CN111175509A

CN111175509A - ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN

Info

Publication number: CN111175509A
Application number: CN202010106727.3A
Authority: CN
Inventors: 翁祖铨; 陈家敏; 黄达
Original assignee: Fuzhou University
Current assignee: Fuzhou University
Priority date: 2020-02-21
Filing date: 2020-02-21
Publication date: 2020-05-19

Abstract

The invention discloses a novel enzyme-linked immunosorbent assay (ELISA) visual detection kit constructed based on Fenton reaction and application thereof in ZEN detection. The kit comprises a ZEN monoclonal antibody, artificial antigens ZEN-CAT and H₂O₂、FeCl₂And Au NRs, which are made by Fe²⁺Acceleration H₂O₂The etching effect on the Au NRs causes the color of the Au NRs to change, so that the approximate concentration of the ZEN can be judged according to the color change degree of the Au NRs, or the accurate quantification of the ZEN concentration is realized by utilizing an ultraviolet absorption curve according to the blue shift of the longitudinal plasma resonance absorption peak of the Au NRs. The preparation method of the Au NRs is simple and easy to obtain, and can realize multiple color development, so that the simple and convenient analysis of the ZEN concentration in the feed can be realized, and the operation is convenientSimple operation, high sensitivity and selectivity, and easy popularization and application.

Description

ELISA visual detection kit constructed based on Fenton reaction and application of kit in detection of ZEN

Technical Field

The invention belongs to the field of food analysis, and particularly relates to a novel enzyme-linked immunosorbent assay (ELISA) visual detection kit constructed based on the etching effect of Fenton reaction on Au NRs and application of the kit in detection of Zearalenone (ZEN).

Background

Zearalenone (ZEN), also known as F-2 toxin, is a non-steroidal substance produced mainly by fusarium roseum and fusarium graminearum. ZEN not only has the action of female hormone of animals, but also has the sex hormone of certain fungi. ZEN can cause abnormal reproductive function such as abortion, stillbirth, and estrus return of animals, and can also cause growth reduction, immunosuppression, infertility, and malformation. As most areas in China have abundant rainfall and high relative humidity, grains and animal feeds are more easily polluted by mycotoxin, and particularly crops such as corn, wheat, oat, barley and the like are easily polluted by ZEN. Through the research on the ZEN toxicity mechanism at home and abroad, the ZEN toxicity mechanism is found to influence the health of animals and human beings mainly through influencing the reproductive performance of organisms, causing the mechanisms of apoptosis, teratogenesis, DNA damage, oxidative damage, influencing the immune function and the like. Through the research on the toxicity of ZEN, the toxicity to the plants is mainly shown in that the toxicity is related to the fertilization and pollination of the plants, and the toxicity to the animals is mainly shown in that the toxicity has reproductive development toxicity, immunotoxicity, hepatorenal toxicity and the influence on endocrine systems.

The investigation found that ZEN was the most contaminated in the samples drawn by the feed mill, and the least contaminated in the food plot, the farm (household). This is associated with the high number of corn samples in the feed mill and the most severe contamination of corn samples by ZEN. After the pigs eat the feed polluted by ZEN, the fattening pigs are mainly manifested as oestrous symptoms of urogenital organs except for slight excitation of spirit and slight reduction of appetite. The detection of ZEN becomes particularly important as animals, once they have consumed ZEN contaminated feed, have no specific drugs to treat them.

The method for detecting ZEN mainly comprises a chromatographic method and an immunological method. The chromatography such as gas chromatography-mass spectrometry combined method, high performance liquid chromatography and the like has the characteristics of sensitive and reliable detection, but needs expensive instruments, has long detection time and has higher requirements on operators. The immunological method has the characteristics of simplicity, sensitivity, rapidness, low cost, suitability for mass detection and the like, and is particularly suitable for self-checking of a plurality of production enterprise units in China. At present, enzyme-linked immunosorbent assay (ELISA) detection of ZEN is researched at home and abroad, and certain results are obtained. However, compared with the imported kit, the domestic detection kit has the defects of narrow detection range and low sensitivity.

Disclosure of Invention

In view of the defects in the prior art, the invention aims to provide a novel enzyme-linked immunosorbent assay (ELISA) visual detection kit constructed based on the fenton reaction, which introduces Au NRs as a chromogenic substrate on the basis of the ELISA reaction to change the problem of single color development of the traditional ELISA. The invention constructs a novel ELISA visual detection kit based on Fenton reaction to detect ZEN, and utilizes the etching effect of Fenton reaction on Au NRs to cause the color change of Au NRs to achieve the color development effect on the basis of the binding specificity of ZEN and artificial antigen ZEN-CAT and antibody, so as to finally carry out visual detection and accurate quantitative determination on ZEN concentration through the color change of Au NRs.

In order to achieve the purpose, the invention adopts the following technical scheme:

ELISA visual detection kit constructed based on Fenton reaction comprises ZEN monoclonal antibody, artificial antigen ZEN-CAT and H₂O₂、FeCl₂And gold nanorods (Au NRs) using Fe²⁺Catalytic acceleration of H₂O₂Etching of Au NRs was performed, and color change of Au NRs was measured.

The synthesis of the artificial antigen ZEN-CAT comprises two parts of synthesis of hapten and artificial antigen, and comprises the following specific steps:

(1) synthesis of hapten: accurately weighing 1 mg of ZEN, dissolving the ZEN in 0.4 mL of pyridine, adding 4 mg of carboxymethyl hydroxylamine hemihydrochloride (CMO), stirring at room temperature for reaction for 24 hours, performing vacuum drying at 45 ℃, dissolving the residue in 0.4 mL of distilled water, adjusting the pH to 8.0 by using 1mol/L NaOH solution, repeatedly extracting for 3 times by using 0.4 mL of benzene to remove unreacted ZEN from the water phase, adjusting the pH of the reaction product to 3.0 by using HCl to generate a precipitate, extracting for 4 times by using 0.5 mL of ethyl acetate, and performing vacuum drying at 45 ℃ to obtain a ZEN-CMO hapten;

(2) synthesis of artificial antigen: dissolving N, N-Dicyclohexylcarbodiimide (DCC), succinimide (NHS) and the hapten obtained in the step (1) in 1 mL of N, N-Dimethylformamide (DMF) according to the mass ratio of 5:2:1, stirring at room temperature for 2 h for reaction, stirring at 4 ℃ for further reaction for 24 h, centrifuging at 3000 r/min for 10min, slowly dripping the supernatant into a precooled Catalase (CAT) solution of 1.5mL and 2.3 mg/mL, stirring at 4 ℃ for reaction for 24 h, and using a Phosphate Buffer Solution (PBS) of 2L and 0.01 mol/L, pH =7.4 as a reaction solution, wherein 4 g of NaCl and 1.45g of Na are added into the reaction solution₂HPO₄•12H₂O，0.1g KCl，0.2 gKH₂PO₄DDH dissolved in double distilled water₂Prepared by O and metering to 1000 mL) for 3 days to obtain the artificial antigen ZEN-CAT.

The preparation of the Au NRs comprises two parts of preparation of a nano gold core and growth of a gold nanorod, and the preparation method comprises the following specific steps:

(1) 250 mu.L of 10 mmol/L chloroauric acid (HAuCl)₄) Adding the solution into 9.75 mL of 0.1 mol/L Cetyl Trimethyl Ammonium Bromide (CTAB) solution, placing in a water bath at 30 ℃, magnetically stirring at the rotating speed of 700r/min, and uniformly mixing; then, the rotation speed of the magnetic stirrer is adjusted to 1200r/min, and 0.9 mL and 10 mmol/L of fresh sodium borohydride (NaBH)₄) Rapidly adding the solution into the above solution, stirring vigorously for 2 min, stopping stirring, taking out the magnetic stirrer, and standing the reaction solution in 30 deg.C water bath for 30 min to obtain nanometer gold core solution (the prepared gold core solution must be used within 2 h);

(2) 3.425 g CTAB was weighed into a 250 mL flat bottom flask, 100 mL of ultrapure water was added, followed by 960. mu.L of 10 mmol/L AgNO₃The solution was allowed to stand at 30 ℃ for 15 min, followed by addition of 5mL of 10 mmol/L HAuCl₄Continuously stirring the solution for 90 min at 700 r/min;

(3) adding 640 mu L of Ascorbic Acid (AA) solution of 100 mmol/L into the solution obtained in the step (2), after vigorously stirring for 30s at 1200r/min, adding 450 mu L of the nano gold core solution prepared in the step (1) into the solution, after continuously and vigorously stirring for 30s at 1200r/min, stopping stirring, taking out a magnetic stirring rod, standing in a water bath at 30 ℃ for more than 12 h, and waiting for the Au NRs to completely grow;

the aspect ratio of the resulting Au NRs was about 3.8.

The ELISA detection kit can be used for detecting ZEN, such as the detection of ZEN in feed. The application method specifically comprises the following steps: 50 μ L of ZEN monoclonal antibody was coated on an ELISA plate, and 50 μ L of ZEN-containing analyte and 50 μ L of artificial antigen ZEN-CAT were added to the wells, and incubated at 37 ℃ for 30 min to allow ZEN and ZEN-CAT to compete for the monoclonal antibody on the ELISA plate, and then treated with phosphate buffer solution containing Tween 20 (PBST, which is prepared by mixing 5ml of Tween 20, 4 g of NaCl, 1.45g of Na₂HPO₄•12H₂O，0.1g KCl，0.2 g KH₂PO₄DDH dissolved in double distilled water₂Prepared by adding O into water and fixing the volume to 1000 mL) for three times, adding 50 mu L of H-containing solution after spin-drying₂O₂1mol/L PBS solution, incubating at 37 deg.C for 10min, washing the plate with PBST three times, spin-drying, adding 50 μ L FeCl 10 mmol/L₂The solution was reacted with 100. mu.L of Au NRs at room temperature for 10min, and then stop solution H was added thereto₂SO₄The reaction was terminated and the concentration of ZEN was judged by observing the color change of Au NRs, or accurately and quantitatively determined by measuring the ultraviolet absorption at 450 to 900 nm.

High concentration of H at room temperature₂O₂A redox reaction occurs with the Au NRs so that the aspect ratio of the Au NRs is changed, and in the case where the CTAB is a soft template, the redox reaction between the Au NRs and the oxidant preferentially proceeds from both ends of the Au NRs, the length of the Au NRs is gradually shortened, and the diameter is not substantially changed. But at room temperature, low concentration of H₂O₂（<100 mmol/L) and Au NRs takes up to 12 h to completely oxidize the Au NRs. And in the Fenton reaction, Fe²⁺As a catalyst, H₂O₂Decomposed into OH and OOH, since OH has a much higher oxidizing power than OHH₂O₂The reaction time can be greatly shortened by reacting the product of the Fenton reaction with Au NRs. Namely, the invention increases H through the hydroxyl free radical generated by Fenton reaction₂O₂And Au NRs.

The invention constructs a novel ELISA visual detection kit based on the etching effect of Fenton reaction on Au NRs, which enables a target object and an artificial antigen ZEN-CAT to compete for a monoclonal antibody on an ELISA plate according to the specific binding effect of the target object ZEN, the artificial antigen ZEN-CAT and the monoclonal antibody on the ZEN monoclonal antibody, and then the artificial antigen ZEN-CAT combined with the antibody is remained on the ELISA plate through operations of plate washing, spin-drying and the like, and then H is added₂O₂CAT will decompose H₂O₂Adding FeCl₂And Au NRs, undecomposed H₂O₂Will be in Fe²⁺The etching of Au NRs is accelerated under the catalysis, after the Au NRs is etched, the longitudinal plasma resonance absorption peak can generate blue shift, the length-diameter ratio can also be changed, and the color of the Au NRs is changed, so that the color change can be visually observed by naked eyes to roughly perform semi-quantitative analysis on the concentration of ZEN, and in addition, the accurate quantitative analysis can be performed by utilizing the size of the light absorption value corresponding to the longitudinal plasma resonance absorption peak of the Au NRs.

Compared with the defects of the prior art, the invention has the advantages that:

1. the invention relates to a novel ELISA visual detection kit constructed by utilizing the etching effect of Fenton reaction on Au NRs, which is prepared by Fe²⁺Acceleration H₂O₂The etching effect on the Au NRs changes the color of the Au NRs, so that the rough semi-quantitative detection and the accurate quantitative analysis of the ZEN concentration can be realized.

2. Au NRs quilt H used in the invention₂O₂After etching, the substrate can be in various colors such as blue, green, light red, yellow and the like, namely has multiple color rendering properties and is an excellent color rendering substrate.

3. The Au NRs used by the invention are prepared by a seed growth method, cetyl dimethyl ammonium bromide is used as a growth solution, the length-diameter ratio of the prepared Au NRs is about 3.8, and the Au NRs have good multiple color rendering property.

4. The method does not need complex instruments and complicated operation in the detection process, the amount of AuNRs required by each detection is very small (50 mu L), and the detection cost is low.

5. The linear range of the ZEN detection of the invention is 0.2-4000 ng/ml, the detection limit is 0.2 ng/ml, the detection sensitivity is high, the selectivity is good, the detection process is simple and convenient, the economic cost is low, the stability is good, and the method is easy to popularize and use.

Drawings

FIG. 1 shows different concentrations of H in the Fenton reaction₂O₂Etching the ultraviolet absorption spectrogram of Au NRs at different time, wherein (A) is 0.04 mmol/L H₂O₂；(B) 0.2 mmol/L H₂O₂；(C) 1 mmol/L H₂O₂；(D) 5 mmol/L H₂O₂。

FIG. 2 shows different concentrations of H in the Fenton reaction₂O₂Ultraviolet absorption spectrum (A), color development image (B) and TEM image (C) for AuNRs, wherein 1-13 represent H₂O₂Are 0, 1, 2, 3, 4, 5, 10, 40, 50, 60, 600, 800 and 1000 mmol/L, respectively, with a TEM scale of 50 nm.

FIG. 3 shows the identification result of the artificial antigen ZEN-CAT; wherein (A) ZEN-CAT was identified for ultraviolet absorption, (B) ZEN-CAT was identified for SDS-PAGE, lane 1 was 260. mu.g/ml CAT,

lanes

2 and 3 were ZEN-CAT, and (C) ZEN-CAT was identified for WB, lane 1 was 260. mu.g/ml CAT, and

lanes

2, 3 and 4 were ZEN-CAT.

FIG. 4 is a graph showing the optimization of reaction time in the reaction process.

FIG. 5 shows FeCl during the reaction₂And (4) concentration optimization chart. Wherein, FeCl with different concentrations is added when the reaction time of (A), (B) and (C) is 5min, 10min and 20min respectively₂The ultraviolet absorption spectrum of (D) is a color developing photograph.

Fig. 6 is a uv absorption spectrum (a), a linear relationship (B) between absorbance and ZEN concentration, and a color rendering image (C) of Au NRs after reaction with ZEN of different concentrations, wherein 1 to 18 represent ZEN concentrations of 0, 0.2, 0.3, 0.6, 0.8, 2, 5, 8, 20, 30, 50, 70, 300, 400, 700, 900, 3000 and 4000 ng/ml, respectively.

Detailed Description

ELISA visual detection kit constructed based on Fenton reaction comprises ZEN monoclonal antibody (purchased from abcam), artificial antigen ZEN-CAT and H₂O₂、FeCl₂And gold nanorods (Au NRs).

(1) adding 250 mu L of 10 mmol/L HAuCl₄Adding the solution into 9.75 mL of 0.1 mol/L CTAB solution, placing in a water bath at 30 ℃, magnetically stirring at the rotating speed of 700r/min, and uniformly mixing; then, the rotation speed of the magnetic stirrer is adjusted to 1200r/min, and 0.9 mL and 10 mmol/L of newly prepared NaBH is frozen₄Rapidly adding the solution into the above solution, stirring vigorously for 2 min, stopping stirring, taking out the magnetic stirrer, and standing the reaction solution in 30 deg.C water bath for 30 min to obtain nanometer gold core solution (the prepared gold core solution must be used within 2 h);

(3) adding 640 mu L of 100 mmol/L AA solution into the solution obtained in the step (2), after violently stirring for 30s at 1200r/min, adding 450 mu L of the nano gold core solution prepared in the step (1) into the solution, after continuously and violently stirring for 30s at 1200r/min, stopping stirring, taking out a magnetic stirring rod, standing in a water bath at 30 ℃ for more than 12 h, and till the Au NRs completely grow; the aspect ratio of the resulting AuNRs was about 3.8.

Novel ELISA visual detection kit constructed based on etching effect of Fenton reaction on Au NRs through Fe²⁺Acceleration H₂O₂The etching effect on the Au NRs causes the color of the Au NRs to change, the approximate concentration of the ZEN can be judged according to the color change degree of the Au NRs, or the accurate quantification of the ZEN concentration can be realized by an ultraviolet absorption curve of blue shift of a longitudinal plasma resonance absorption peak of the Au NRs.

FIG. 1 shows different concentrations of H in the Fenton reaction₂O₂And etching the ultraviolet absorption curve graph of the Au NRs at different times. As shown in FIG. 1, at H₂O₂At a concentration of 5 mmol/L, in the form of Fe²⁺The product of Fenton reaction as catalyst can completely oxidize Au NRs within 10min, and simultaneously, high-concentration H₂O₂H with a concentration lower than the etching degree of Au NRs₂O₂More severe, i.e. high concentrations of H₂O₂So that the blue shift of the absorption peak of Au NRs is more obvious and the color change is faster.

In order to make the present invention more comprehensible, the technical solutions of the present invention are further described below with reference to specific embodiments, but the present invention is not limited thereto.

EXAMPLE 1 Synthesis and characterization of Au NRs

(1) 250 mu.L of 10 mmol/L HAuCl₄Adding the solution into 9.75 mL of 0.1 mol/L CTAB solution, placing in a water bath at 30 ℃, magnetically stirring at the rotating speed of 700r/min, and uniformly mixing; then, the rotation speed of the magnetic stirrer is adjusted to 1200r/min, and 0.9 mL and 10 mmol/L of newly prepared NaBH is frozen₄Rapidly adding the solution into the solution (at the moment, the solution is brown yellow, if the solution is red, the preparation is wrong, the solution is required to be prepared again), stirring for 2 min vigorously, stopping stirring, taking out the magnetic stirrer, and standing the reaction solution in a water bath at 30 ℃ for 30 min to obtain a nano gold core solution (the prepared gold core solution is required to be used within 2 h);

(3) and (3) adding 640 mu L of 100 mmol/L AA solution into the solution obtained in the step (2), after vigorously stirring for 30s at 1200r/min, adding 450 mu L of the nano gold core solution prepared in the step (1) into the solution, after continuously and vigorously stirring for 30s at 1200r/min, stopping stirring, taking out a magnetic stirring rod, standing in a water bath at 30 ℃ for more than 12 h, and till the Au NRs completely grows.

In Fe²⁺Under catalysis, H₂O₂The UV absorption spectrum of the Au oxide NRs is shown in FIG. 2. As shown in FIG. 2, H is not present₂O₂In the process, an ultraviolet absorption spectrogram of Au NRs has two absorption peaks, namely a major axis plasma resonance absorption peak (765 nm) and a minor axis plasma resonance absorption peak (510 nm). When H is added₂O₂Then, long-axis plasmon resonance absorption of Au NRsThe peak begins to blue shift, while the intensity of the absorption peak also gradually decreases, and with H₂O₂The increase in concentration increases the degree of blue shift in the peak position of the major axis plasmon resonance absorption peak of Au NRs. Therefore, Au NRs with different concentrations of H₂O₂After the reaction, the color of the solution begins to change from light purple to light red, blue, green, colorless, light yellow, ginger yellow and other colors. It can be seen that the solutions of different colors are mixed with H₂O₂Exhibit a certain correlation between the concentrations of (A) and (B), and thus can be directed to H by the color change of the solution₂O₂The concentration of (a) is subjected to visual semi-quantitative analysis.

Further, to demonstrate that the color of the solution was changed due to Au NRs and H₂O₂The length-diameter ratio is changed after the reaction, and the geometrical shapes of Au NRs before and after the reaction are represented by adopting TEM. As shown in FIG. 2 (C), the original AuNRs solution was reddish brown in color with an aspect ratio of 3.8. When Au NRs and H₂O₂After the reaction, the aspect ratio of Au NRs decreased, and the color of the solution also changed from pale purple to yellow. When H is present₂O₂At a higher concentration of (a), the Au NRs starts to change from the original rod-like nanoparticles to a spherical shape, and the color of the solution also changes to yellow accordingly.

Example 2 Synthesis and identification of Artificial antigen ZEN-CAT

(2) synthesis of artificial antigen: dissolving N, N-Dicyclohexylcarbodiimide (DCC), succinimide (NHS) and the hapten obtained in the step (1) in 1 mL of N, N-Dimethylformamide (DMF) according to the mass ratio of 5:2:1, stirring at room temperature for 2 h for reaction, stirring at 4 ℃ for further reaction for 24 h, then centrifuging at 3000 r/min for 10min, then slowly dripping the supernatant into precooled 1.5mL and 2.3 mg/mL of Catalase (CAT) solution, stirring at 4 ℃ for reaction for 24 h, and dialyzing the reaction solution for 3 days by using 2L and 0.01 mol/L, pH =7.4 Phosphate Buffer Solution (PBS) to obtain the artificial antigen ZEN-CAT.

In order to identify the synthetic result of the artificial antigen ZEN-CAT, the concentration of the artificial antigen ZEN-CAT is firstly detected by a Coomassie brilliant blue kit, and finally the concentration of the ZEN-CAT is detected to be 260 mug/ml.

①, ultraviolet absorption preliminary identification of artificial antigen ZEN-CAT

CAT solution and ZEN solution of 260. mu.g/ml and synthesized ZEN-CAT sample are prepared respectively, 200. mu.l of each sample is put in an enzyme-labeled hole, and an absorption curve of 230-400 nm is measured. As can be seen from FIG. 3 (A), ZEN has 2 characteristic peaks of ultraviolet absorption at 274 nm and 316 nm, respectively; the ultraviolet absorption characteristic peak of the carrier protein is mainly 278 nm, however, the ultraviolet absorption characteristic peak and the absorption peak height of the artificial antigen ZEN-CAT are different from those of the ZEN and the carrier protein, superposition and translation are generated on the basis of the ultraviolet absorption characteristic peak and the absorption peak height, the absorption peaks respectively appear at 276 nm and 316 nm, due to the fact that the unconjugated ZEN is removed through extraction after the artificial antigen ZEN-CAT is conjugated, and the artificial antigen ZEN-CAT after the artificial antigen is conjugated appears at 316 nm and has the absorption peak which is unique to the ZEN and is not possessed by the carrier protein, the success of the ZEN artificial antigen coupling is preliminarily proved.

② SDS-PAGE identification of artificial antigens

The UV absorption spectrum only preliminarily identifies the successful synthesis of the artificial antigen, and further demonstrates the successful coupling of ZEN-CAT by SDS-PAGE, as shown in FIG. 3 (B), wherein lane 1 is a CAT standard at 260. mu.g/ml, and

lanes

2 and 3 are synthesized ZEN-CAT. The result shows that CAT has a band at about 60KDa, the band of the synthesized ZEN-CAT is weaker than that of a CAT standard product, and the coupling amount of CAT in the synthesis process is possibly less. However, the results of SDS-PAGE can also further demonstrate that the coupling of ZEN-CAT was successful.

③ identifying artificial antigen by Western Blot

The first two identification methods only carry out preliminary verification on the coupling success of ZEN-CAT, and whether the coupling of ZEN and CAT is really completed needs to be checked by using Western-blot to really identify whether the coupling of ZEN-CAT is successful or not. The results are shown in FIG. 3 (C), wherein lane 2 is CAT standard, and

lanes

3, 4 and 5 are synthetic ZEN-CAT with bands around 60KD, indicating that the ZEN-CAT coupling is successful.

Example 3: determination of ZEN Standard Curve

FIG. 4 and FIG. 5 show the reaction time and FeCl of the visual detection system₂And (4) concentration optimization chart. The optimal FeCl with the optimal reaction time of 10min is determined by a controlled variable method₂The concentration was 10 mmol/L.

Adding 50 μ L of ZEN standard diluted to a certain concentration into a reaction well coated with 50 μ L of ZEN monoclonal antibody, subsequently adding 50 μ L of artificial antigen ZEN-CAT, mixing well, covering with a membrane, reacting at 37 deg.C for 30 min, and washing with PBST (5 ml of Tween 20, 4 g of NaCl, 1.45g of Na, etc.)₂HPO₄•12H₂O，0.1g KCl，0.2 g KH₂PO₄DDH dissolved in double distilled water₂O and constant volume 1000 mL), repeatedly washing 3-4 times, patting to dry, adding H dissolved in PBS (0.1 mol/LpH = 7.8)₂O₂(45. mu.L, 1 mol/L), mixing well, covering a membrane, and reacting at 37 ℃ for 10 min. Subsequently, Au NRs (100. mu.L), FeCl was added₂Adding (50 μ L, 10 mmol/L) solution into the enzyme-labeled well, covering with membrane, reacting at room temperature for 10min, adding stop solution H₂SO₄(10. mu.l, 2M), finally, the UV absorption spectrum was scanned and photographed.

Fig. 6 is a uv absorption spectrum (a), a linear relationship (B) between absorbance and ZEN concentration, and a color rendering image (C) of Au NRs after reaction with ZEN of different concentrations. As can be seen from the figure, the linear range of the visual detection kit for detecting ZEN is 0.2-4000 ng/ml, and as shown in (C) in FIG. 6, ZEN with different concentrations can present multiple colors such as light red, light green, light yellow, yellow and the like, which proves that the visual detection kit has good color rendering property.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

Claims

1. The ELISA visual detection kit constructed based on the Fenton reaction is characterized in that: the kit comprises a ZEN monoclonal antibody, artificial antigens ZEN-CAT and H₂O₂、FeCl₂And Au NRs using Fe²⁺Catalytic acceleration of H₂O₂Etching of AuNRs was performed and color change by Au NRs was measured.

2. The visual ELISA detection kit constructed based on Fenton's reaction according to claim 1, wherein the synthesis of the artificial antigen ZEN-CAT comprises two parts of synthesis of hapten and synthesis of artificial antigen, and the specific steps are as follows:

(1) synthesis of hapten: accurately weighing 1 mg of ZEN, dissolving in 0.4 mL of pyridine, adding 4 mg of carboxymethyl hydroxylamine hemihydrochloride, stirring at room temperature for reaction for 24 hours, performing vacuum drying at 45 ℃, dissolving the residue in 0.4 mL of distilled water, adjusting the pH to 8.0 by using 1mol/L NaOH solution, repeatedly extracting for 3 times by using 0.4 mL of benzene to remove unreacted ZEN from the water phase, adjusting the pH of the reaction product to 3.0 by using HCl to generate a precipitate, extracting for 4 times by using 0.5 mL of ethyl acetate, and performing vacuum drying at 45 ℃ to obtain a ZEN-CMO hapten;

(2) synthesis of artificial antigen: dissolving N, N-dicyclohexylcarbodiimide, succinimide and hapten obtained in the step (1) in 1 mL of DMF according to the mass ratio of 5:2:1, stirring and reacting for 2 h at room temperature, stirring and continuing to react for 24 h at 4 ℃, centrifuging for 10min at 3000 r/min, slowly dripping supernate into precooled CAT solution of 1.5mL and 2.3 mg/mL, stirring and reacting for 24 h at 4 ℃, and dialyzing the reaction solution for 3 days by using 2L of phosphate buffer solution of 0.01 mol/L, pH =7.4 to obtain the artificial antigen ZEN-CAT.

3. The ELISA visual detection kit constructed based on Fenton's reaction according to claim 1, wherein the preparation of Au NRs comprises two parts of preparation of nano gold cores and growth of gold nanorods, and the specific steps are as follows:

(1) 250 mu.L of 10 mmol/L HAuCl₄Adding the solution into 9.75 mL of 0.1 mol/L CTAB solution, placing in a water bath at 30 ℃, magnetically stirring at the rotating speed of 700r/min, and uniformly mixing; then, the rotation speed of the magnetic stirrer is adjusted to 1200r/min, and 0.9 mL and 10 mmol/L of newly prepared NaBH is frozen₄Rapidly adding the solution into the solution, stirring vigorously for 2 min, stopping stirring, and standing the reaction solution in water bath at 30 deg.C for 30 min to obtain nano gold core solution;

(2) 3.425 g CTAB was added with 100 mL of ultrapure water, followed by 960. mu.L of 10 mmol/L AgNO₃The solution was allowed to stand at 30 ℃ for 15 min, followed by addition of 5mL of 10 mmol/L HAuCl₄Continuously stirring the solution for 90 min at 700 r/min;

(3) adding 640 mu L of ascorbic acid solution with the concentration of 100 mmol/L into the solution obtained in the step (2), stirring vigorously for 30s, adding 450 mu L of the nano gold core solution prepared in the step (1), stirring continuously and vigorously for 30s, stopping stirring, and standing in a water bath at the temperature of 30 ℃ for more than 12 h to generate Au NRs;

the aspect ratio of the resulting Au NRs was 3.8.

4. Use of an ELISA test kit according to claim 1 for the detection of ZEN, wherein: the method comprises the detection of ZEN in the feed.

5. The use of the ELISA detection kit of claim 4 for the detection of ZEN, wherein: the application method comprises the steps of coating 50 mu L of ZEN monoclonal antibody on an enzyme label plate, adding 50 mu L of ZEN-containing substance to be detected and 50 mu L of artificial antigen ZEN-CAT into the hole, incubating for 30 min at 37 ℃, washing out unreacted ZEN and artificial antigen ZEN-CAT, and sequentially adding 50 mu L of 1mol/L H₂O₂Solution, 50. mu.L of 10 mmol/L FeCl₂The reaction was terminated after the reaction was carried out at room temperature for 10min with 100. mu.L of Au NRs, and the concentration of ZEN was judged by observing the color change of Au NRs, or the concentration of ZEN was accurately and quantitatively determined by measuring the change of ultraviolet absorption value.