linear relationship, without the need for complicated data post-processing, which meets the requirements of high stability, simple operation and rapid detection of vomitoxin. Detailed Implementation
[0018] To make the above-mentioned objects, features, and advantages of the present invention more apparent and understandable, specific embodiments of the present invention are described in detail below. Many specific details are set forth in the following description to provide a thorough understanding of the present invention. However, the present invention can be practiced in many other ways different from those described herein, and those skilled in the art can make similar modifications without departing from the spirit of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.
[0019] As used herein, the terms “prepared from” and “comprising” are synonymous. The terms “comprising,” “including,” “having,” “containing,” or any other variations thereof, as used herein, are intended to cover non-exclusive inclusion. For example, a composition, step, method, article, or apparatus that includes the listed elements is not necessarily limited to those elements, but may include other elements not expressly listed or elements inherent to such composition, step, method, article, or apparatus.
[0020] When a quantity, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pair of any upper or preferred value with any lower or preferred value, regardless of whether the range is disclosed individually. For example, when the range “1 to 5” is disclosed, the described range should be interpreted as including the ranges “1 to 4”, “1 to 3”, “1 to 2”, “1 to 2 and 4 to 5”, “1 to 3 and 5”, etc. When numerical ranges are described herein, unless otherwise stated, the range is intended to include its endpoints and all integers and fractions within that range.
[0021] Furthermore, the indefinite articles “a” and “an” preceding the elements or components of this invention do not impose any limitation on the quantity requirement (i.e., the number of times) of the elements or components. Therefore, “an” or “a” should be interpreted as including one or at least one, and the singular form of an element or component also includes the plural form, unless the quantity clearly refers to the singular form.
[0022] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.
[0023] Preparation of working electrode: The glassy carbon electrode was polished on the surface of deer hide containing 0.05 μm alumina slurry, ultrasonically cleaned in ethanol and deionized water for 5 min in sequence, and dried under infrared lamp. 1 mg of carboxylated carbon nanotubes were dispersed in 1 mL of dimethylformamide solution containing 0.3 mg of carboxymethyl cellulose, vortexed for 2 min and sonicated for 15 min, and 5 μL was dropped onto the electrode surface and dried by infrared drying to obtain the modified electrode. Using vomitoxin as a template molecule (1 mM) and arginine as a functional monomer (5 mM), in 0.1 M In an electrolyte solution, a molecularly imprinted polymer film was formed by polymerization at a scan rate of 150 mV / s for 5 cycles in a potential range of -1.5 V to +2.3 V using cyclic voltammetry. The electrode was eluted in acetonitrile for 15 min to remove template molecules, thus obtaining the working electrode. Example 1
[0024] A corn sample known to be free of vomitoxin was crushed, 5 g of sample powder was weighed, 25 mL of methanol aqueous solution was added, the mixture was vortexed and shaken for 3 min, centrifuged at 4000 rpm for 2 min, the supernatant was collected, and 0.1 ng / g, 1 ng / g, and 10 ng / g of vomitoxin standard were added respectively. Three parallel samples were prepared for each concentration as the test sample solution. The glucose oxidase-labeled vomitoxin (GOD-DON) was diluted to 1 μg / mL with phosphate buffer and mixed with an equal volume of the sample solution to form a competitive reaction system. The working electrode was immersed in the competitive reaction system and incubated for 10 min to allow free DON to compete with GOD-DON for binding to the recognition sites on the electrode surface. Remove the electrode, wash it with PBS buffer to remove unbound material, and then transfer it to a detection solution containing 5 mM o-phenylenediamine and 10 mM glucose. Let it stand for 5 min to allow the GOD bound to the electrode to catalyze the formation of glucose. This initiates in-situ electropolymerization of o-phenylenediamine, forming a poly-o-phenylenediamine film; The peak current change before and after the formation of the poly(o-phenylenediamine) film was measured using the square wave voltammetry method, and the concentration of free vomitoxin was quantified based on the peak current. Example 2
[0025] The concentration of GOD-DON was 2 μg / mL, and the incubation time was 15 min. The electropolymerization monomer was a phenylenediamine derivative (3,4-diaminobenzoic acid) with a concentration of 8 mM and a glucose concentration of 15 mM. The polymerization time was 8 min. The remaining steps were the same as in Example 1. Example 3
[0026] The concentration of GOD-DON was 0.5 μg / mL, and the incubation time was 20 min. The electropolymerization monomers were pyrrole (20 mM) and glucose (5 mM), and the polymerization time was 3 min (potential constant polymerization, +0.8 V). The peak current change was measured by differential pulse voltammetry. The remaining steps were the same as in Example 1.
[0027] Comparative Example 1 Compared with Example 1, Comparative Example 1 used DON monoclonal antibody instead of vomitoxin molecularly imprinted polymer as recognition unit and was fixed on the electrode surface by physical adsorption; the remaining steps were the same as in Example 1.
[0028] Comparative Example 2 Compared to Example 1, the test solution in Comparative Example 2 contained only glucose (without electropolymerized monomers), and the production of glucose catalyzed by GOD was directly measured using chronoamperometry. The concentration of free vomitoxin is quantified based on the oxidation current on the electrode; the remaining steps are the same as in Example 1.
[0029] Table 1. Spike recoveries and RSDs of Examples 1 to 3 and Comparative Examples 1 to 2
[0030] As shown in Table 1, Examples 1 to 3 have better performance in terms of recovery rate and RSD than Comparative Examples 1 to 2, indicating that Examples 1 to 3 have better accuracy and precision. Example 4
[0031] Compared with Example 1, in Example 4, five commercially available corn samples containing vomitoxin (numbered N1~N5, and the concentration of vomitoxin in each sample was verified by HPLC-MS / MS) were pulverized, 5 g of sample powder was weighed from each sample, 25 mL of methanol aqueous solution was added, the mixture was vortexed and shaken for 3 min, centrifuged at 4000 rpm for 2 min, and the supernatant was collected as the sample solution to be tested; the remaining steps were the same as in Example 1.
[0032] Comparative Example 3 Compared with Example 4, Comparative Example 3 weighed 5g of each of the corn sample powder pulverized in Example 4 and used enzyme-linked immunosorbent assay (ELISA) instead of this method. The ELISA used vomitoxin monoclonal antibody as the biological antibody.
[0033] Table 2 Comparison of results from Example 4 and ELISA method (using HPLC-MS / MS as reference method)
[0034] As shown in Table 2, compared with the HPLC-MS / MS reference method, Example 4 exhibits a relative error range of -4.4% to +1.6%, with an average absolute error of only 2.1%, indicating high accuracy and close proximity to the internationally recognized reference method. The ELISA (Comparative Example 3) showed a relative error range of -17.6% to -3.6%, with an average absolute error of 7.5%, especially with low-concentration sample S4 (0.91 ng / g), where the error reached -17.6%, significantly lower than this method. This demonstrates that the accuracy and precision of this method in real-world sample detection are significantly superior to the traditional ELISA method.
[0035] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.
[0036] The specific embodiments described above further illustrate the purpose, technical solution, and beneficial effects of this application. It should be understood that the above descriptions are merely specific embodiments of this application and are not intended to limit the scope of protection of this application. In particular, it should be noted that any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of protection of this application for those skilled in the art.
Claims
1. A rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals, characterized in that, include: S1, the sample solution to be tested is mixed with enzyme-labeled vomitoxin of a predetermined concentration to form a competitive reaction system, wherein the enzyme in the enzyme-labeled vomitoxin can catalyze the enzyme-labeled substrate to produce an active substance that initiates the electropolymerization of monomers; S2, the working electrode is placed in the competitive reaction system for incubation, so that the free vomitoxin in the sample solution to be tested competes with the enzyme-labeled vomitoxin to bind to the specific recognition site on the working electrode, thereby obtaining the target working electrode, wherein the working electrode is modified with a vomitoxin molecularly imprinted polymer. S3, after removing the unbound material from the target working electrode, place it in a detection solution containing electropolymerized monomers and enzyme-labeled substrate, so that the enzyme-labeled vomitoxin on the target working electrode catalyzes the enzyme-labeled substrate to produce the active substance, and the active substance initiates the in-situ electropolymerization of the electropolymerized monomers on the surface of the target working electrode to form a polymer film; S4. Electrochemical methods are used to measure the changes in electrochemical signals of the polymer film before and after its formation, and based on the changes in electrochemical signals, the concentration of free vomitoxin in the sample solution to be tested is quantitatively detected.
2. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 1, characterized in that, The sample solution to be tested in S1 is prepared based on the following steps: The grain sample to be tested was crushed into sample powder; Add methanol-water solution or ethyl acetate to the sample powder, vortex to mix, and then perform extraction by shaking for 3 min to 5 min, followed by centrifugation at 2000 rpm to 4000 rpm for 1 min to 3 min. Collect the supernatant to obtain the sample solution to be tested.
3. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 1, characterized in that, The enzyme-labeled vomitoxin in S1 is glucose oxidase-labeled vomitoxin, the enzyme-labeled substrate is glucose, and the active substance is hydrogen peroxide.
4. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 3, characterized in that, The predetermined concentration of enzyme-labeled vomitoxin in S1 is 0.1 μg / mL to 5 μg / mL.
5. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 3, characterized in that, The electropolymerization monomer in S3 is o-phenylenediamine, phenylenediamine derivatives, or pyrrole.
6. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 1, characterized in that, The working electrode in S2 is prepared based on the following steps: On the surface of a bare electrode or an electrode modified with nanomaterials, a molecularly imprinted polymer film is formed by electropolymerization using vomitoxin as a template molecule and functional monomers. The template molecules are eluted to remove them, forming a cavity that has a specific ability to recognize vomitoxin, thus obtaining the working electrode.
7. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 6, characterized in that, The functional monomer is one or more of arginine, acrylamide, o-phenylenediamine, dopamine, and pyrrole, and the nanomaterial is one or more of carboxylated carbon nanotubes, graphene oxide, and gold nanoparticles.
8. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 1, characterized in that, The incubation time in S2 is 5 min to 20 min; the polymer film formation time in S3 is 1 min to 10 min.
9. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 1, characterized in that, The electrochemical method in S4 is square wave voltammetry, differential pulse voltammetry, or AC impedance spectroscopy; the change in the electrochemical signal is a decrease in peak current or an increase in electron transport impedance.
10. The rapid electrochemical detection method for vomitoxin based on enzyme-catalyzed electropolymerization signals as described in claim 1, characterized in that, The quantitative detection of free vomitoxin concentration in the test sample solution based on the electrochemical signal change in step S4 includes: Based on a preset correspondence between changes in electrochemical signals and the amount of enzyme-labeled vomitoxin, the amount of enzyme-labeled vomitoxin corresponding to the changes in electrochemical signals is determined, wherein the amount of enzyme-labeled vomitoxin is the amount of enzyme-labeled vomitoxin that binds to specific recognition sites on the working electrode. Based on the linear relationship between the amount of enzyme-labeled vomitoxin and the concentration of free vomitoxin, the concentration of free vomitoxin in the test sample solution is determined, wherein the expression for the linear relationship is: ; The concentration of free vomitoxin in the sample solution to be tested. The concentration of enzyme-labeled vomitoxin in the competitive reaction system. This represents the total number of specific recognition sites on the working electrode. The amount of enzyme-labeled vomitoxin that binds to the specific recognition site on the working electrode.