Electrochemical detection method of melamine

Abstract

The invention relates to an electrochemical detection method of melamine, and relates to the technical field of food detection and analysis detection. The method comprises the following steps: S10, obtaining a solution to be detected, a protein-labeled melamine antigen and an electrochemical sensor; the surface of a working electrode of the electrochemical sensor is modified with a melamine antibody; s20, mixing a solution to be detected with a quantitative protein-labeled melamine antigen, and adding the mixture to the surface of a working electrode of the electrochemical sensor; s30, measuring a first detection current value of the working electrode by adopting a differential pulse voltammetry; and S40, according to the first detection current value and a first standard current-melamine concentration curve of the electrochemical sensor, obtaining the detection concentration of melamine in the solution to be detected. According to the method provided by the invention, efficient and rapid melamine detection can be realized.

Description

Technical Field

[0001] The present disclosure relates to the technical field of food detection and analysis, and in particular to an electrochemical detection method for melamine. Background Art

[0002] Melamine is a chemical substance with a high nitrogen content, which is widely used in the production of industrial products such as plastics, coatings, and adhesives. In the production of dairy products, infant food, and animal feed, if melamine is added to increase the apparent value of protein detection, it will cause great food safety risks. Melamine is potentially toxic to the human body, especially after combining with uric acid to form an insoluble complex, it is easy to form stones in the kidneys, and in severe cases may lead to renal failure or even death. Therefore, the rapid and accurate detection of melamine is crucial to ensure the safety of food and feed. At present, the detection of melamine mainly relies on high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS). The samples to be tested need to undergo complex pre-treatment steps and high detection costs, which makes it difficult to meet the needs of rapid detection scenarios.

[0003] Therefore, there is an urgent need for a sensitive, low-cost, portable and rapid melamine detection technology to meet the needs of the food and feed industry for melamine detection. Electrochemical detection technology can convert and detect the presence of target molecules through electrical signals, without the need for complex sample preparation and processing, and has a high potential for on-site application.

[0004] It should be noted that the information disclosed in the above background technology section is only used to enhance the understanding of the background of the present disclosure, and therefore may include information that does not constitute the prior art known to ordinary technicians in the field. Summary of the invention

[0005] The purpose of the present invention is to overcome the deficiencies of the prior art and provide an electrochemical detection method for melamine to achieve rapid detection of melamine.

[0006] According to one aspect of the present disclosure, there is provided a method for electrochemical detection of melamine, comprising the following steps:

[0007] S10, obtaining a test solution, a protein-labeled melamine antigen, and an electrochemical sensor; the surface of the working electrode of the electrochemical sensor is modified with a melamine antibody;

[0008] S20, mixing the test solution with a quantitative amount of protein-labeled melamine antigen, and adding the mixture to the working electrode surface of the electrochemical sensor, so that the melamine antigen in the test solution and the protein-labeled melamine antigen competitively bind to the melamine antibody modified on the surface of the working electrode;

[0009] S30, measuring a first detection current value of the working electrode by using differential pulse voltammetry;

[0010] S40, obtaining the detection concentration of melamine in the test solution according to the first detection current value and a first standard current-melamine concentration curve of the electrochemical sensor.

[0011] According to one embodiment of the present disclosure, in step S10, the melamine antibody is modified on the surface of the working electrode by the following method:

[0012] forming a first modification layer on the surface of the working electrode, wherein the first modification layer comprises a gold modification layer;

[0013] A second modification layer is formed on the side of the first modification layer away from the surface of the working electrode, and the second modification layer includes melamine antibody.

[0014] According to one embodiment of the present disclosure, forming the second modified layer includes:

[0015] Drop-coating a mercaptopropionic acid solution on the gold modified layer of the working electrode for incubation, so that the surface of the gold modified layer is bound to mercaptopropionic acid through a gold-sulfur bond;

[0016] dripping a carboxylic acid activator on the surface of the working electrode so that the carboxyl group of mercaptopropionic acid forms an active ester functional group;

[0017] A melamine antibody solution is drop-coated on the surface of the working electrode to allow the melamine antibody to react with the active ester functional group and be fixed on the surface of the gold modified layer of the working electrode to form a second modified layer.

[0018] According to one embodiment of the present disclosure, forming the second modified layer further comprises:

[0019] After the surface of the gold modification layer is modified with mercaptopropionic acid, a 6-mercaptohexanol solution is drop-coated on the surface of the working electrode for incubation to seal the gold modification layer.

[0020] According to an embodiment of the present disclosure, the first standard current-melamine concentration curve is obtained by the following method: obtaining first standard current values ​​corresponding to a plurality of melamine standard solutions of different concentrations, and determining the first standard current-melamine concentration curve;

[0021] Obtaining the first standard current value corresponding to a melamine standard solution of any concentration includes:

[0022] Prepare a melamine standard solution with a preset concentration;

[0023] Mixing the melamine standard solution with a quantitative protein-labeled melamine antigen to form a standard mixed solution;

[0024] The standard mixed solution is added to the surface of the working electrode of the electrochemical sensor, and the current value corresponding to the working electrode is measured by differential pulse voltammetry to obtain a first standard current value corresponding to the melamine standard solution of a preset concentration.

[0025] According to one embodiment of the present disclosure, obtaining a protein-labeled melamine antigen comprises:

[0026] Mix the protein solution and excess sodium periodate solution evenly and react for 20 to 40 minutes in the dark;

[0027] Add excess ethylene glycol to the solution and react for 20 to 40 minutes in the dark;

[0028] Add excess melamine to the solution and allow to react for 100 to 160 minutes in the dark;

[0029] Add an excess of sodium borohydride to the solution, the molar amount of sodium borohydride being more than 10 times the molar amount of sodium periodate, and react for 100-160 minutes in the dark;

[0030] The protein-tagged melamine antigen was purified by dialysis;

[0031] The dialysis-purified protein-labeled melamine antigen was freeze-dried.

[0032] According to one embodiment of the present disclosure, in the protein-labeled melamine antigen, the protein is one of an enzyme or a serum protein.

[0033] According to one embodiment of the present disclosure, in the protein-labeled melamine antigen, the protein is horseradish peroxidase.

[0034] According to an embodiment of the present disclosure, step S30 further includes obtaining a second detection current value;

[0035] The obtaining of the second detection current value comprises: adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine to the surface of the working electrode, so that 3,3',5,5'-tetramethylbenzidine and horseradish peroxidase undergo a catalytic reaction, and measuring the second detection current value of the working electrode by differential pulse voltammetry.

[0036] Step S30 also includes obtaining the detection concentration of melamine in the test solution according to the second detection current value and a second standard current-melamine concentration curve of the electrochemical sensor.

[0037] According to an embodiment of the present disclosure, the second standard current-melamine concentration curve is obtained by the following method: obtaining second standard current values ​​corresponding to a plurality of melamine standard solutions of different concentrations, and determining the second standard current-melamine concentration curve;

[0038] Obtaining the second standard current value corresponding to a melamine standard solution of any concentration includes:

[0039] Prepare a melamine standard solution with a preset concentration;

[0040] Mixing the melamine standard solution with a quantitative protein-labeled melamine antigen to form a standard mixed solution;

[0041] Adding the standard mixed solution to the surface of the working electrode of the electrochemical sensor, incubating and cleaning the working electrode, and then adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine to the surface of the working electrode;

[0042] The current value corresponding to the working electrode is measured by differential pulse voltammetry, thereby obtaining a second standard current value corresponding to a melamine standard solution of a preset concentration.

[0043] The electrochemical detection method of melamine provided by the present invention is based on the immune principle. When applied to the detection of foods such as dairy products, it does not require complicated sample preparation and processing, and has high application potential. In the process of detecting melamine in milk using the method provided by the present invention: on the one hand, the working electrode with melamine antibodies modified on the surface itself has good anti-interference performance against other substances in dairy products, and can reflect the melamine content in milk; on the other hand, the melamine antibodies are directly fixed on the working electrode, and can be directly tested after reacting with the solution to be tested. The overall test process does not exceed 10 minutes at the fastest, and it has the characteristics of easy integration and high efficiency, and can be used for rapid detection in the food industry.

[0044] It is to be understood that the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the present disclosure. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] The accompanying drawings herein are incorporated into the specification and constitute a part of the specification, illustrate embodiments consistent with the present disclosure, and together with the specification are used to explain the principles of the present disclosure. Obviously, the accompanying drawings described below are only some embodiments of the present disclosure, and for ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without creative work.

[0046] Figure 1 This is a schematic diagram of DPV detection results of an electrochemical sensor for melamine standard solutions of different concentrations in one embodiment of the present disclosure.

[0047] Figure 2 The figure is a fitting curve diagram of melamine concentration-changing current at different concentrations in one embodiment of the present disclosure.

[0048] Figure 3 This is a schematic diagram of DPV detection results of an electrochemical sensor for melamine standard solutions of different concentrations in one embodiment of the present disclosure.

[0049] Figure 4 This is a schematic diagram of the results of specific detection of melamine by an electrochemical sensor in one embodiment of the present disclosure. DETAILED DESCRIPTION

[0050] Example embodiments will now be described more fully with reference to the accompanying drawings. However, example embodiments can be implemented in many forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be comprehensive and complete and will fully convey the concept of the example embodiments to those skilled in the art.

[0051] The present disclosure provides an electrochemical detection method for melamine, comprising the following steps:

[0052] S10, obtaining a test solution, a protein-labeled melamine antigen, and an electrochemical sensor; the surface of the working electrode of the electrochemical sensor is modified with a melamine antibody;

[0053] S20, mixing the test solution with a quantitative amount of protein-labeled melamine antigen, and adding the mixture to the working electrode surface of the electrochemical sensor, so that the melamine antigen in the test solution and the protein-labeled melamine antigen competitively bind to the melamine antibody modified on the surface of the working electrode;

[0054] S30, measuring a first detection current value of the working electrode by using differential pulse voltammetry;

[0055] S40, obtaining the detection concentration of melamine in the test solution according to the first detection current value and a first standard current-melamine concentration curve of the electrochemical sensor.

[0056] In the electrochemical detection method of melamine provided in the present disclosure, melamine can be detected by electrochemical method, which can not only realize the quantitative analysis of melamine in the sample to be detected (such as food), but also greatly shorten the detection time to meet the requirements of on-site rapid screening.

[0057] The electrochemical detection method of melamine detects melamine in a sample to be tested (e.g., food such as dairy products) based on the immune principle. It fixes a quantitative melamine antibody on a working electrode, and then allows the solution to be tested and a quantitative protein-labeled melamine antigen mixed solution to be fully immunologically bound to the working electrode; the working electrode after immunological binding is subjected to a differential pulse voltammetry (DPV) test to obtain a first detection current value, and the melamine concentration in the sample to be tested is determined according to the peak value of the first detection current value. Melamine has a small molecular weight, and after immunological binding with the modified antibody on the working electrode, the steric hindrance of the electron transfer interface changes little, and the change of the electrical signal is not obvious. In order to improve the sensitivity, the embodiment of the present disclosure is mixed with a quantitative protein-labeled melamine antigen in the solution to be tested; the protein-labeled melamine antigen can be combined with the melamine antibody, and has a large volume to cause a large degree of change in the electrical signal, thereby achieving the effect of amplifying the electrical signal and improving the sensitivity of the detection.

[0058] The electrochemical detection method of melamine provided by the present invention is based on the immune principle. When applied to the detection of foods such as dairy products, it does not require complicated sample preparation and processing, and has high application potential. In the process of detecting melamine in milk using the method of the present invention: on the one hand, the working electrode with melamine antibodies modified on the surface itself has good anti-interference performance against other substances in dairy products, and can reflect the melamine content in milk. On the other hand, the melamine antibody is directly fixed on the working electrode, and can be directly tested after reacting with the solution to be tested. The overall test process does not exceed 10 minutes at the fastest. It has the characteristics of easy integration and high efficiency, and can be used for rapid detection in the food industry.

[0059] In one embodiment of the present disclosure, in step S10, the solution to be tested may be a dairy solution. For example, the solution to be tested may be one of fresh milk, pasteurized pure milk, yogurt, milk powder solution, infant milk powder solution, etc. In some other embodiments, the solution to be tested may also be an extract or a dissolve of a product such as food or feed. For example, the solution to be tested may be an animal feed extract or an animal feed suspension.

[0060] In one embodiment of the present disclosure, in step S10, obtaining protein-labeled melamine antigen includes:

[0061] Step S11, mixing the protein solution and an excess of sodium periodate solution evenly, and reacting in the dark for 20 to 40 minutes; for example, reacting in the dark for 20 minutes, 22.5 minutes, 25 minutes, 27.5 minutes, 30 minutes, 32.5 minutes, 35 minutes, 37.5 minutes or 40 minutes;

[0062] Step S12, adding excess ethylene glycol to the solution, and reacting in the dark for 20 to 40 minutes; for example, reacting in the dark for 20 minutes, 22.5 minutes, 25 minutes, 27.5 minutes, 30 minutes, 32.5 minutes, 35 minutes, 37.5 minutes or 40 minutes;

[0063] Step S13, adding excess melamine to the solution, and reacting in the dark for 100 to 160 minutes; for example, reacting in the dark for 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes or 160 minutes;

[0064] Step S14, adding an excess of sodium borohydride to the solution, wherein the molar amount of sodium borohydride is more than 10 times the molar amount of sodium periodate, and reacting in the dark for 100-160 minutes; for example, reacting in the dark for 100 minutes, 110 minutes, 120 minutes, 130 minutes, 140 minutes, 150 minutes or 160 minutes;

[0065] Step S15, purifying the protein-labeled melamine antigen by dialysis;

[0066] Step S16, freeze-drying the protein-labeled melamine antigen after dialysis purification.

[0067] Thus, through the treatment of step S11) to step S14), the protein in the protein solution can be connected to melamine through a chemical bond, for example, the protein and melamine are connected through an ethylene group. After the treatment of step S15) and step S16), a purified solid powder can be obtained: protein-labeled melamine antigen. On the one hand, the impurities in the protein-labeled melamine antigen are reduced by dialysis, which is conducive to ensuring the accuracy of the quality of the protein-labeled melamine antigen added to the solution to be tested in step S120. On the other hand, the protein-labeled melamine antigen that is freeze-dried in powder form is conducive to storage, transportation and weighing, and is suitable as a part of a melamine detection kit.

[0068] In one example, in the protein-labeled melamine antigen, the protein is an enzyme or a serum protein, for example, the protein is horseradish peroxidase.

[0069] In one example, in the protein-labeled melamine antigen, the molecular weight of the protein is between 35 kD and 50 kD, such as 35 kD, 37.5 kD, 40 kD, 42.5 kD, 45 kD, 47.5 kD or 50 kD.

[0070] In one example, in step S14, the reaction solution is mixed every 25 to 35 minutes, for example, by shaking the bottle containing the reaction solution or stirring the reaction solution with a stirring rod. For example, the reaction solution is stirred every 25 minutes, 30 minutes or 35 minutes.

[0071] In one embodiment of the present disclosure, in step S10, the melamine antibody is modified on the surface of the working electrode by the following method:

[0072] Step S21, forming a first modification layer on the surface of the working electrode, wherein the first modification layer includes a gold modification layer;

[0073] Step S22, forming a second modification layer on a side of the first modification layer away from the working electrode surface, wherein the second modification layer includes melamine antibodies.

[0074] In one example, the main body of the working electrode can be one of a glassy carbon electrode or a screen-printed electrode.

[0075] In one example, a gold modification layer can be formed on the surface of the working electrode by a deposition method, for example, by electrodeposition of a chloroauric acid solution.

[0076] In one example, when preparing the gold modification layer by electro-depositing a chloroauric acid solution, a multi-step voltammetry method can be used, with a deposition voltage of ±0.1 V and a cycle number of 200 to 600 cycles, for example, the cycle number is 200, 250, 300, 350, 400, 450, 500, 550 or 600 cycles. Further, the concentration of the chloroauric acid solution is 0.5%.

[0077] In one embodiment of the present disclosure, forming the second modified layer includes:

[0078] Step S31), dripping mercaptopropionic acid solution on the gold modified layer of the working electrode for incubation, so that the surface of the gold modified layer is bound to mercaptopropionic acid through a gold-sulfur bond; thus, the deposited gold electrode is connected by mercaptopropionic acid to form a carboxyl terminal;

[0079] Step S33), dripping a carboxylic acid activator on the surface of the working electrode so that the carboxyl group of mercaptopropionic acid forms an active ester functional group;

[0080] Step S34), dripping melamine antibody solution on the surface of the working electrode, so that the melamine antibody reacts with the active ester functional group and is fixed on the surface of the gold modification layer of the working electrode to form a second modification layer.

[0081] In one example, in step S31), when the mercaptopropionic acid solution is dripped on the gold modified layer of the working electrode for incubation, the size and incubation time of each working electrode are the same, and the volume and concentration of the mercaptopropionic acid solution dripped on a single working electrode are the same. In this way, different working electrodes can have substantially the same electrochemical properties.

[0082] In one example, in step S33, a solution of 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide can be dripped onto the surface of the working electrode to activate the free carboxyl groups into active ester functional groups.

[0083] In one embodiment of the present disclosure, the forming of the second modified layer further comprises:

[0084] Step S32, after the surface of the gold modification layer is modified with mercaptopropionic acid, a 6-mercaptohexanol solution is dripped onto the surface of the working electrode for incubation to seal the gold modification layer. In this way, the anti-interference property of the working electrode is improved to prevent small molecule proteins in the sample to be tested from binding to the gold modification layer and interfering with the measurement of melamine.

[0085] In one embodiment of the present disclosure, the forming of the second modified layer further comprises:

[0086] Step S35, after forming the second modification layer, gently rinse the working electrode with ultrapure water, and purge the surface with nitrogen to obtain a working electrode with melamine antibody modified on the surface.

[0087] In one embodiment of the present disclosure, in step S20, mixing the test solution with a quantitative protein-labeled melamine antigen comprises:

[0088] The test solution is mixed with a quantitative amount of protein-labeled melamine antigen, so that the concentration of the protein-labeled melamine antigen in the mixture is a set concentration.

[0089] In other words, the larger the volume of the solution to be tested, the larger the amount of protein-labeled melamine antigen added, and the concentration of the protein-labeled melamine antigen in the solution to be tested after the protein-labeled melamine antigen is added is used as the set concentration. In this way, the accuracy of the detection can be guaranteed. For example, in the solution to be tested, the concentration of the protein-labeled melamine antigen (i.e., the set concentration) is between 1 mg / L and 10 g / L, and in particular, can be between 0.5 g / L and 2.5 g / L, for example, 0.5 g / L, 1 g / L, 1.5 g / L, 2 g / L or 2.5 g / L.

[0090] In one embodiment of the present disclosure, in step S20, the solution to be tested is mixed with a quantitative protein-labeled melamine antigen and then added to the working electrode surface of the electrochemical sensor. The solution can be dripped or applied to the working electrode surface (the surface modified with melamine antibodies), or the working electrode can be inserted into the mixed solution of the solution to be tested and the quantitative protein-labeled melamine antigen. Further, when the solution to be tested and the quantitative protein-labeled melamine antigen mixed solution are dripped or applied to the working electrode surface, the volume of the mixed solution added to each working electrode surface is consistent to ensure the accuracy of the detection.

[0091] In one embodiment of the present disclosure, in step S20, the test solution and the quantitative protein-labeled melamine antigen are mixed and added to the surface of the working electrode, incubated for a period of time, for example, 5 to 10 minutes (for example, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes or 10 minutes), and then rinsed with a phosphate buffer solution.

[0092] In one embodiment of the present disclosure, in step S30, measuring the first detection current value of the working electrode by differential pulse voltammetry includes:

[0093] The working electrode obtained in step S30 is immersed in an electrolytic cell containing an electron mediator solution to perform a DPV test to obtain a current value I2 corresponding to the concentration, and the current value I2 is a first detection current value corresponding to the solution to be tested.

[0094] In one example, the electron mediator solution is an aqueous solution of potassium ferrocyanide, Prussian blue, and ferrocene, and more preferably, an aqueous solution of potassium ferrocyanide.

[0095] In one example, the electrolytic cell includes a working electrode, a counter electrode and a reference electrode. The reference electrode is a saturated silver / silver chloride electrode, and the counter electrode is a Pt electrode or a carbon electrode.

[0096] In one embodiment of the present disclosure, in step S40, the first standard current-melamine concentration curve is obtained by the following method: obtaining first standard current values ​​corresponding to a plurality of melamine standard solutions of different concentrations, and determining the first standard current-melamine concentration curve.

[0097] In one embodiment of the present disclosure, the first standard current-melamine concentration curve may be presented as a curve fitted by a plurality of data points, for example, as a linear curve fitted by a plurality of data points, wherein each data point may include the concentration of the melamine standard solution and the first standard current value corresponding to the melamine standard solution.

[0098] In another embodiment of the present disclosure, the first standard current-melamine concentration curve can be presented as two or more discrete data points; when determining the detection concentration of melamine in the test solution according to the first standard current-melamine concentration curve, two data points in the first standard current-melamine concentration curve can be selected together with the first detection current value corresponding to the test solution to determine the detection concentration of melamine in the test solution through a linear interpolation algorithm.

[0099] Optionally, in an embodiment of the present disclosure, each data point in the first standard current-melamine concentration curve is determined by using the same method as that for obtaining the first detection current value corresponding to the test solution when the concentration of the melamine standard solution is known.

[0100] In one example, obtaining a first standard current value corresponding to a melamine standard solution of any concentration includes:

[0101] Prepare a melamine standard solution with a preset concentration;

[0102] Mixing the melamine standard solution with a quantitative protein-labeled melamine antigen to form a standard mixed solution;

[0103] The standard mixed solution is added to the surface of the working electrode of the electrochemical sensor, and the first standard current value of the working electrode is measured by differential pulse voltammetry to obtain the first standard current value corresponding to the melamine standard solution of preset concentration.

[0104] As an example, a quantitative protein-labeled melamine antigen and a melamine standard solution with a quantitative concentration of C0 can be mixed (the concentration of the protein-labeled melamine antigen is a set concentration) and incubated on the surface of a working electrode modified with a melamine antibody for a period of time, rinsed with a phosphate buffer solution, and then immersed in an electrolytic cell containing an electron mediator solution for DPV testing to obtain a current value I0 of the corresponding concentration; a quantitative protein-labeled melamine antigen and a melamine standard solution with a quantitative concentration of C1 can be mixed (the concentration of the protein-labeled melamine antigen is a set concentration) and incubated on the surface of a working electrode modified with a melamine antibody for a period of time, rinsed with a phosphate buffer solution, and then immersed in an electrolytic cell containing an electron mediator solution for DPV testing to obtain a current value I1 of the corresponding concentration. After the test solution is mixed with a quantitative protein-labeled melamine antigen (the concentration of the protein-labeled melamine antigen is a set concentration), it is incubated on the surface of a working electrode modified with melamine antibodies for a period of time, rinsed with a phosphate buffer solution, and immersed in an electrolytic cell containing an electron mediator solution for DPV testing to obtain a current value I2 (i.e., a first detection current value) corresponding to the concentration. The corresponding melamine concentration C2 of the test solution is converted by the following formula.

[0105]

[0106] Wherein, C2 represents the detection concentration of the solution to be tested.

[0107] In one embodiment of the present disclosure, step S30 also includes obtaining a second detection current value; the obtaining the second detection current value includes: adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine to the surface of the working electrode, so that 3,3',5,5'-tetramethylbenzidine and horseradish peroxidase catalyze the reaction, and using differential pulse voltammetry to measure the second detection current value of the working electrode. Step S30 also includes obtaining the detection concentration of melamine in the test solution according to the second detection current value and the second standard current-melamine concentration curve of the electrochemical sensor.

[0108] In one example, after the test solution and a quantitative amount of horseradish peroxidase-labeled melamine (HRP-melamine) antigen are incubated on the surface of the working electrode, the working electrode is cleaned, hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine are added to the surface of the working electrode, a DPV test is performed, and the catalytic reaction rate of 3,3',5,5'-tetramethylbenzidine (TMB) and horseradish peroxidase (HRP) is measured to obtain a second detection current value. In this way, the concentration of melamine in the test sample can be obtained according to the second detection current value.

[0109] In another example, the solution to be tested can be incubated with a quantitative protein-labeled melamine antigen on the surface of the working electrode, and after the DPV test is performed to obtain the first detection current value, the working electrode is cleaned. In the process of obtaining the first detection current value by the DPV test, there is no effect on horseradish peroxidase, and then hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine are added to the surface of the working electrode, and the DPV test is performed to obtain the second detection current value. In this way, the dual current values ​​are obtained by the dual electrical signal reaction on the electrode, and the melamine concentration in the sample to be tested is measured, which increases the anti-interference ability of the melamine concentration measurement.

[0110] In one embodiment of the present disclosure, the second standard current-melamine concentration curve is obtained by the following method: obtaining second standard current values ​​corresponding to a plurality of melamine standard solutions of different concentrations, and determining the second standard current-melamine concentration curve;

[0111] Obtaining the second standard current value corresponding to a melamine standard solution of any concentration includes:

[0112] Prepare a melamine standard solution with a preset concentration;

[0113] Mixing the melamine standard solution with a quantitative protein-labeled melamine antigen to form a standard mixed solution;

[0114] Adding the standard mixed solution to the surface of the working electrode of the electrochemical sensor, incubating and cleaning the working electrode, and then adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine to the surface of the working electrode;

[0115] The current value corresponding to the working electrode is measured by differential pulse voltammetry, thereby obtaining a second standard current value corresponding to a melamine standard solution of a preset concentration.

[0116] As follows, the electrochemical detection method of melamine provided by the present disclosure is described in more detail using an exemplary embodiment.

[0117] Example 1 Preparation of working electrode

[0118] The glassy carbon electrode was polished to a bright finish with 30-50nm alumina powder slurry on 5000-grit sandpaper, and then ultrasonically cleaned with nitric acid: water (v / v = 1:1), acetone and pure water before use. It was immersed in a 0.5% chloroauric acid solution, and a multi-step voltammetry method was used with a deposition voltage of ±0.1V and 200 to 600 cycles to obtain an electrode surface with a metallic luster (gold modification layer). The glassy carbon electrode with a gold modification layer was gently rinsed with ultrapure water and the surface was purged with nitrogen. A 75% mercaptopropionic acid solution was drop-coated on the electrode with a gold modification layer and incubated for 20 minutes to form a carboxyl terminal. The unreacted deposited gold on the surface of the gold modification layer reacted with 6-mercaptohexanol for 20 minutes, and the closed electrode had an anti-pollution effect. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride) and N-hydroxysuccinimide were drop-coated on the electrode surface, incubated in the dark for 30 minutes, melamine antibody was added for secondary incubation for 1 hour, gently rinsed with ultrapure water, and the surface was purged with nitrogen to obtain a working electrode with melamine antibody modified on the surface.

[0119] Example 2 Preparation of protein-labeled melamine antigen

[0120] Take 100 μL of 20 mg / L sodium periodate solution and 100 μL of 20 mg / mL horseradish peroxidase solution, mix them evenly, and let them stand in the dark for 30 minutes; add 2 μL of ethylene glycol to the mixed solution, and let them stand in the dark for 30 minutes; add excess melamine to the mixed solution, and let them stand in the dark for 2 hours; add 0.4 mg of sodium borohydride solution to the mixed solution, and let them stand in the dark for 2 hours, shaking every 30 minutes. The prepared solution is fully dialyzed for 48 hours to remove excess melamine, and the remaining liquid is freeze-dried to obtain horseradish catalase-labeled melamine antigen.

[0121] Example 3 Electrochemical Detection of Melamine

[0122] (1) The working electrode prepared in Example 1 was immersed in an electrolytic cell containing potassium ferrocyanide solution to perform a DPV test to obtain a DPV curve of the bare electrode (see Figure 1 The surface of the working electrode prepared in Example 1 was incubated with phosphate buffer for 10 min, and a DPV test was performed in a potassium ferrocyanide solution to obtain a DPV curve of the bare electrode treated with phosphate (see Figure 1 ). Figure 1 In the figure, the dotted line and the black line almost coincide with each other, which proves that the working electrode of the present invention has excellent test stability.

[0123] An equal amount of horseradish peroxidase-labeled melamine prepared in Example 2 was added to 1 mg / L, 2 mg / L, 3 mg / L, 4 mg / L, and 5 mg / L melamine standard solutions, respectively. After each melamine standard solution was fully mixed with the horseradish peroxidase-labeled melamine, they were incubated on the working electrode for 10 minutes, and then rinsed with a phosphate buffer solution. The working electrode rinsed with the phosphate buffer solution was subjected to a DPV test in a potassium ferrocyanide solution to obtain a current value corresponding to the concentration. The first standard current was the peak value of the DPV curve. The results are as follows: Figure 1 As shown. Compared with small molecular weight melamine, the steric hindrance formed by horseradish peroxidase increases significantly, which will make the electrical signal mediated by the electron mediator solution (potassium ferrocyanide solution) smaller, and the peak current corresponding to the measured DPV becomes smaller. The lower the concentration of the melamine standard solution, the more horseradish peroxidase-labeled melamine will bind to the electrode surface when competing with horseradish peroxidase-labeled melamine for binding to the melamine antibody on the working electrode surface, and the DPV current peak will also decrease accordingly.

[0124] See also Figure 1 As the concentration of the melamine standard solution decreases, the DPV current peak decreases. The DPV peak corresponding to each concentration of melamine is taken as the melamine concentration-first change current fitting curve as shown in Figure 2 As shown; wherein, the first change current is the difference between the peak value of the DPV curve of the bare electrode and the first standard current. As the concentration of melamine decreases, the first change current increases and shows a regular change, which proves that the method has a regular response to melamine and can detect the content of melamine in samples of unknown concentration by establishing a standard curve. The first change current is a variable that changes with the first standard current, so the melamine concentration-first change current can fit the melamine concentration-first standard current curve.

[0125] The solution to be tested was mixed with an equal amount of horseradish peroxidase-labeled melamine prepared in Example 2, incubated on the working electrode for 10 min, and then rinsed with a phosphate buffer solution. The working electrode rinsed with the phosphate buffer solution was subjected to a DPV test in a potassium ferrocyanide solution to obtain a first detection current value I corresponding to the concentration. x , then the change current ΔI of the sample to be tested x =I y -I x ;I y is the DPV peak current of the bare electrode. Figure 2 The concentration of melamine in the test solution is calculated based on the fitting curve of melamine concentration-first change current shown.

[0126] (2) Take 1 mg / L melamine standard solution and 5 mg / L melamine standard solution and mix them with the quantitative horseradish peroxidase labeled melamine prepared in Example 2, then incubate them on the working electrode for 10 min and rinse gently. Add hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine (TMB) to the surface of the working electrode and react for 2 min, and test their DPV curves respectively. The results are as follows: Figure 3 As shown in the figure, when the melamine concentration is high, there is less horseradish peroxidase bound to the electrode surface (less HRP-melamine binds to the melamine antibody), the catalytic rate of TMB is slower, and therefore the peak current signal detected by DPV is weaker; conversely, when the melamine concentration is low, there will be more HRP-melamine bound to the melamine antibody on the electrode surface, the catalytic rate of TMB is faster, and the peak current signal detected by DPV is stronger. In this way, by preparing melamine standard solutions of multiple concentrations, a fitting curve of melamine concentration-second standard current can be obtained, and then the melamine concentration can be detected.

[0127] After the test solution and HRP-melamine competitively bind to the melamine antibody on the electrode, a DPV test is performed to obtain a first electrochemical signal. This process (i.e., the process of obtaining the first standard current) has no effect on the activity and binding amount of the horseradish peroxidase bound to the electrode surface. In this way, by utilizing the catalytic properties of horseradish peroxidase, a substrate of horseradish peroxidase can be added for a catalytic reaction to obtain a second electrochemical signal (i.e., a second standard current), thereby realizing dual electrical signal detection of the system and improving the anti-interference property of melamine detection.

[0128] Example 4 Specificity of the electrochemical detection method for melamine based on the immune principle

[0129] Step 100: Immerse the working electrode prepared in Example 1 in an electrolytic cell containing potassium ferrocyanide solution to perform a DPV test to obtain Figure 4 The DPV curve of the bare electrode shown in Figure 4 medium black curve).

[0130] Step 200: After adding 0.1 mg / L horseradish peroxidase-labeled melamine standard solution to the working electrode surface and incubating for 10 min, a DPV curve test of potassium ferrocyanide solution is performed. The results are as follows: Figure 4 Shown as the red curve in the middle.

[0131] After the DPV test in step 300 and step 200 is completed, 0.1 mg / L horseradish peroxidase-labeled aflatoxin standard solution is added to the surface of the working electrode, and after incubation for 10 minutes, a DPV curve test of potassium ferrocyanide solution is performed. Figure 4The test results show that when other detection components in dairy products exist in the detection environment, for example, aflatoxin in this embodiment, the detection result of melamine by the working electrode does not change significantly (the red curve and the blue curve almost overlap), indicating that the melamine antibody on the electrode surface specifically binds to melamine and exhibits good anti-interference.

[0132] After the test in step 400 and step 300 is completed, a milk sample containing HRP-melamine standard solution is added to the surface of the working electrode ( Figure 4 The melamine spiked sample was incubated for 10 min and the DPV curve of potassium ferrocyanide solution was tested. The results are as follows Figure 4 Shown as the green curve in the middle.

[0133] After the test in step 500 and step 400 is completed, a milk sample containing HRP-aflatoxin standard solution is added to the surface of the working electrode ( Figure 4 The aflatoxin spiked sample shown in the figure was incubated for 10 minutes and the DPV curve test of potassium ferrocyanide solution was performed. The results are shown in Figure 4 Shown by the purple curve.

[0134] like Figure 4 As shown, the peak current corresponding to the DPV test curve in step 400 is significantly lower than the peak current corresponding to the DPV test curve in steps 200 and 300. This is because, in step 200, the amount of HRP-melamine added is small, and there are still more melamine antibodies on the surface of the working electrode that are not bound. In this way, the working electrode has a higher peak current when placed in a potassium ferrocyanide solution for testing. In step 300, the newly added HRP-aflatoxin cannot specifically bind to the melamine antibody, so that the DPV measurement result of step 300 is basically unchanged from the DPV measurement result of step 200. In step 400, a new milk sample containing HRP-melamine is added, so that more melamine antibodies are bound to the working electrode, thereby causing the peak current corresponding to the DPV test curve to decrease.

[0135] In step 500, a milk sample containing HRP-aflatoxin is newly added. The DPV measurement curve of step 500 is compared with the DPV measurement curve of step 400, and there is only a 3% fluctuation, indicating that small molecule proteins such as lactoferrin contained in milk will not bind to the surface of the working electrode to interfere with DPV detection. The method provided by the present disclosure has good anti-interference and detection specificity, and has high detection accuracy in practical application environments. In this way, different specific antibodies can also be modified on the working electrode to achieve the detection of antibiotics in other foods such as dairy products.

[0136] Those skilled in the art will readily appreciate other embodiments of the present disclosure after considering the specification and practicing the invention disclosed herein. This application is intended to cover any modification, use or adaptation of the present disclosure, which follows the general principles of the present disclosure and includes common knowledge or customary techniques in the art that are not disclosed in the present disclosure. The specification and examples are intended to be exemplary only, and the true scope and spirit of the present disclosure are indicated by the appended claims.

Claims

1. An electrochemical detection method for melamine, characterized in that: The following steps are involved: S10, obtaining a test solution, a protein-labeled melamine antigen, and an electrochemical sensor; the surface of the working electrode of the electrochemical sensor is modified with a melamine antibody; S20, mixing the test solution with a quantitative amount of protein-labeled melamine antigen, and adding the mixture to the working electrode surface of the electrochemical sensor, so that the melamine antigen in the test solution and the protein-labeled melamine antigen competitively bind to the melamine antibody modified on the surface of the working electrode; S30, measuring a first detection current value of the working electrode by using differential pulse voltammetry; S40, obtaining the detection concentration of melamine in the test solution according to the first detection current value and a first standard current-melamine concentration curve of the electrochemical sensor.

2. The electrochemical detection method of melamine according to claim 1, characterized in that: In step S10, the melamine antibody is modified on the surface of the working electrode by the following method: forming a first modification layer on the surface of the working electrode, wherein the first modification layer comprises a gold modification layer; A second modification layer is formed on the side of the first modification layer away from the surface of the working electrode, and the second modification layer includes melamine antibody.

3. The electrochemical detection method of melamine according to claim 2, characterized in that: The forming of the second modification layer comprises: drop-coating a mercaptopropionic acid solution on the gold modified layer of the working electrode for incubation, so that the surface of the gold modified layer is bound to mercaptopropionic acid through a gold-sulfur bond; dripping a carboxylic acid activator on the surface of the working electrode so that the carboxyl group of mercaptopropionic acid forms an active ester functional group; A melamine antibody solution is drop-coated on the surface of the working electrode to allow the melamine antibody to react with the active ester functional group and be fixed on the surface of the gold modified layer of the working electrode to form a second modified layer.

4. The electrochemical detection method of melamine according to claim 3, characterized in that: The forming of the second modification layer further comprises: After the surface of the gold modification layer is modified with mercaptopropionic acid, a 6-mercaptohexanol solution is drop-coated on the surface of the working electrode for incubation to seal the gold modification layer.

5. The electrochemical detection method of melamine according to any one of claims 1 to 4, characterized in that: The first standard current-melamine concentration curve is obtained by the following method: obtaining first standard current values ​​corresponding to a plurality of melamine standard solutions of different concentrations, and determining the first standard current-melamine concentration curve; Obtaining the first standard current value corresponding to a melamine standard solution of any concentration includes: Prepare a melamine standard solution with a preset concentration; Mixing the melamine standard solution with a quantitative protein-labeled melamine antigen to form a standard mixed solution; The standard mixed solution is added to the surface of the working electrode of the electrochemical sensor, and the current value corresponding to the working electrode is measured by differential pulse voltammetry to obtain a first standard current value corresponding to the melamine standard solution of a preset concentration.

6. The electrochemical detection method of melamine according to claim 1, characterized in that: Obtain protein-labeled melamine antigens including: Mix the protein solution and excess sodium periodate solution evenly and react for 20 to 40 minutes in the dark; Add excess ethylene glycol to the solution and allow to react for 20 to 40 minutes in the dark; Add excess melamine to the solution and allow to react for 100 to 160 minutes in the dark; Add an excess of sodium borohydride to the solution, the molar amount of sodium borohydride being more than 10 times the molar amount of sodium periodate, and react for 100-160 minutes in the dark; The protein-tagged melamine antigen was purified by dialysis; The dialysis-purified protein-labeled melamine antigen was freeze-dried.

7. The electrochemical detection method of melamine according to claim 6, characterized in that: In the protein-labeled melamine antigen, the protein is one of an enzyme and a serum protein.

8. The electrochemical detection method of melamine according to claim 7, characterized in that: In the protein-labeled melamine antigen, the protein is horseradish peroxidase.

9. The electrochemical detection method of melamine according to claim 8, characterized in that: Step S30 also includes obtaining a second detection current value; The obtaining of the second detection current value comprises: adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine to the surface of the working electrode, so that 3,3',5,5'-tetramethylbenzidine and horseradish peroxidase undergo a catalytic reaction, and measuring the second detection current value of the working electrode by differential pulse voltammetry; Step S30 also includes obtaining the detection concentration of melamine in the test solution according to the second detection current value and a second standard current-melamine concentration curve of the electrochemical sensor.

10. The electrochemical detection method of melamine according to claim 9, characterized in that: The second standard current-melamine concentration curve is obtained by the following method: obtaining second standard current values ​​corresponding to a plurality of melamine standard solutions with different concentrations, and determining the second standard current-melamine concentration curve; Obtaining the second standard current value corresponding to any concentration of melamine standard solution includes: Prepare a melamine standard solution with a preset concentration; Mixing the melamine standard solution with a quantitative protein-labeled melamine antigen to form a standard mixed solution; Adding the standard mixed solution to the surface of the working electrode of the electrochemical sensor, incubating and cleaning the working electrode, and then adding hydrogen peroxide and 3,3',5,5'-tetramethylbenzidine to the surface of the working electrode; The current value corresponding to the working electrode is measured by differential pulse voltammetry, thereby obtaining a second standard current value corresponding to a melamine standard solution of a preset concentration.

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