An electrochemical aptamer sensor for detecting mycotoxins in agricultural products, and its preparation method and detection method

Abstract

This invention relates to the field of electrochemical sensing, specifically disclosing an electrochemical aptamer sensor for detecting mycotoxins in agricultural products, its preparation method, and detection method. The sensor includes a working electrode, a reference electrode, and a counter electrode modified with a silica nanoporous membrane. The detection solution contains a composite probe of graphene oxide-mycotoxin aptamer-hexaammineruthenium trichloride. This invention uses hexaammineruthenium trichloride and mycotoxin aptamers as the electrochemical signal molecule and specific recognition element, respectively. They are adsorbed onto the surface of graphene oxide through electrostatic interactions or π-π interactions, forming the graphene oxide-mycotoxin aptamer-hexaammineruthenium trichloride composite probe. Combined with the signal amplification effect of the silica nanoporous membrane, the constructed homogeneous electrochemical aptamer sensor exhibits high detection sensitivity, a wide detection linear range, and a low detection limit, showing significant application potential in the field of food safety monitoring.

Description

Technical Field

[0001] This invention relates to the field of electrochemical sensing, specifically to an electrochemical aptamer sensor for detecting mycotoxins in agricultural products, and its preparation and detection methods. Background Technology

[0002] Mycotoxins are a group of chemical substances produced by fungal species and are among the causes of disease and even death. Zearalenone (ZEN), ochratoxin (OTA), and aflatoxin B1 (AFB1) are highly toxic and widely distributed mycotoxins, often found in corn, wheat, oats, sorghum, rice, and beer. These mycotoxins are among the most common contaminants in agricultural products, causing adverse effects on humans and animals, such as teratogenicity, miscarriage, hepatotoxicity, hemotoxicity, and carcinogenicity. Furthermore, ZEN has estrogenic activity, disrupting the balance of reproductive hormones and causing reproductive dysfunction; the International Agency for Research on Cancer has classified ZEN as a Group 3 carcinogen. However, these mycotoxins are extremely poorly soluble in water and have high thermal stability, making their detection challenging.

[0003] Currently, widely used methods for detecting mycotoxins include thin-layer chromatography (TLC), gas chromatography-mass spectrometry (GC-MS), and enzyme-linked immunosorbent assay (ELISA). TLC and GC-MS are cumbersome, time-consuming, and require expensive equipment. ELISA is simple to operate and highly sensitive, but its susceptibility to false positives limits its practical application. Therefore, establishing a low-cost, efficient, sensitive, and accurate analytical method is crucial for the effective monitoring of mycotoxins in crops.

[0004] Nanoporous silica (VMSF), also known as vertically ordered silica membrane, is a mesoporous thin film material with highly ordered pores perpendicular to the electrode substrate. VMSF possesses high permeability, molecular selectivity, and excellent molecular sieving capabilities, making it suitable as an anti-fouling and anti-interference layer, demonstrating significant advantages in direct electrochemical analysis of complex real-world samples. In recent years, researchers have reported various methods for successfully preparing VMSF on electrode surfaces, such as... Solution growth method (Angew. Chem. Int. Ed. 2012, 51, 2173-2177), electrochemically assisted self-assembly method (Nat. Mater. 2007, 6, 602-608), two-phase layered growth method (Adv. Mater. 2017, 29, 1702-274), organic molecule-induced co-assembly method (J. Am. Chem. Soc., 2015, 137, 3779-3782), etc. Solution growth and electrochemical-assisted self-assembly are the most commonly used methods. The prepared silica nanoporous membranes have highly uniform pore size (2-3 nm), are ultrathin and have adjustable thickness (20-200 nm), have high porosity, good stability and biocompatibility, and have great application potential in catalysis, adsorption, sensing and other fields.

[0005] On the one hand, most biomolecules, such as proteins, in complex samples have difficulty entering the ultrasmall nanochannels of VMSF; on the other hand, VMSF is rich in Si-OH groups (pK). a (2) In conventional solution media, its surface carries a negative charge, which can avoid interference from cells and proteins through electrostatic repulsion. At the same time, it can electrostatically adsorb positively charged probe molecules, requiring only a small number of molecules to achieve signal amplification. Therefore, VMSF-modified electrodes not only exhibit good anti-contamination / interference capabilities, but also have the ability to enhance sensitivity and reduce detection costs.

[0006] Patent document CN105241944A reports the enrichment and detection of hydrophobically active small molecule antibiotics in milk using the hydrophobic effect of cetyltrimethylammonium bromide (CTAB), a surfactant present inside silica nanopores. However, there are currently no reports on the application of the aforementioned VMSF-modified electrode-binding aptamer for the detection of mycotoxins in agricultural products. Summary of the Invention

[0007] This invention addresses the issues in existing technologies concerning agricultural products. Mycomycin To address the issues of low detection sensitivity and high detection difficulty in traditional methods, this paper provides a method for preparing an electrochemical aptamer sensor for detecting mycotoxins in agricultural products. The electrochemical aptamer sensor prepared by this method is used to detect aflatoxin, ochratoxin, or zearalenone, and has the advantages of high detection sensitivity and specificity, good selectivity, and low cost. Moreover, the detection process does not require a complex pretreatment process.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] An electrochemical aptamer sensor for detecting mycotoxins in agricultural products includes a working electrode, a reference electrode, and a counter electrode modified with a silica nanoporous membrane, and the detection solution contains a composite probe of graphene oxide-mycotoxin aptamer-hexaammineruthenium trichloride.

[0010] This invention uses hexaammineruthenium trichloride and a mycotoxin aptamer as the electrochemical signal molecule and specific recognition element, respectively. They are adsorbed onto the surface of graphene oxide via electrostatic interactions or π-π interactions, forming a graphene oxide-mycotoxin aptamer-hexaammineruthenium trichloride composite probe. Combined with the signal amplification effect of a silica nanoporous membrane, a homogeneous electrochemical aptamer sensor is constructed for the highly sensitive detection of mycotoxins in agricultural products. This sensor features a low detection limit, a wide detection range, and eliminates the need for complex pretreatment and separation processes, reducing detection costs. Its simple operation makes it suitable for large-scale applications.

[0011] Taking zearalenone as an example, the detection principle is explained as follows: When zearalenone is absent in agricultural products, hexaammineruthenium trichloride (HJT) passes through. At this time, the amount of free HJT in the solution is very small, so the electrode modified with a silica nanoporous membrane can only detect a low electrochemical signal of HJT. When zearalenone is present in agricultural products, the zearalenone aptamer specifically binds to the target analyte to form a complex and detaches from the graphene oxide surface. This causes the HJT probe molecules adsorbed on the graphene oxide to detach, become free in the solution, and be enriched by the negatively charged silica nanoporous membrane, thus amplifying the electrochemical signal. Finally, the electrochemical detection of zearalenone in agricultural products is achieved by recording the changes in the electrochemical signal of the HJT probe molecules.

[0012] The concentration of graphene oxide in the detection solution is 0.01–2 mg / mL, the concentration of fungicides is 0.1–10 μmol / L, and the concentration of hexaammineruthenium trichloride is 1–1000 μmol / L. Too low or too high a concentration of the composite probe will result in insignificant changes in the signal of hexaammineruthenium trichloride released into the solution after the target analyte binds to the composite probe, thus affecting the detection sensitivity.

[0013] The preparation method of the graphene oxide-fungus toxin aptamer-hexaammineruthenium trichloride composite probe includes the following steps:

[0014] Step 1: Dissolve graphene oxide, hexaammineruthenium trichloride and mycotoxin aptamers in buffer solutions to prepare graphene oxide solution, hexaammineruthenium trichloride solution and mycotoxin aptamer solution respectively;

[0015] Step 2: Mix the graphene oxide solution, hexaammineruthenium trichloride solution, and fungal toxin aptamer solution, and incubate to obtain the composite probe.

[0016] Both excessively low and excessively high concentrations of hexaammineruthenium trichloride (HJT) will result in insignificant changes in the HJT signal after the target analyte binds to the composite probe and is released into the solution, thus affecting detection sensitivity. The composite probe contains graphene oxide at a concentration of 0.01–2 mg / mL, mycotoxin at a concentration of 0.1–10 μmol / L, and HJT at a concentration of 1–1000 μmol / L. Because the amount of analyte added to the composite probe during detection is very small, the concentrations of graphene oxide, mycotoxin, and HJT in the final detection solution are essentially equivalent to the concentrations in the initially prepared composite probe.

[0017] Preferably, the concentration of hexaammineruthenium trichloride is 0.08–0.2 mg / mL; the concentration of the fungicide aptamer is 0.8–2 μmol / L; and the concentration of hexaammineruthenium trichloride is 50–200 μmol / L.

[0018] If the incubation temperature is too low or too high, or the incubation time is too short, the composite probe will not bind completely, affecting the detection sensitivity. If the incubation time is too long, it will affect the detection efficiency. The incubation conditions in step 2 are 0-60℃ for 5 min to 24 h; more preferably, incubation at 30-40℃ for 1-2 h.

[0019] The buffer solution comprises one of sodium chloride aqueous solution, sodium sulfate aqueous solution, potassium chloride aqueous solution, potassium hydrogen phthalate aqueous solution, phosphate buffer solution, and acetate-sodium acetate buffer solution, wherein the concentration of the buffer solution is 0.005-0.5 mol / L, more preferably 0.008-0.2 mol / L; and the pH is 6.0-8.0, more preferably 6.5-7.5.

[0020] The fungal aptamers include any one of ochratoxin aptamers, aflatoxin aptamers, or zearalenone aptamers. Different aptamers are adaptively selected for different fungal aptamers to be tested.

[0021] The method for preparing the electrode modified with the silica nanoporous film involves growing a porous silica structure with pore sizes in the nanometer range on the electrode surface, including electrochemically assisted self-assembly. Any one of the following methods: solution growth, two-phase layered growth, evaporation-induced self-assembly, π-π interaction-induced self-assembly, epitaxial growth, strong magnetic field method, electric field method, and organic solvent-induced self-assembly.

[0022] Preferably, the electrode modified with the silica nanoporous membrane is prepared by an electrochemically assisted self-assembly method, specifically, the preparation method includes the following steps:

[0023] The working electrode was placed in a mixed solution containing hexadecyltrimethylammonium bromide (CTAB), tetraethoxysilane, water, ethanol, sodium nitrate, and hydrochloric acid, and a constant current density of –0.08 mA cm⁻¹ was applied. –2 ~–1.2mA cm –2 Growth time is 3–30 seconds.

[0024] The mixed solution includes 1-2g of CTAB, 2-4mL of tetraethoxysilane, 20mL of water, 20mL of ethanol, 0.05-0.3g of sodium nitrate, and 2-5μL of hydrochloric acid, and is stirred and mixed for 1-4 hours in advance.

[0025] The electrode includes any one of indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, and carbon fiber electrode. More preferably, it is an indium tin oxide electrode or a glassy carbon electrode.

[0026] The present invention also provides a method for preparing the electrochemical aptamer sensor for detecting mycotoxins in agricultural products, comprising the following steps:

[0027] Step 1: Dissolve graphene oxide, hexaammineruthenium trichloride and mycotoxin aptamers in buffer solutions to prepare graphene oxide solution, hexaammineruthenium trichloride solution and mycotoxin aptamer solution respectively;

[0028] Step 2: Mix the graphene oxide solution, hexaammineruthenium trichloride solution and fungal toxin aptamer solution, and incubate to obtain the composite probe of graphene oxide-fungin aptamer-hexaammineruthenium trichloride;

[0029] Step 3: Growing a porous silica structure with pore size in the nanometer range on the electrode surface to prepare an electrode modified with a silica nanoporous film.

[0030] Step 4: Using an electrode modified with a silica nanoporous membrane as the working electrode, a composite probe of graphene oxide-mycotoxin aptamer-hexaammineruthenium trichloride is added to the detection solution, and an electrochemical method is used to detect mycotoxins in agricultural products in the detection solution.

[0031] This invention also provides a method for detecting mycotoxins in agricultural products using the aforementioned electrochemical aptamer sensor, comprising the following steps:

[0032] Step 1: Prepare a test solution for agricultural products containing mycotoxins, add the composite probe to the test solution, and use an electrode modified with a silica nanoporous membrane as the working electrode to detect mycotoxins in agricultural products.

[0033] The detection method of this invention exhibits a linear relationship between the logarithm of the detection concentration of fungimycin in the range of 1 pg / mL to 1 μg / mL. This detection method has a wide linear range, high selectivity, high sensitivity, low detection limit, and does not require complex pretreatment and separation processes, thus reducing detection costs. It is simple to operate and suitable for large-scale applications.

[0034] Preferably, the minimum detectable concentration of ochratoxin by the detection method of the present invention is 10 pg / mL.

[0035] The preparation of the test solution for the agricultural product includes: taking 1g of agricultural product, grinding it into powder, adding 20mL of a mixed solution of sodium chloride and acetonitrile, sonicating and centrifuging, and taking the supernatant to dissolve in a buffer solution; centrifugation is preferably performed at a speed of 4000-8000rpm / s, and more preferably at 6000rpm / s.

[0036] The concentration of sodium chloride in the sodium chloride and acetonitrile mixed solution is 1–100 mmol / L; the dilution factor of the supernatant is 1–500 times.

[0037] Preferably, the concentration of sodium chloride in the sodium chloride and acetonitrile mixed solution is 5-20 mmol / L, and the dilution factor of the supernatant is 20-80 times.

[0038] The agricultural products containing mycotoxins include corn or chestnuts containing any one of ochratoxin, aflatoxin, or zearalenone.

[0039] Corn or chestnuts containing zearalenone are preferred.

[0040] Compared with the prior art, the present invention has the following beneficial effects:

[0041] (1) The homogeneous electrochemical aptamer sensor of the present invention enables the detection of mycotoxin content in agricultural products. The electrochemical signal can be measured after the target substance is incubated with the composite probe without the need for separation and extraction steps. Compared with other label-fixed aptamer sensors, it is simpler to operate and has a lower cost.

[0042] (2) The detection method of the present invention shows a linear relationship between the logarithm of the zearalenone detection concentration and 1 pg / mL to 1 μg / mL, and the detection limit is 1.2 fg / mL. The detection method has a wide linear range, high selectivity, high detection sensitivity and low detection limit.

[0043] (3) The zearalenone aptamer in this invention has the advantages of low cost, good stability and high affinity, and can specifically recognize zearalenone.

[0044] (4) The silica nanoporous membrane in this invention is simple to prepare, does not require a complicated electrode modification process, and has an amplification effect on the positively charged hexaammine trichloride signal molecule, which can achieve highly sensitive detection of fungal mycotoxins, especially zearalenone.

[0045] (5) The silica nanoporous membrane of the present invention has ultra-small nanochannels (2-3 nm), and the surface of the channels is rich in Si-OH groups (pK). a ~2) The surface carries a negative charge in conventional solution media, which can not only amplify the electrochemical probe signal and improve the detection sensitivity, but also prevent the entry of negatively charged cells and proteins as well as most biological macromolecules in complex samples, and has good anti-fouling and anti-interference capabilities.

[0046] (6) By simply changing the type of aptamer, the homogeneous electrochemical aptamer sensing strategy of the present invention can be extended to the high-sensitivity detection of various toxins such as ochratoxin and aflatoxin, and has great application potential in the field of food safety monitoring. Attached Figure Description

[0047] Figure 1 The images shown are transmission electron microscopy (TEM) image (A) and scanning electron microscopy (SEM) image (B) of the ITO electrode modified with a silica nanoporous membrane in Example 1.

[0048] Figure 2 The figures show the cyclic voltammetry curves of the bare ITO electrode (Bare ITO), the ITO electrode modified with a silica nanoporous membrane containing CTAB micelles (SM@VMSF / ITO), and the ITO electrode modified with a silica nanoporous membrane (VMSF / ITO) in Example 1, respectively, in 0.5 mM K3Fe(CN)6 (A) and Ru(NH3)6Cl3 (B) solutions. The electrolyte solution was potassium hydrogen phthalate (0.05 mol / L, pH = 7.4).

[0049] Figure 3 The X-ray photoelectron spectrum (A), Fourier transform infrared spectrum (B), and ultraviolet-visible spectrum (C) of graphene oxide (GO) in Example 1 are shown.

[0050] Figure 4 The images show the cyclic voltammetry curves (A), differential pulse voltammetry curves (B), zeta potential intensity graphs (C), and bar graphs (D) of VMSF / ITO in different composite solutions in Example 1.

[0051] Figure 5This is an optimization graph showing the concentration of Ru(NH3)6Cl3 in the composite probe in Example 1. ΔI is the difference in oxidation peak current values ​​in the differential pulse voltammetry curves before (I0) and after (I) the addition of the target ZEN to the composite probe solutions containing 0.1 mg / mL GO, 1 μM ZEN aptamer and different concentrations of Ru(NH3)6Cl3. The error bars in the graph represent the standard deviation of the three measurements.

[0052] Figure 6 In Example 1, (A) shows the differential pulse voltammetry curves of the VMSF / ITO electrode with a GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe and different concentrations of ZEN (1 pg / mL to 1 μg / mL). (B) shows the linear relationship between the oxidation peak current and the logarithmic concentration of ZEN. The concentrations of GO, Ru(NH3)6Cl3, and ZEN aptamer in the GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe were 0.1 mg / mL, 100 μM, and 1 μM, respectively. The error bars in the figure represent the standard deviation of three measurements.

[0053] Figure 7 The figure shows the DPV signal difference of the VMSF / ITO electrode in Example 1 before (I0) and after (I) the presence of the GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe and 0.1 μg / mL starch, 0.1 ng / mL ZEN, 10 ng / mL ochratoxin (OTA), 10 ng / mL aflatoxin B1 (AFB1), or a mixture of the above substances. The concentrations of GO, Ru(NH3)6Cl3, and ZEN aptamer in the GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe were 0.1 mg / mL, 100 μM, and 1 μM, respectively. The error bars in the figure represent the standard deviation of the three measurements.

[0054] Figure 8 The images show the cyclic voltammetry (A) and differential pulse voltammetry (B) curves of VMSF / ITO in different composite solutions in Example 2.

[0055] Figure 9 In Example 2, (A) shows the differential pulse voltammetry curves of the VMSF / ITO electrode with a GO-OTA aptamer-Ru(NH3)6Cl3 composite probe and different concentrations of OTA (10 pg / mL to 1 μg / mL). (B) shows the linear relationship between the oxidation peak current and the logarithmic concentration of OTA. The concentrations of GO, Ru(NH3)6Cl3, and OTA aptamer in the GO-OTA aptamer-Ru(NH3)6Cl3 composite probe were 0.1 mg / mL, 200 μM, and 1 μM, respectively. The error bars in the figure represent the standard deviation of three measurements. Detailed Implementation

[0056] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Modifications or equivalent substitutions made by those skilled in the art based on their understanding of the technical solutions of this invention, without departing from the spirit and scope of the invention, should be covered within the protection scope of this invention.

[0057] Unless otherwise specified, all raw materials used in the following specific embodiments were purchased commercially and used directly without special processing. The main raw materials used in the embodiments are as follows:

[0058] Potassium ferricyanide: Shanghai Aladdin Biotechnology Co., Ltd.;

[0059] Ruthenium trichloride (III): Shanghai Aladdin Biotechnology Co., Ltd.

[0060] Hexadecyltrimethylammonium bromide: Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0061] Tetraethoxysilane: Shanghai Aladdin Biochemical Technology Co., Ltd.;

[0062] Potassium hydrogen phthalate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0063] Zearalenone: Shanghai Maclean Biochemical Technology Co., Ltd.;

[0064] DNA nucleic acid sequence: Bioengineering (Shanghai) Co., Ltd.;

[0065] Disodium hydrogen phosphate and sodium dihydrogen phosphate: Shanghai Aladdin Biotechnology Co., Ltd.;

[0066] Example 1

[0067] Using an ITO electrode modified with a silica nanoporous membrane as the working electrode, a graphene oxide-zearalenone aptamer-hexaammineruthenium trichloride composite probe was added to the test solution to detect the electrochemical process of zearalenone in chestnuts.

[0068] (1) Preparation of graphene oxide-zearalenone aptamer-hexaammineruthenium trichloride composite probe: Graphene oxide, hexaammineruthenium trichloride, and zearalenone aptamer were dissolved in 0.01 mol / L phosphate buffer (pH = 7.4) to prepare graphene oxide solution, hexaammineruthenium trichloride solution, and zearalenone aptamer solution, respectively. The hexaammineruthenium trichloride solution, graphene oxide solution, and zearalenone aptamer solution were mixed and incubated at 37℃ for 2 h to obtain the graphene oxide-zearalenone aptamer-hexaammineruthenium trichloride composite probe, wherein the concentrations of graphene oxide, hexaammineruthenium trichloride, and zearalenone aptamer were 0.1 mg / mL, 100 μM, and 1 μM, respectively.

[0069] (2) Preparation of electrodes modified with silica nanoporous membranes: 1.585 g cetyltrimethylammonium bromide and 2.833 g tetraethoxysilane were added to a mixed solution of 20 mL ethanol and 20 mL sodium nitrate (concentration 0.1 mol / L, pH = 2.6) and stirred at room temperature for 2.5 h to obtain a precursor solution.

[0070] A three-electrode system was used, with an ITO electrode as the working electrode, a platinum electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode, placed in a precursor solution for growing silica nanoporous films; a constant current (current density of –0.7 μA / cm²) was applied. 2 After growing for 10 seconds, the working electrode was quickly removed, rinsed with a large amount of flowing ultrapure water, dried with nitrogen, and aged overnight at 120°C to obtain ITO modified with a micelle-containing silica nanoporous membrane (SM@VMSF / ITO).

[0071] Finally, the obtained SM@VMSF / ITO electrode was immersed in an ethanol solution containing 0.1 mol / L hydrochloric acid and stirred for 5 min to remove the hydrophobic micelles in the pores, thus obtaining an ITO electrode (VMSF / ITO) modified with a silica nanoporous membrane.

[0072] (3) Using the VMSF / ITO prepared in step (2) as the working electrode, the zearalenone in chestnuts was detected by an electrochemical method. The specific process includes the following steps:

[0073] (a) Accurately weigh 1 mg ZEN, dissolve it in 1 mL of methanol, and store it in a brown vial at –20°C protected from light. Transfer 100 μL of 1 mg / mL ZEN stock solution and dissolve it in 900 μL of methanol to prepare a 100 μg / mL ZEN standard working solution. Store it at 4°C protected from light for later use.

[0074] (b) Take 1g of chestnut powder, add 20mL of a mixture of sodium chloride and acetonitrile, sonicate, centrifuge at 6000rpm / s, take the supernatant and dissolve it in 0.01mol / L phosphate buffer (pH=7.4) and dilute it 50 times. Add different volumes of ZEN standard working solution to obtain a series of test solutions (where the ZEN concentrations are 0.001ng / mL, 0.01ng / mL, 0.1ng / mL, 1ng / mL, 10ng / mL, 100ng / mL and 1000ng / mL, respectively).

[0075] (c) Using the VMSF / ITO working electrode and platinum electrode prepared in step (2) as the counter electrode and silver / silver chloride electrode as the reference electrode, add the GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe to the above test solution, wherein the concentrations of GO, Ru(NH3)6Cl3 and ZEN aptamer are 0.1 mg / mL, 100 μM and 1 μM, respectively, and use differential pulse voltammetry to test the above test solution.

[0076] The electron microscope image of the ITO electrode (VMSF / ITO) modified with a silica nanoporous membrane prepared in step (2) of the above method is shown in the figure. Figure 1 As shown.

[0077] Figure 1 (A) is a top view of a transmission electron microscope (TEM) image of an ITO electrode modified with a silica nanoporous membrane. The image clearly shows that the silica nanoporous membrane has uniformly arranged hexagonal pores without large-area defects, and the measured pore size is 2–3 nm. Figure 1 The scanning electron microscope image shown in (B) reveals that the silica nanoporous film has a uniform thickness, and the electrode cross-section exhibits a distinct three-layer structure, corresponding from bottom to top to glass, ITO, and the silica nanoporous film layer, respectively. The thickness of the silica nanoporous film is approximately 125 nm.

[0078] Figure 2 Cyclic voltammetry curves are shown for a bare ITO electrode, an ITO electrode modified with a silica nanoporous membrane containing CTAB micelles (SM@VMSF / ITO), and an ITO electrode modified with a silica nanoporous membrane (VMSF / ITO), respectively, in 0.5 mM K3Fe(CN)6(A) and Ru(NH3)6Cl3(B) solutions. The 0.5 mM K3Fe(CN)6(A) and Ru(NH3)6Cl3(B) solutions were prepared from the electrolyte solution potassium hydrogen phthalate (0.05 mol / L, pH = 7.4).

[0079] Depend on Figure 2It was found that neither the charged K3Fe(CN)6 nor Ru(NH3)6Cl3 probes exhibited a significant Faraday current signal on the SM@VMSF / ITO electrode, only showing a charging current. This is because the micelles in the pores are hydrophobic, hindering the hydrophilic probes K3Fe(CN)6 and Ru(NH3)6Cl3 from reaching the electrode surface. After removing the micelles, the signals of both probes on the VMSF / ITO electrode became significant. Compared to the bare ITO electrode, the electrochemical signal of K3Fe(CN)6 was suppressed, while the signal of Ru(NH3)6Cl3 was significantly enhanced. This is because, under these experimental conditions, the deprotonation of the silanol groups in the pores made the inner wall of the pores negatively charged, which could repel the negatively charged K3Fe(CN)6 probe and attract the positively charged Ru(NH3)6Cl3 probe. The above results demonstrate that the prepared VMSF was intact and crack-free, and that VMSF / ITO exhibits significant charge selectivity, enriching positively charged substances while having no amplification effect on the negatively charged K3Fe(CN)6 probe.

[0080] Figure 3 The images show the X-ray photoelectron spectroscopy (XPS) spectrum (A), Fourier transform infrared (FIR) spectrum (B), and ultraviolet-visible (UV-Vis) spectrum of graphene oxide (GO). The XPS spectrum of GO exhibits four characteristic peaks: 284.5 eV, 286.3 eV, 287.5 eV, and 288.4 eV, corresponding to the C / C=C bond, CO bond, carbon-oxygen double bond (C=O), and carboxyl group (OC=O) of GO. The Fourier transform infrared spectrum shows a peak at 3431 cm⁻¹. –1 Absorption bands with -OH groups are present at 1629 and 1726 cm⁻¹. –1 The absorption band corresponds to the carbonyl stretching vibration. The UV-Vis spectrum shows an absorption peak at 228 nm and a shoulder peak at 300 nm, representing the π→π transition of the aromatic C=C bond and the n→π transition of the C=O bond, respectively. These results demonstrate that GO possesses oxygen-containing functional groups and an aromatic structure, and can adsorb positively charged π-conjugated molecules through electrostatic interactions or π-π interactions. That is, it can complex with aptamers possessing π-conjugation, and also with positively charged hexaammineruthenium trichloride.

[0081] Figure 4 The images show the cyclic voltammetry (A), differential pulse voltammetry (B), zeta potential intensity plot (C), and bar chart (D) of VMSF / ITO in different composite solutions.

[0082] like Figure 4As shown in (A), Ru(NH3)6Cl3 exhibits a pair of distinct redox peaks on the VMSF / ITO electrode. The electrochemical signal of Ru(NH3)6Cl3 significantly decreases upon the addition of GO or ZEN aptamers. This is because positively charged Ru(NH3)6Cl3 in the solution adsorbs onto the negatively charged GO or ZEN aptamer surface via electrostatic interactions, leading to a reduction in the number of free Ru(NH3)6Cl3 molecules in the solution. When both GO and ZEN aptamers are present in the solution, the electrochemical signal of Ru(NH3)6Cl3 decreases further. This is due to the formation of a GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe, and the ZEN aptamer further adsorbing Ru(NH3)6Cl3 via electrostatic interactions.

[0083] like Figure 4 As shown in (B), after adding the target ZEN to the GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe solution, the ZEN aptamer preferentially binds to ZEN, leading to Ru(NH3)6Cl3... 3+ The molecules detach and become free in the solution, where they are enriched by the negatively charged VMSF channels, generating a significant electrochemical signal. This demonstrates that the constructed sensor can be used for the detection of ZEN.

[0084] Depend on Figure 4 (CD) shows that GO has a relatively negative zeta potential. GO and the ZEN aptamer complex via π-π interactions. Due to the weakly electronegative DNA coating on the GO surface, its zeta potential is somewhat increased. Compared to GO, the ZEN aptamer, and their complexes, complexation with Ru(NH3)6Cl3 significantly increases the zeta potential, indicating that Ru(NH3)6Cl3... 3+ Successful electrostatic adsorption onto the GO surface. The above results demonstrate the successful preparation of the GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe in step (1).

[0085] Figure 5 Ru(NH3)6 in electrochemical composite probe 3+ The concentration optimization graph shows that the sensor exhibits the highest sensitivity when the probe concentration is 100 μM. Therefore, the optimal range is 100-150 μM.

[0086] Figure 6 (A) is a differential pulse voltammogram of a VMSF / ITO electrode under optimal experimental conditions, detecting a GO-ZEN aptamer-Ru(NH3)6Cl3 composite probe and different concentrations of ZEN. It can be seen that the peak current increases with increasing ZEN concentration. The graph is plotted with the oxidation peak current as the ordinate and the logarithm of the ZEN concentration (logconcentration) as the ordinate. ZEN Plot a graph with the x-axis as the horizontal axis, such as... Figure 6(B). The peak current value showed a good linear relationship with the logarithmic concentration of ZEN in the range of 1 pg / mL to 1 μg / mL, and the detection limit was 1.2 fg / mL, indicating that its sensitivity was good.

[0087] Figure 7 Selectivity data for the constructed homogeneous electrochemical aptamer sensor are presented. Ochratoxin (OTA), aflatoxin B1 (AFB1), and starch (Starch) were used as interfering substances, and the difference in oxidation peak current before and after the addition of different interfering substances or target compounds was recorded. The results showed that only the addition of the target compound ZEN produced a significant change in electrochemical signal, indicating that the prepared homogeneous electrochemical aptamer sensor has excellent specificity and selectivity.

[0088] The above results demonstrate that combining the signal amplification effect of the silica nanoporous membrane with the graphene oxide-zearalenone aptamer-hexaammineruthenium trichloride composite probe can achieve highly sensitive and selective detection of zearalenone in chestnuts.

[0089] Example 2

[0090] An ITO electrode modified with a silica nanoporous membrane was used as the working electrode. A graphene oxide-ochratoxin aptamer-hexaamminetrimonium trichloride composite probe was added to the test solution to detect the electrochemical process of ochratoxin in chestnuts.

[0091] (1) Preparation of graphene oxide-ochratoxin aptamer-hexaammine trichloride composite probe: Graphene oxide, hexaammine trichloride, and ochratoxin aptamer were dissolved in 0.01 mol / L phosphate buffer (pH = 7.4) to prepare graphene oxide solution, hexaammine trichloride solution, and ochratoxin aptamer solution, respectively. The hexaammine trichloride solution, graphene oxide solution, and ochratoxin aptamer solution were mixed and incubated at 37℃ for 2 h to prepare the graphene oxide-ochratoxin aptamer-hexaammine trichloride composite probe, wherein the concentrations of graphene oxide, hexaammine trichloride, and ochratoxin aptamer were 0.1 mg / mL, 200 μM, and 1 μM, respectively.

[0092] (2) Preparation of electrodes modified with silica nanoporous membranes: 1.585 g cetyltrimethylammonium bromide and 2.833 g tetraethoxysilane were added to a mixed solution of 20 mL ethanol and 20 mL sodium nitrate (concentration 0.1 mol / L, pH = 2.6) and stirred at room temperature for 2.5 h to obtain a precursor solution.

[0093] A three-electrode system was used, with an ITO electrode as the working electrode, a platinum electrode as the counter electrode, and a silver / silver chloride electrode as the reference electrode, placed in a precursor solution for growing silica nanoporous films; a constant current (current density of –0.7 μA / cm²) was applied. 2 After growing for 10 seconds, the working electrode was quickly removed, rinsed with a large amount of flowing ultrapure water, dried with nitrogen, and aged overnight at 120°C to obtain ITO modified with a micelle-containing silica nanoporous membrane (SM@VMSF / ITO).

[0094] Finally, the obtained SM@VMSF / ITO electrode was immersed in an ethanol solution containing 0.1 mol / L hydrochloric acid and stirred for 5 min to remove the hydrophobic micelles in the pores, thus obtaining an ITO electrode (VMSF / ITO) modified with a silica nanoporous membrane.

[0095] (3) Using the VMSF / ITO prepared in step (2) as the working electrode, ochratoxin in chestnuts was detected by electrochemical method. The specific process includes the following steps:

[0096] (a) Accurately weigh 1 mg OTA, dissolve it in 1 mL of methanol, and store it in a brown vial at –20°C protected from light. Transfer 100 μL of 1 mg / mL OTA stock solution, dissolve it in 900 μL of methanol to prepare a 100 μg / mL OTA standard working solution, and store it at 4°C protected from light for later use.

[0097] (b) Take 1g of chestnut powder, add 20mL of sodium chloride and acetonitrile mixed solution, sonicate and centrifuge at 6000rpm / s, take the supernatant and dissolve it in 0.01mol / L phosphate buffer (pH=7.4) diluted 50 times, add different volumes of OTA standard working solution to obtain a series of test solutions (where the concentration of OTA is 0.01ng / mL, 0.1ng / mL, 1ng / mL, 10ng / mL, 100ng / mL and 1000ng / mL respectively).

[0098] (c) Using the VMSF / ITO working electrode and platinum electrode prepared in step (2) as the counter electrode and silver / silver chloride electrode as the reference electrode, a GO-OTA aptamer-Ru(NH3)6Cl3 composite probe was added to the above test solution, wherein the concentrations of GO, Ru(NH3)6Cl3 and OTA aptamer were 0.1 mg / mL, 200 μM and 1 μM respectively. The above test solution was tested by differential pulse voltammetry.

[0099] Figure 8 Cyclic voltammetry (A) and differential pulse voltammetry (B) curves of VMSF / ITO in different composite solutions.

[0100] like Figure 8 As shown in (A), Ru(NH3)6Cl3 exhibits a pair of distinct redox peaks on the VMSF / ITO electrode. The electrochemical signal of Ru(NH3)6Cl3 significantly decreases upon the addition of GO or OTA aptamers. This is because positively charged Ru(NH3)6Cl3 in the solution adsorbs onto the negatively charged GO or OTA aptamer surface via electrostatic interactions, leading to a reduction in the number of free Ru(NH3)6Cl3 molecules in the solution. When both GO and OTA aptamers are present in the solution, the electrochemical signal of Ru(NH3)6Cl3 decreases further. This is due to the formation of a GO-OTA aptamer-Ru(NH3)6Cl3 composite probe, and the further adsorption of Ru(NH3)6Cl3 by the OTA aptamer via electrostatic interactions.

[0101] like Figure 8 As shown in (B), after adding the target OTA to the GO-OTA aptamer-Ru(NH3)6Cl3 composite probe solution, the OTA aptamer preferentially binds to OTA, leading to Ru(NH3)6Cl3... 3+ The molecules detach and become free in the solution, where they are enriched by the negatively charged VMSF channels, generating a significant electrochemical signal. This demonstrates that the constructed sensor can be used for the detection of OTA.

[0102] Figure 9 (A) is a differential pulse voltammogram of a VMSF / ITO electrode detecting a GO-OTA aptamer-Ru(NH3)6Cl3 composite probe and different concentrations of OTA. It can be seen that the peak current increases with increasing OTA concentration. The peak current value is plotted on the ordinate, and the logarithm of the OTA concentration is plotted on the other side. OTA Plot a graph with the x-axis as the horizontal axis, such as... Figure 9 (B). The peak current value showed a good linear relationship with the logarithmic concentration of OTA in the range of 10 pg / mL to 1 μg / mL, and the detection limit was 8.9 pg / mL, indicating that its sensitivity was good.

[0103] The above results demonstrate that combining the signal amplification effect of the silica nanoporous membrane with the graphene oxide-ochratoxin aptamer-hexaamminetrimonium chloride composite probe can achieve highly sensitive detection of ochratoxin in chestnuts.

Claims

1. An electrochemical aptamer sensor for detecting mycotoxins in agricultural products, characterized in that, The electrode includes a working electrode, a reference electrode, and a counter electrode modified with a silica nanoporous membrane, and a composite probe containing graphene oxide-fungi toxin aptamer-hexaammineruthenium trichloride in the detection solution. The preparation method of the graphene oxide-fungus toxin aptamer-hexaammineruthenium trichloride composite probe includes the following steps: Step 1: Dissolve graphene oxide, hexaammineruthenium trichloride and mycotoxin aptamers in buffer solutions to prepare graphene oxide solution, hexaammineruthenium trichloride solution and mycotoxin aptamer solution respectively; Step 2: Mix the graphene oxide solution, hexaammineruthenium trichloride solution, and fungal toxin aptamer solution, and incubate to obtain the composite probe.

2. The electrochemical aptamer sensor for detecting mycotoxins in agricultural products according to claim 1, characterized in that, The concentration of graphene oxide in the detection solution is 0.01~2 mg / mL, the concentration of mycotoxins is 0.1~10 μmol / L, and the concentration of hexaammineruthenium trichloride is 1~1000 μmol / L.

3. The electrochemical aptamer sensor for detecting mycotoxins in agricultural products according to claim 1, characterized in that, In step 2, the incubation conditions are 0~60℃ for 5 min~24 h; The buffer solution includes one of the following: sodium chloride aqueous solution, sodium sulfate aqueous solution, potassium chloride aqueous solution, potassium hydrogen phthalate aqueous solution, phosphate buffer solution, and acetate-sodium acetate buffer solution. The concentration of the buffer solution is 0.005~0.5 mol / L, and the pH is 6.0~8.

0.

4. The electrochemical aptamer sensor for detecting mycotoxins in agricultural products according to claim 1, characterized in that, The fungal toxin aptamers include any one of ochratoxin aptamers, aflatoxin aptamers, or zearalenone aptamers.

5. The electrochemical aptamer sensor for detecting mycotoxins in agricultural products according to claim 1, characterized in that, The method for preparing the electrode modified with the silica nanoporous membrane is to grow a porous silica structure with a pore size in the nanometer size on the electrode surface, including any one of the following: electrochemical assisted self-assembly, Stöber solution growth, two-phase layered growth, evaporation-induced self-assembly, π-π interaction-induced self-assembly, epitaxial growth, strong magnetic field method, electric field method, and organic solvent-induced self-assembly.

6. The electrochemical aptamer sensor for detecting mycotoxins in agricultural products according to claim 5, characterized in that, The electrode includes any one of indium tin oxide electrode, glassy carbon electrode, fluorine-doped tin oxide electrode, gold electrode, screen-printed electrode, graphite electrode, and carbon fiber electrode.

7. The method for preparing an electrochemical aptamer sensor for detecting mycotoxins in agricultural products according to any one of claims 1-6, characterized in that, Including the following steps: Step 1: Dissolve graphene oxide, hexaammineruthenium trichloride and mycotoxin aptamers in buffer solutions to prepare graphene oxide solution, hexaammineruthenium trichloride solution and mycotoxin aptamer solution respectively; Step 2: Mix the graphene oxide solution, hexaammineruthenium trichloride solution and fungal toxin aptamer solution, and incubate to obtain the composite probe of graphene oxide-fungin aptamer-hexaammineruthenium trichloride; Step 3: Growing a porous silica structure with pore size in the nanometer range on the electrode surface to prepare an electrode modified with a silica nanoporous film. Step 4: Using an electrode modified with a silica nanoporous membrane as the working electrode, a composite probe of graphene oxide-mycotoxin aptamer-hexaammineruthenium trichloride is added to the detection solution, and the mycotoxins in agricultural products in the detection solution are detected by an electrochemical method.

8. The method for detecting mycotoxins in agricultural products using an electrochemical aptamer sensor according to any one of claims 1-6, characterized in that, Including the following steps: Step 1: Prepare a test solution of agricultural products containing mycotoxins, add the composite probe to the test solution, and use an electrode modified with a silica nanoporous membrane as the working electrode to detect mycotoxins in agricultural products.

9. The method for detecting mycotoxins in agricultural products using an electrochemical aptamer sensor according to claim 8, characterized in that, The preparation of the test solution for the agricultural product includes: taking 1 g of agricultural product, grinding it into powder, adding 20 mL of a mixed solution of sodium chloride and acetonitrile, sonicating and centrifuging, and taking the supernatant to dissolve in a buffer solution; The concentration of sodium chloride in the sodium chloride and acetonitrile mixed solution is 1~100 mmol / L; the dilution factor of the supernatant is 1~500 times.

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