Electrochemical immunosensor for detecting fumonisin and its preparation and application

Abstract

The present invention discloses an electrochemical immunosensor for detecting fumonisin and its preparation and application, and relates to the field of medicinal plant quality safety monitoring. The preparation method of the electrochemical immunosensor includes: (1) preparation of N-G@CS composite material; (2) preparation of N-G@PANI@CS modified electrode; (3) preparation of AuNPs / N-G@PANI@CS / GCE modified electrode; preparation of BSA / Anti-FB1 / AuNPs / N-G@PANI@CS / GCE electrochemical immunosensor. The BSA / Anti-FB1 / AuNPs / N-G@PANI@CS / GCE electrochemical immunosensor of the present invention can realize the highly sensitive and rapid detection of fumonisin (FB1), a marker of root rot of medicinal plants, with a lower detection limit and a wider detection range, and has good specificity and stability, and also has the advantages of simple operation and short detection time.

Description

Technical Field

[0001] The invention relates to the field of medicinal plant quality safety monitoring, and in particular to an electrochemical immunosensor for detecting fumonisin and the preparation and application thereof. Background Art

[0002] Root rot is a disease caused by fungi, which causes the roots of plants to rot and the leaves to turn yellow and wither, thereby weakening the plant's ability to absorb water and nutrients, and ultimately causing the death of the plant. At the same time, root rot is one of the important reasons for the reduction in the yield of medicinal plants (referring to a class of plants that contain medicinal ingredients and can be developed and utilized as botanical drugs). With the development of the economy, people's acceptance of herbal health products has gradually increased, and the demand for medicinal plants has also increased year by year. According to the newly revised ICD-11 of the World Health Organization (WHO), traditional medical disease categories for medicinal plants have been added, and the global demand for medicinal plants is increasing. However, pests and diseases seriously restrict their yield and quality. Among them, root rot, as a common disease of medicinal plants, is known as "plant cancer". The disease is extremely easy to infect and has a wide range of occurrence. Studies have shown that its incidence rate on medicinal plants such as Astragalus is 35%-92%, and on licorice it is 5%-10%, and in severe cases it reaches 80%. Therefore, achieving early warning and prevention of root rot is of great significance for increasing the quantity and quality of medicinal plants.

[0003] In recent years, as researchers have continued to deepen their research on the root rot of medicinal plants, a large number of studies have shown that after Fusarium fungi infect plants, they cause the metabolism of harmful compounds such as fumonisin (FB), zearalenone (ZEA), and aflatoxin (AFT), which causes the medicinal plants to rot, which is also the main cause of the root rot of medicinal plants. Therefore, researchers have selected some plants that appear normal in appearance but are actually slightly infected with root rot pathogens as research objects, and screened for abnormally expressed compounds in their bodies as "signals" for early warning of root rot. We call it the Root Rot Early Warning Marker (RREWM) for medicinal plants. Fumonisin, as a highly representative secondary metabolite of the metabolic output of Fusarium, has attracted the attention of many researchers due to its abnormal expression in the early stage of root rot of medicinal plants. Among them, fumonisin B1 (FB1) accounts for 70% of the total content of fumonisins. It not only causes damage and rot to plants, but also damages the human liver and immune system, leading to immunosuppression and low immune function, and then induces diseases such as esophageal cancer, liver cancer, and gastric cancer. Therefore, the sensitive, efficient, and accurate detection of FB1 is not only of great significance for the early warning and prevention of root rot of medicinal plants, but also has far-reaching significance for ensuring the safety of Chinese medicinal materials and food and the prevention of related diseases.

[0004] There are many methods for detecting FB1, such as high performance liquid chromatography (HPLC), HPLC mass spectrometry (LC-MS), immunoassay, etc. Although these methods are widely used and highly operable, they have problems such as complex sample pretreatment, long time consumption and high labor input. Electrochemical methods have gradually attracted widespread attention from researchers due to their high sensitivity, low cost and high precision. Among them, electrochemical immunosensor analysis based on highly specific antigen-antibody recognition has been deeply studied and applied in the detection and identification of many disease-related markers in recent years. At the same time, fumonisin antibodies have high specificity and stability, are easy to be fixed on the active sites on the electrode surface, and can specifically bind to fumonisin antigens to detect fumonisin in root rot. Therefore, based on the principle of antigen-antibody specific recognition, we use nanocomposites to amplify the response signal and construct an electrochemical immunosensor that can be used for high-sensitivity and rapid detection of FB1 in actual samples. Summary of the invention

[0005] The purpose of the present invention is to provide an electrochemical immunosensor for detecting fumonisin and its preparation and application in view of the defects of the prior art in the above-mentioned background technology.

[0006] In order to achieve the above object, the present invention adopts the following technical solution:

[0007] The present invention provides a method for preparing an electrochemical immunosensor for detecting fumonisin, comprising the following steps:

[0008] (1) Preparation of NG@CS composite materials

[0009] Nitrogen-doped graphene (NG) powder is taken, added into chitosan (CS) solution, and ultrasonically dispersed to make nitrogen-doped graphene (NG) completely dispersed in chitosan (CS) solution, thereby obtaining a suspension of NG@CS composite material;

[0010] (2) Preparation of NG@PANI@CS modified electrodes

[0011] Polyaniline (PANI) was added to the NG@CS suspension and assembled by ultrasonic method to obtain the NG@PANI@CS dispersion. 10 μL of the prepared NG@PANI@CS dispersion was drop-coated on the glassy carbon electrode (GCE). After infrared drying, the NG@PANI@CS / GCE modified electrode was obtained.

[0012] (3) Preparation of AuNPs / NG@PANI@CS / GCE modified electrodes

[0013] AuNPs / NG@PANI@CS / GCE modified electrode was obtained by electrochemical deposition of gold nanoparticles on NG@PANI@CS / GCE modified electrode;

[0014] (4) Preparation of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor

[0015] FB1 antibody was adsorbed on the surface of AuNPs / NG@PANI@CS / GCE modified electrode, rinsed with PBST solution, and then the active sites were blocked with BSA solution. After rinsing with PBST solution again, the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor was obtained after natural drying and stored in a 4°C refrigerator for future use.

[0016] Furthermore, in step (1), the chitosan (CS) solution is prepared by weighing 0.2 g of chitosan (CS) and adding 100 mL of 1 wt% acetic acid solution, stirring and mixing continuously until the solution becomes clear and transparent to obtain a 2.0 mg / mL chitosan solution, which is then stored at 4° C. for later use; the suspension concentration of the NG@CS composite material is 1 mg / mL.

[0017] Furthermore, the ultrasonic dispersion time is 2 hours.

[0018] Furthermore, in step (2), before dropping the NG@PANI@CS dispersion on the glassy carbon electrode (GCE), the glassy carbon electrode (GCE) was continuously polished on a smooth suede using alumina powders with particle sizes of 0.3 μm and 0.05 μm, and then the glassy carbon electrode was ultrasonically cleaned using methanol and distilled water, respectively.

[0019] Furthermore, in step (2), the ultrasonic assembly time is 4 hours.

[0020] Furthermore, in step (3), the specific method for preparing the AuNPs / NG@PANI@CS / GCE modified electrode is to place the NG@PANI@CS / GCE modified electrode in a 0.5M H2SO4 solution containing 5mM HAuCl4, and to conduct the reaction at a potential range of -0.4 to 0.8V at 60mVs -1 The CV method was scanned for 15 cycles at a rate of 1.5 to deposit AuNPs, thus obtaining the AuNPs / NG@PANI@CS / GCE modified electrode.

[0021] Furthermore, in step (4), the specific method for preparing the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor is as follows: at 4°C, the AuNPs / NG@PANI@CS / GCE modified electrode is incubated with a 30 μL, 6 μg / mL fumonisin antibody solution (Anti-FB1) for 4 hours, and after the incubation is completed, it is rinsed with a 0.1 mmol / L PBST solution, and the FB1 antibody that is not firmly adsorbed is rinsed clean, and then placed in a 1 wt% BSA solution, incubated at 37°C for 40 minutes to block the active site, and after the blocking is completed, it is rinsed again with a PBST solution, and after natural drying, the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor is obtained and stored in a 4°C refrigerator; the PBST solution is a phosphate buffer solution with a pH of 7.4 containing 0.05 wt% Tween-80.

[0022] The present invention also provides an electrochemical immunosensor for detecting fumonisin, which is prepared by the above-mentioned preparation method.

[0023] The present invention also provides an application of an electrochemical immunosensor for detecting fumonisin in preparing a product for detecting root rot of medicinal plants. The detection range of the electrochemical immunosensor for detecting fumonisin is 0.10 ng / mL to 800.00 ng / mL.

[0024] Beneficial effects of the present invention:

[0025] (1) The present invention utilizes nitrogen-doped graphene (NG) and polyaniline (PANI) with good conductivity and chitosan (CS) with good stability and film-forming properties as electrode substrate materials, and uses an electrodeposition method to modify gold nanoparticles (AuNPs) with good biocompatibility and high conductivity on the electrode surface. Due to the high specific surface area of ​​NG and PANI, more active sites can be provided for AuNPs to better amplify the corresponding response signal. By utilizing the covalent action of the gold-thiol bond between AuNPs and antibodies, an electrochemical immunosensor with high sensitivity, high selectivity and rapid detection based on antigen-antibody recognition is constructed, which is used for the detection of fumonisin (FB1), an early warning biomarker for root rot of medicinal plants.

[0026] (2) The BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor of the present invention can achieve highly sensitive and rapid detection of fumonisin (FB1), a marker of medicinal plant root rot, with a detection limit of 0.094 ng / mL and a good linear relationship in the range of 0.10 ng / mL to 800.00 ng / mL. Compared with other existing detection methods, it has a lower detection limit and a wider detection range.

[0027] (3) When the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor of the present invention is used for actual sample detection, it exhibits good specificity and stability, and has the advantages of simple operation and short detection time, and can achieve high sensitivity and rapid detection of trace FB1 in actual samples. The present invention not only provides an effective means for convenient, efficient and accurate analysis of root rot warning markers, but also provides a novel strategy and scientific reference basis for the construction of early warning prevention and control technology for medicinal plant diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The present invention is a schematic diagram of the preparation principle of an electrochemical immunosensor for detecting fumonisin;

[0029] Figure 2 FTIR spectra of NG, PANI, CS, NG@CS, and NG@PANI@CS in a test example of the present invention;

[0030] Figure 3 The Raman spectra of NG, NG@CS, and NG@PANI@CS in a test example of the present invention;

[0031] Figure 4 The scanning electron microscope images in a test example of the present invention, wherein (D) is a scanning electron microscope image of NG@CS, (E) is a scanning electron microscope image of NG@PANI@CS, and (F) is a scanning electron microscope image of AuNPs / NG@PANI@CS / GCE;

[0032] Figure 5 In a test example of the present invention, [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- CV curves of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE detected by cyclic voltammetry (CV) in solution;

[0033] Figure 6In a test example of the present invention, [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- Electrochemical impedance spectroscopy (EIS) curves of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE in solution;

[0034] Figure 7 AuNPs / NG@PANI@CS / GCE in [Fe(CN)6] 3- / 4- CV curves detected at different scan rates (10V / s~290V / s) in solution;

[0035] Figure 8 is a graph showing the relationship between the peak current and the logarithm of the scan rate in a test example of the present invention;

[0036] Fig. 9 Qt curves of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE detected by chronocoulometry (CC) in a 0.10 mmol / L K3[Fe(CN)6] solution containing 0.01 mmol / L KCl in a test example of the present invention;

[0037] Fig.10 The Q-t1 / 2 linearity graphs of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE detected by chronocoulometry (CC) in a 0.10 mmol / L K3[Fe(CN)6] solution containing 0.01 mmol / L KCl in a test example of the present invention;

[0038] Fig.11 In a test example of the present invention, [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- CV curves of (a) AuNPs / NG@PANI@CS / GCE, (b) Anti-FB1 / AuNPs / NG@PANI@CS / GCE, (c) BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE, and (d) FB1 / BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE detected by cyclic voltammetry (CV) in solution;

[0039] Fig.12In a test example of the present invention, [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- Electrochemical impedance spectroscopy (EIS) curves of (a) AuNPs / NG@PANI@CS / GCE, (b) Anti-FB1 / AuNPs / NG@PANI@CS / GCE, (c) BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE, and (d) FB1 / BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE in solution;

[0040] Fig.13 This is a DPV curve diagram of the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor detected at different FB1 concentrations in a test example of the present invention;

[0041] Fig.14 This is a standard curve diagram of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor for different concentrations of FB1 in a test example of the present invention;

[0042] Fig.15 The result diagram of FB1 specificity test in the presence of various interfering substances (zearalenone (ZEN), ochratoxin A (OTA), deoxynivalenol (DON), aflatoxin (AFT)) in a test example of the present invention;

[0043] Fig.16 This is a result diagram of 8 DPV tests performed on the same electrode in a test solution in a test example of the present invention;

[0044] Fig.17 This is a graph showing the results of the preparation and sealing of the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor in a test example of the present invention, and the current response was measured by the DPV method on the 0th, 3rd, 7th, 14th and 28th days respectively. DETAILED DESCRIPTION

[0045] The technical solution of the present invention will be described clearly and completely in conjunction with the embodiments below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0046] Example

[0047] Please see attached Figure 1As shown, the present embodiment provides a method for preparing an electrochemical immunosensor for detecting fumonisin, comprising the following steps:

[0048] (1) Preparation of NG@CS composite materials

[0049] Weigh 0.2 g of chitosan (CS) and add 100 mL of 1 wt% acetic acid solution, continue stirring and mixing until clear and transparent to obtain a 2.0 mg / mL chitosan solution, which is stored at 4°C for later use; take nitrogen-doped graphene (NG) powder, add it to the chitosan (CS) solution, and ultrasonically disperse it for 2 h to completely disperse the nitrogen-doped graphene (NG) in the chitosan (CS) solution, thereby obtaining a suspension of NG@CS composite material with a concentration of 1 mg / mL;

[0050] (2) Preparation of NG@PANI@CS modified electrodes

[0051] Polyaniline (PANI) was added to the NG@CS suspension and ultrasonically assembled to obtain a NG@PANI@CS dispersion. Alumina powders with particle sizes of 0.3 μm and 0.05 μm were used to continuously polish the glassy carbon electrode (GCE) on a smooth suede. The glassy carbon electrode was then ultrasonically cleaned with methanol and distilled water, respectively. After cleaning, 10 μL of the NG@PANI@CS dispersion was droplet-coated on the glassy carbon electrode (GCE). After infrared drying, the NG@PANI@CS / GCE modified electrode was obtained.

[0052] (3) Preparation of AuNPs / NG@PANI@CS / GCE modified electrodes

[0053] The NG@PANI@CS / GCE modified electrode was placed in a 0.5 M H2SO4 solution containing 5 mM HAuCl4 and the potential was adjusted at 60 mVs in the range of -0.4 to 0.8 V. -1 The rate was scanned by CV method for 15 cycles to deposit AuNPs, thus obtaining AuNPs / NG@PANI@CS / GCE modified electrode;

[0054] (4) Preparation of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor

[0055] At 4°C, the AuNPs / NG@PANI@CS / GCE modified electrode was incubated with a 30 μL, 6 μg / mL fumonisin antibody solution (Anti-FB1) for 4 h. After incubation, it was rinsed with a 0.1 mmol / L PBST solution to rinse off the FB1 antibody that was not firmly adsorbed, and then placed in a 1 wt% BSA solution and incubated at 37°C for 40 min to block the active sites. After blocking, it was rinsed again with a PBST solution and naturally dried to obtain the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor, which was stored in a 4°C refrigerator. The PBST solution is a phosphate buffer solution with a pH of 7.4 containing 0.05 wt% Tween-80, and the BSA solution is the abbreviation of bovine serum albumin solution.

[0056] Test Example 1

[0057] In the process of preparing AuNPs / NG@PANI@CS / GCE modified electrode in the example, infrared spectroscopy, Raman spectroscopy and SEM electron microscopy were performed.

[0058] The characterization results are as follows Figure 2-4 As shown, Figure 2 FTIR spectra of NG, PANI, CS, NG@CS, and NG@PANI@CS; Figure 3 Raman spectra of NG, NG@CS, and NG@PANI@CS; Figure 4 (C) is the scanning electron microscope image of NG. Figure 4 (D) is the scanning electron microscope image of NG@CS. Figure 4 (E) is the scanning electron microscope image of NG@PANI@CS. Figure 4 (F) SEM image of AuNPs / NG@PANI@CS / GCE.

[0059] The test results show that NG@PANI@CS composite materials, NG@PANI@CS modified electrodes and AuNPs / NG@PAN I@CS / GCE modified electrodes were all successfully prepared.

[0060] Test Example 2

[0061] The electrochemical behaviors of GCE, NG@CS / GCE, NG@PANI@CS / GCE and AuNPs / NG@PANI@CS / GCE were characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS) and chronocoulometry.

[0062] The electrolyte was a 4.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.01 M KCl. The CV method was used to characterize the basic electrode under the following conditions: voltage range -0.4 V to 0.8 V; scan rate 60 mVs -1 The EIS test setting frequency range is: 1Hz~106Hz, the sine wave signal amplitude is 5mV, and the voltage is 0.22V.

[0063] The electrochemical characterization results are shown in Figure 5-10 As shown, Figure 5 [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- CV curves of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE detected by cyclic voltammetry (CV) in solution; Figure 6 [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- Electrochemical impedance spectroscopy (EIS) curves of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE in solution; Figure 7 AuNPs / NG@PANI@CS / GCE in [Fe(CN)6] 3- / 4- CV curves detected at different scan rates (10V / s~290V / s) in solution; Figure 8 is the relationship between the peak current and the logarithm of the scan rate; Fig. 9 Qt curves of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE detected by chronocoulometry (CC) in 0.10 mmol / L K3[Fe(CN)6] solution containing 0.01 mmol / L KCl; Fig.10 Q-t1 / 2 linearity plots of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE detected using chronocoulometry (CC) in 0.10 mmol / L K3[Fe(CN)6] solution containing 0.01 mmol / L KCl.

[0064] The test results show that NG@PANI@CS composite materials, NG@PANI@CS modified electrodes, and AuNPs / NG@PANI@CS / GCE modified electrodes were successfully prepared; and the effective surface areas of (a) unmodified GCE, (b) NG@CS / GCE, (c) NG@PANI@CS / GCE, and (d) AuNPs / NG@PANI@CS / GCE were 0.408 cm 2 、0.621cm 2 、1.021cm 2 、2.426cm 2 .

[0065] Test Example 3

[0066] The electrochemical behaviors of AuNPs / NG@PANI@CS / GCE, Anti-FB1 / AuNPs / NG@PANI@CS / GCE, BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE, and FB1 / BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE were characterized by cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS).

[0067] The electrolyte was a 4.0 mM K3[Fe(CN)6] / K4[Fe(CN)6] solution containing 0.01 M KCl. The CV method characterization measurement conditions for each electrode were: voltage range -0.4 V to 0.8 V; scan rate 60 mVs-1. The EIS method test setting frequency range: 1 Hz to 106 Hz, sine wave signal amplitude 5 mV, voltage 0.22 V.

[0068] The electrochemical characterization results are shown in Figure 11-12 As shown, Fig.11 [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4- CV curves of (a) AuNPs / NG@PANI@CS / GCE, (b) Anti-FB1 / AuNPs / NG@PANI@CS / GCE, (c) BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE, and (d) FB1 / BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE detected by cyclic voltammetry (CV) in solution; Fig.12 [Fe(CN)6] in 4.0mmol / L containing 0.01mmol / L KCl 3- / 4-Electrochemical impedance spectroscopy (EIS) curves of (a) AuNPs / NG@PANI@CS / GCE, (b) Anti-FB1 / AuNP s / NG@PANI@CS / GCE, (c) BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE, and (d) FB1 / BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE in solution;

[0069] The test results show that AuNPs / NG@PANI@CS / GCE, Anti-FB1 / AuNPs / NG@PANI@CS / GCE, BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE, and FB1 / BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE modified electrodes in the embodiments of the present invention were all successfully prepared.

[0070] Test Example 4

[0071] Weigh an appropriate amount of FB1 and dissolve it in anhydrous methanol to obtain an FB1 solution. Then take a certain volume of FB1 solution in PBS (the solution ratio is 1:19) to prepare 0.10, 0.50, 1.00, 10.00, 50.00, 100.00, 200.00, 400.00, 600.00, and 800.00 ng / mL FB1 standards for quantitative analysis.

[0072] The electrochemical response of the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor prepared in the embodiment was investigated by differential pulse voltammetry (DPV), and the initial current I0 was recorded; the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor was taken out and washed with PBS (pH=7.6) buffer, and then placed in different concentrations of FB1 standards for 10 minutes, and then DPV scanning was performed to record the current In; the inhibition rate was calculated according to the inhibition rate = (I0-In) / I0×100%; the DPV measurement conditions were: voltage range of 0 to 0.416; potential increment of 0.004V; amplitude of 0.05V; pulse width of 0.05s; sampling width of 0.0167s; pulse period of 0.5s, and standing time of 2s; finally, the linear functional relationship between the inhibition rate and the electrochemical response value was obtained by mathematical modeling.

[0073] Test results such as Fig.13 and 14 As shown, Fig.13DPV curves of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor at different FB1 concentrations; Fig.14 This is the standard curve of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor for different concentrations of FB1.

[0074] The test results show that: in the concentration range of a→k: 0, 0.10, 0.50, 1.00, 10.00, 50.00, 100.00, 200.00, 400.00, 600.00, 800.00 ng / mL, the logarithm of FB1 concentration and the current response show a good linear relationship, and the linear equation is: Y = -6.131 × LgC FB1 +57.863(R 2 =0.991), and the detection limit was 0.094 ng / mL (S / N=3).

[0075] Test Example 5

[0076] In order to verify the practicality of the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor of the present invention, the specificity, repeatability and stability of the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor prepared in the example were tested.

[0077] Test results such as Figure 15-17 As shown, Fig.15 The results of FB1 specificity test in the presence of various interfering substances (zearalenone (ZEN), ochratoxin A (OTA), deoxynivalenol (DON), aflatoxin (AFT)); Fig.16 This is the result of 8 DPV tests on the same electrode in the test solution; Fig.17 The BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor was prepared and sealed for storage, and the current response was measured by the DPV method on the 0th, 3rd, 7th, 14th and 28th days respectively.

[0078] The test results show that the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor has excellent specificity, repeatability and stability.

[0079] Test Example 6

[0080] In order to verify the feasibility of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor in actual sample detection, actual lily samples were analyzed.

[0081] The standard addition method was used to determine FB1 in actual lily samples. Fresh disease-free lilies were crushed, juiced and filtered, and then 1.00 mL of the prepared filtrate was taken, diluted 10 times with deionized water, and different concentrations of FB1 (5.00, 50.00, 250.00, 500.00 ng / mL) were added to the prepared filtrate. Finally, the filtrate containing different concentrations of FB1 was dropped on the surface of the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor and incubated at room temperature for 60 minutes, and detected using SWV in a three-electrode system. At the same time, under the same experimental conditions, lily samples with different concentrations of FB1 were detected by ELISA. A total of 4 groups of samples were tested, and 3 parallel experiments were performed for each group of samples. The results are shown in Table 1.

[0082] Table 1 Determination of FB1 in actual samples and comparison with Elisa kit test results

[0083]

[0084] The results show that the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor of the present invention has high sensitivity, and the detection results are better than those of the Elisa kit, and can realize real-time detection of FB1 in actual samples.

Claims

1. A method for preparing an electrochemical immunosensor for detecting fumonisin, characterized in that: The steps include: (1) Preparation of NG@CS composite materials Nitrogen-doped graphene (NG) powder is taken, added into chitosan (CS) solution, and ultrasonically dispersed to make nitrogen-doped graphene (NG) completely dispersed in chitosan (CS) solution, thereby obtaining a suspension of NG@CS composite material; (2) Preparation of NG@PANI@CS modified electrodes Polyaniline (PANI) was added to the NG@CS suspension and assembled by ultrasonic method to obtain the NG@PAN I@CS dispersion. 10 μL of the prepared NG@PANI@CS dispersion was drop-coated on the glassy carbon electrode (GCE). After infrared drying, the NG@PANI@CS / GCE modified electrode was obtained. (3) Preparation of AuNPs / NG@PANI@CS / GCE modified electrodes AuNPs / NG@PANI@CS / GCE modified electrode was obtained by electrochemical deposition of gold nanoparticles on NG@PANI@CS / GCE modified electrode; (4) Preparation of BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor FB1 antibody was adsorbed on the surface of AuNPs / NG@PANI@CS / GCE modified electrode, rinsed with PBST solution, and then the active sites were blocked with BSA solution. After rinsing with PBST solution again, the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor was obtained after natural drying and stored in a 4°C refrigerator for future use.

2. The method for preparing an electrochemical immunosensor for detecting fumonisin according to claim 1, characterized in that: In step (1), the chitosan (CS) solution is prepared by weighing 0.2 g of chitosan (CS) and adding 100 mL of 1 wt% acetic acid solution, stirring and mixing continuously until the solution becomes clear and transparent to obtain a 2.0 mg / mL chitosan solution, which is then stored at 4° C. for later use; the suspension concentration of the NG@CS composite material is 1 mg / mL.

3. The method for preparing an electrochemical immunosensor for detecting fumonisin according to claim 1, characterized in that: In step (1), the ultrasonic dispersion time is 2 hours.

4. The method for preparing an electrochemical immunosensor for detecting fumonisin according to claim 1, characterized in that: In step (2), before dropping the NG@PANI@CS dispersion on the glassy carbon electrode (GCE), the glassy carbon electrode (GCE) was polished continuously on a smooth suede using alumina powders with particle sizes of 0.3 μm and 0.05 μm, and then ultrasonically cleaned using methanol and distilled water, respectively.

5. The method for preparing an electrochemical immunosensor for detecting fumonisin according to claim 1, characterized in that: In step (2), the ultrasonic assembly time is 4 hours.

6. The method for preparing an electrochemical immunosensor for detecting fumonisin according to claim 1, characterized in that: In step (3), the specific method for preparing the AuNPs / NG@PANI@CS / GCE modified electrode is to place the NG@PANI@CS / GCE modified electrode in a 0.5M H2SO4 solution containing 5mM HAuCl4, and scan 15 cycles by CV method at a rate of 60mVs-1 in the potential range of -0.4 to 0.8V to deposit AuNPs, thereby obtaining the AuNPs / NG@PANI@CS / GCE modified electrode.

7. The method for preparing an electrochemical immunosensor for detecting fumonisin according to claim 1, characterized in that: In step (4), the specific method for preparing the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor is as follows: at 4°C, the AuNPs / NG@PANI@CS / GCE modified electrode is incubated with a 30 μL, 6 μg / mL fumonisin antibody solution (Anti-FB1) for 4 hours, and after the incubation is completed, it is rinsed with a 0.1 mmol / L PBST solution to rinse the FB1 antibody that is not firmly adsorbed, and then placed in a 1 wt% BSA solution, incubated at 37°C for 40 minutes to block the active site, and after the blocking is completed, it is rinsed again with a PBST solution, and after natural drying, the BSA / Anti-FB1 / AuNPs / NG@PANI@CS / GCE electrochemical immunosensor is obtained and stored in a 4°C refrigerator; the PBST solution is a phosphate buffer solution with a pH of 7.4 containing 0.05 wt% Tween-80.

8. An electrochemical immunosensor for detecting fumonisin prepared by the preparation method according to any one of claims 1 to 7.

9. Use of an electrochemical immunosensor for detecting fumonisin as claimed in claim 8 in the preparation of a product for detecting root rot of medicinal plants, wherein the detection range of the electrochemical immunosensor for detecting fumonisin is 0.10 ng / mL to 800.00 ng / mL.

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