Label-free electrochemical aptamer sensor for detecting ochratoxin A as well as construction method and application of label-free electrochemical aptamer sensor

Abstract

The invention discloses a label-free electrochemical aptamer sensor for detecting ochratoxin A as well as a construction method and application of the label-free electrochemical aptamer sensor. The sensor comprises a three-electrode system of a modified working electrode, a reference electrode and a counter electrode, and exonuclease I and potassium ferricyanide are contained in electrolyte; the modified working electrode is obtained by sequentially covalently immobilizing cDNA and an ochratoxin A aptamer on a VMSF modified electrode. According to the invention, potassium ferricyanide and an ochratoxin A aptamer are respectively used as an electrochemical signal molecule and a specific recognition element; a label-free electrochemical aptamer sensor is constructed by combining a signal amplification strategy of exonuclease I assisted target circulation, is used for detecting ochratoxin A in samples such as wheat samples and wine and has the characteristics of high sensitivity, wide linear range, simplicity in operation and the like.

Description

Technical Field

[0001] The present invention relates to the technical field of biological detection, and in particular to a label-free electrochemical aptamer sensor for detecting ochratoxin A, and a construction method and application thereof. Background Art

[0002] Ochratoxins are a group of common contaminants produced by Aspergillus and Penicillium molds. Among them, ochratoxin A (OTA) is the most toxic, the most widely distributed, the most toxic, the most serious contamination of agricultural products, and the one that is most closely related to human health. Ochratoxin A is a stable compound that can maintain its structural integrity under most processing conditions. It is commonly found in contaminated foods and crops such as corn, wheat, oats, coffee, wine and vegetables. Studies have shown that it is also nephrotoxic, hepatotoxic, teratogenic, carcinogenic and immunosuppressive. The International Agency for Research on Cancer has classified it as a Class 2B carcinogen.

[0003] Conventional ochratoxin A detection methods include liquid chromatography (LC), high performance liquid chromatography, LC tandem mass spectrometry, and gas chromatography-tandem mass spectrometry. Although these methods are highly sensitive and accurate, they require cumbersome sample pretreatment operations, expensive instruments, and professional requirements for operators, which limits their application in rapid OTA analysis. In addition to traditional chromatography, some antibody-based rapid detection sensors have also been developed for quantitative analysis of OTA, such as immunochromatography, enzyme-linked immunosorbent assay, electrochemical and optical immunosensors. These methods can achieve rapid and sensitive OTA detection. However, due to the shortcomings of antibodies themselves, these immunosensors have problems such as poor repeatability, semi-quantification, and false positive results. In contrast, aptamers are artificially synthesized and show great potential in the field of sensing analysis due to their high affinity and specificity for targets, simple preparation and easy modification, good stability, non-immunogenicity, low cost, and a wide range of targets.

[0004] In order to improve the detection performance of aptamer sensors, various signal amplification strategies have been proposed, such as the introduction of nanomaterials, the design of energy transfer strategies, enzyme-assisted target cycles, and DNA-based signal amplification technology. Among them, enzyme-assisted target cycles have the advantages of simple operation and no need for complex preparation processes. Therefore, signal amplification strategies based on nuclease assistance are widely used in the field of biosensing. For example, researchers have developed an electrochemical aptamer sensor for sensitive detection of OTA based on exonuclease-assisted digestion.

[0005] For example, CN101936940 A discloses a method for detecting ochratoxin A using an electrochemiluminescent aptamer sensor, which is used for detecting the content of ochratoxin A in wheat, grains, feed and their products. The basis of this invention is the specific recognition of ochratoxin A by the aptamer and the electrochemiluminescent reaction of hydrogen peroxide (H2O2) and isoluminol (ABEI) under alkaline conditions. Nanogold particles are modified to the surface of a bare gold electrode to amplify the electrochemiluminescent signal, and then single-stranded DNA is modified on the surface of the working electrode, and the aptamer labeled with isoluminol (ABEI) is modified to the electrode surface through base pairing, hydrogen peroxide (H2O2) is added, and the electroluminescent signal is detected, which is a quantitative detection method for ochratoxin A with high sensitivity and strong operability.

[0006] Silica nanochannel array film (SNF) is a mesoporous film material composed of ultra-small nanochannels oriented perpendicular to the substrate. The SNF obtained by conventional methods has highly uniform pore size (2-3nm), ultra-thin and adjustable thickness (20-200nm), high porosity, good stability and biocompatibility. In addition, the surface of SNF is rich in silanol (pK a ~2), in a neutral solution environment, deprotonation is negatively charged. Therefore, SNF has high permeability, excellent size screening ability and significant ion selective permeability, and can be used as an electrode antifouling layer, showing great potential in direct electrochemical or electrochemiluminescent analysis of complex actual samples. The outer surface of SNF has silanol functional groups, which can be further modified with biorecognition elements (such as antibodies, antigen aptamers or enzymes) to construct specific electrochemical or electrochemiluminescent sensing platforms. Summary of the invention

[0007] Aiming at the problems of low detection sensitivity and great detection difficulty of ochratoxin A in food samples, the present invention provides a label-free electrochemical aptamer sensor for detecting ochratoxin A. The sensor is used for ochratoxin A in wheat and wine. The method has the advantages of wide detection range, low lower limit, high accuracy, small sample requirement, no need for complicated sample preparation, and easy use.

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

[0009] A label-free electrochemical aptamer sensor for detecting ochratoxin A, the label-free electrochemical aptamer sensor comprising a three-electrode system of a modified working electrode, a reference electrode and a counter electrode, wherein the electrolyte contains exonuclease I and potassium ferrocyanide;

[0010] The modified working electrode is a VMSF modified electrode obtained by sequentially covalently fixing cDNA and ochratoxin A aptamer.

[0011] The present invention uses potassium ferrocyanide and ochratoxin A aptamer as an electrochemical signal molecule and a specific recognition element, respectively; combines the signal amplification strategy of exonuclease I-assisted target cycle, and constructs a label-free electrochemical aptamer sensor for detecting ochratoxin A in samples such as wheat samples and wine. The aptamer sensor has the characteristics of high sensitivity, wide linear range, simple operation, etc.

[0012] The detection principle is as follows: when ochratoxin A is present in the test, ochratoxin A binds to the ochratoxin A aptamer on the electrode surface to form an ochratoxin A-aptamer complex and falls off from the electrode surface, resulting in a reduction in the steric hindrance effect. At this time, the content of potassium ferrocyanide probe entering the silica nanochannel increases. The increase in the electrochemical signal of potassium ferrocyanide is associated with the amount of ochratoxin A, thereby achieving quantitative analysis of ochratoxin A. In addition, exonuclease I can cut the exposed single-stranded DNA (cDNA) complementary to ochratoxin A on the electrode surface and the aptamer in the ochratoxin A-aptamer complex, releasing ochratoxin A to continue to participate in the reaction on the electrode surface, achieving the effect of signal amplification, and achieving highly sensitive detection of ochratoxin A.

[0013] The preparation process of the modified working electrode comprises the following steps:

[0014] Step 1, drop-coating glutaraldehyde solution on the surface of the micelle NH2-VMSF modified electrode, and incubating in the dark to obtain a surface aldehyde functionalized VMSF modified electrode;

[0015] Step 2, after removing the micelle template from the aldehyde-functionalized VMSF modified electrode, the surface is dripped with a cDNA buffer solution for incubation; a blocking agent solution is then dripped on the electrode to block the remaining aldehyde sites, and then an ochratoxin A aptamer buffer solution is dripped and incubated to obtain the modified working electrode.

[0016] The amino group in the micelle-containing NH2-VMSF modified electrode is aminosilane modified on the surface of the micelle-containing VMSF modified electrode by a one-step modification method or a post-modification method;

[0017] The micelle VMSF modified electrode is prepared by growing a nanometer-sized silica porous membrane on the electrode surface, and the growth method includes an electrochemical assisted method, Any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method;

[0018] The electrode in the micellar NH2-VMSF modified electrode includes any one of an indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode;

[0019] The aminosilane includes 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane or 11-aminoundecyltrimethoxysilane, preferably 3-aminopropyltriethoxysilane.

[0020] The blocking agent includes one or more of ethanolamine, diethanolamine, acetamide, propionamide, mercaptoethanol, and mercaptopropionic acid.

[0021] The solvent of the glutaraldehyde solution in step 1 includes any one or more of ethanol, water, and ether, wherein the volume proportion of glutaraldehyde is 0.02% to 10%, preferably 5%;

[0022] In step 1, the incubation time is 0.1 to 2 hours, and the temperature is 4 to 40°C.

[0023] The molar concentration of the ochratoxin A aptamer in the buffer solution of the ochratoxin A aptamer is 0.02 to 2 μM;

[0024] The molar concentration of cDNA in the cDNA buffer solution is 0.02 to 2 μM;

[0025] The molar concentration of ethanolamine in the blocking agent solution is 0.02 to 2 mM;

[0026] The buffer solution includes any one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution;

[0027] In step 2, each incubation time is independently 0.1 to 2 hours, and the incubation temperature is 0-5°C;

[0028] Preferably, the ochratoxin A aptamer sequence is 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3';

[0029] Preferably, the cDNA is an amino-modified single-stranded DNA chain, and its sequence is 5'-NH2-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3'.

[0030] The molar concentration of exonuclease I in the test solution is 2 to 50 U, more preferably 20 U;

[0031] The molar concentration of potassium ferrocyanide is 0.1-5 mM; more preferably 2-4 mM;

[0032] The pH of the electrolyte is 6-8.

[0033] The present invention also provides a method for constructing a label-free electrochemical aptamer sensor for detecting ochratoxin A, comprising:

[0034] Step 1: drop-coat glutaraldehyde solution on the surface of the micelle NH2-VMSF modified electrode, incubate in dark to obtain a surface aldehyde functionalized VMSF modified electrode;

[0035] Step 2, after removing the micelle template from the aldehyde-functionalized VMSF modified electrode, drip-coat the surface with a cDNA buffer solution and incubate; then drip-coat the remaining aldehyde sites on the electrode with a blocking agent solution, and then drip-coat the ochratoxin A aptamer buffer solution and incubate to obtain the modified working electrode;

[0036] Step 3, using the three-electrode system formed by the modified working electrode, the reference electrode and the counter electrode, and using a solution containing exonuclease I and potassium ferrocyanide as an electrolyte, to construct a label-free electrochemical aptamer sensor for detecting ochratoxin A.

[0037] The present invention also provides the use of the label-free electrochemical aptamer sensor in detecting ochratoxin A, comprising the steps of:

[0038] The exonuclease I and the test solution are dripped on the working electrode and incubated to obtain the test electrode; the test electrode, the reference electrode and the counter electrode form a three-electrode system, and the electrochemical luminescence signal is detected in a potassium ferrocyanide buffer solution.

[0039] The incubation time is 35-38°C for 0.5-2h.

[0040] The solution to be tested is an extract of food and crops such as corn, wheat, oats, coffee, wine and vegetables.

[0041] The liquid to be tested contains any one or more interfering substances selected from aflatoxin B1, amine oxide and zearalenone, and the interfering ion concentration is below 1 ng / mL. Since the aptamer sensor in the present invention is covalently grafted with an aptamer of ochratoxin A, it has specific selection for ochratoxin A, is not easily affected by other interfering substances, has high detection stability, strong specificity, and a wide range of applications.

[0042] Compared with the prior art, the present invention has the following beneficial effects:

[0043] (1) The label-free electrochemical aptamer sensor of the present invention fixes a single-stranded DNA chain complementary to the ochratoxin A aptamer on the surface of a silica nanochannel array film, further combines the ochratoxin A aptamer, and constructs an aptamer sensing interface. The modified electrode is used as a working electrode. Nuclease exonuclease I is added to the test solution to perform cyclic amplification on the target substance ochratoxin A, thereby achieving highly sensitive detection of ochratoxin A.

[0044] (2) The detection method of the present invention is to measure the electrochemical signal after incubating the sample to be tested with the label-free electrochemical aptamer sensor, without the need for separation and purification steps, which is simpler to operate and low in cost. In addition, the detection method has the advantages of a wide linear detection range, high accuracy, high detection sensitivity, and a low detection limit.

[0045] (3) The label-free electrochemical aptamer sensor of the present invention does not require a complicated labeling process for the aptamer and the corresponding complementary DNA single strand. The DNA single strand is covalently fixed on the electrode surface and then further combined with the ochratoxin A aptamer. Compared with the free ochratoxin A aptamer system, the usage of the aptamer and the DNA single strand is reduced, thereby reducing the economic cost.

[0046] (4) The label-free electrochemical aptamer sensor of the present invention introduces exonuclease I, which can cyclically cleave the aptamer bound to ochratoxin A, release the target ochratoxin A, and continue to participate in the reaction on the electrode surface, thereby achieving the effect of signal sensitization.

[0047] (5) The amino-functionalized silica nanochannel array film of the present invention has an ultra-small pore size (2-3 nm), and the surface of the pores is rich in amino groups. It is protonated and positively charged in a neutral solution. On the one hand, it can enrich the electrochemiluminescent probes through electrostatic attraction to achieve signal enhancement and improve detection sensitivity. On the other hand, it can hinder the influence of large-sized negatively charged pollutants (such as pulp, fruit shells and other macromolecules) in complex samples on the base electrode through electrostatic repulsion, hydrophilic effect and size screening effect, and has good anti-fouling and anti-interference capabilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] Figure 1 This is a flow chart of the preparation of the electrochemical aptamer sensor electrode for detecting ochratoxin A in Example 1 and a diagram of the detection mechanism of ochratoxin A.

[0049] Figure 2 TEM image (A) and SEM cross-sectional image (B) of the amino-functionalized silica nanochannel array film in Example 1.

[0050] Figure 3 The bare ITO (ITO), the ITO electrode modified with amino-functionalized silica nanochannel array thin film (NH2-SNF / ITO), and the ITO electrode modified with amino-functionalized silica nanochannel array thin film with micelles retained in the pores (SM@NH2-SNF / ITO) in Example 1 were heated to 0.5 mM Ru(NH3)6 3+ Cyclic voltammetry curves in potassium hydrogen phthalate solution (concentration 50 mM).

[0051] Figure 4These are the differential pulse voltammetry (DPV) curves of NH2-SNF / ITO, GA / NH2-SNF / ITO, cDNA / GA / NH2-SNF / ITO, MEA / cDNA / GA / NH2-SNF / ITO, and MEA / Apt-cDNA / GA / NH2-SNF / ITO in Example 1 before and after incubation with ochratoxin (1 ng / mL OTA) in 0.1 M KCl solution containing 1 mM K3[Fe(CN)6].

[0052] Figure 5 To verify the effect of exonuclease I (Exo I) in Example 1, the CV curves of NH2-SNF / ITO in 0.01 M PBS (pH 7.4) solution containing or not containing 5 μM DNA, 20 U EXO I, and 5 μM DNA and 20 U EXO I at the same time are shown.

[0053] Figure 6 This is the optimization diagram of K3[Fe(CN)6] concentration in Application Example 2. The DPV signal intensity and signal change rate in PBS solution (0.01M, pH 7.4) containing 20UEXO I and different concentrations of K3[Fe(CN)6] after Apt-cDNA / GA / NH2-SNF / ITO, GA / NH2-SNF / ITO, and Apt-cDNA / GA / NH2-SNF / ITO were incubated with 10 pg / mL ochratoxin A (OTA).

[0054] Figure 7 The DPV curves for detecting different concentrations of OTA in Application Example 3. The detection solution is a PBS solution (0.01 M, pH 7.4) containing 20 UEXO I, 1 mM K3[Fe(CN)6] and different concentrations of OTA.

[0055] Figure 8 This is the DPV response of the Apt-cDNA / GA / NH2-SNF / ITO electrode in Application Example 4 after incubation with interfering substances (aflatoxin B1, okadaic acid and zearalenone) and OTA.

[0056] Fig. 9 DPV responses of the Apt-cDNA / GA / NH2-SNF / ITO electrode after incubation with OTA solutions of different concentrations without (left) or with (right) 20U EXO I. DETAILED DESCRIPTION

[0057] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described in detail below in conjunction with embodiment. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention. Those skilled in the art can make modifications or equivalent substitutions based on the understanding of the technical scheme of the present invention without departing from the spirit and scope of the technical scheme of the present invention, and all should be included in the protection scope of the present invention.

[0058] Unless otherwise specified, the raw materials used in the following specific embodiments were purchased from commercial sources and used directly without special treatment. The main raw materials used in the examples are as follows:

[0059] Hexaammineruthenium(III) chloride ([Ru(NH3)6]Cl3, 98.0%): Sigma-Aldrich (Shanghai, China).

[0060] Cetyltrimethylammonium bromide (CTAB, 99%): Sigma-Aldrich (Shanghai, China).

[0061] Hydrochloric acid (HCl, 99.7%): Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0062] Ethanol (EtOH, ≥98.0%): Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China).

[0063] Potassium ferrocyanide (K3[Fe(CN)6], ≥98%): Aladdin Chemical Co., Ltd. (Shanghai, China).

[0064] Tetraethyl orthosilicate (TEOS, ≥99.0%): Aladdin Chemical Co., Ltd. (Shanghai, China).

[0065] 3-Aminopropyltriethoxysilane: Shanghai MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0066] Sodium nitrate (NaNO3, ≥98%): Aladdin Chemical Co., Ltd. (Shanghai, China).

[0067] Glutaraldehyde (GA, ≥99.0%): Aladdin Chemical Co., Ltd. (Shanghai, China).

[0068] Starch soluble matter (≥98%): Aladdin Chemical Co., Ltd. (Shanghai, China).

[0069] Ethanolamine (MEA, 98.0%): McLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0070] Potassium hydrogen phthalate (KHP, 99.0%): MacLean Biochemical Technology Co., Ltd. (Shanghai, China).

[0071] Exonuclease I (Exo I): Sangon Biotech (Shanghai, China)

[0072] Aflatoxin B1 (AFB1): Sangon Biotech (Shanghai, China).

[0073] Zearalenone (ZEN): Sangon Biotech (Shanghai, China).

[0074] Okadaic acid (OA): Sangon Biotech (Shanghai, China).

[0075] OTA aptamer and complementary DNA (HPLC purification, ≥99%): Sangon Biotech (Shanghai, China).

[0076] The OTA aptamer sequence is:

[0077] 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3';

[0078] The cDNA sequence table is:

[0079] 5'-NH2-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3'.

[0080] Example 1

[0081] The single-stranded DNA chain complementary to the ochratoxin A aptamer was fixed on the surface of the silica nanochannel array film, and the ochratoxin A aptamer was further combined to construct an aptamer sensing interface, and the ITO electrode modified by it was used as the working electrode (Apt-cDNA / GA / NH2-SNF / ITO). The preparation process is as follows:

[0082] (1) An amino-functionalized silica nanochannel array film (NH2-SNF / ITO) was prepared on the surface of indium tin oxide (ITO) by electrochemically assisted self-assembly (EASA). 4.35 mM CTAB was added to a mixed solution of 20 mL ethanol and 20 mL 0.1 M NaNO3. After stirring until completely dissolved, a mixed solution of 13.6 mM TEOS and APTES (molar ratio of 9:1) was added. The pH value of the above solution was adjusted to 3.0 with hydrochloric acid, and then stirred at room temperature for 2.5 h before use. Clean ITO (0.5 × 1 cm 2) was immersed in the above precursor solution and electrodeposited for 10s at a constant current of -0.35mA. After the electrodeposition, the electrode was immediately taken out, rinsed thoroughly with ultrapure water, dried under nitrogen, and aged overnight at 120°C. The electrode directly obtained retained CTAB surfactant micelles (SM) inside the silica nanopores, named SM@NH2-SNF / ITO, and after removing the micelles, it was NH2-SNF / ITO.

[0083] (2) Using glutaraldehyde (GA) as a coupling agent, the outer surface of the SM@NH2-SNF / ITO electrode prepared in step (1) was modified with a single-stranded DNA chain (c-DNA) complementary to the OTA aptamer. In order to avoid the clogging problem of the silica nanochannel array film during the GA modification process, the SM was removed after GA modification. The specific operation was to drop 50μL of 5% GA solution on the surface of SM@NH2-SNF / ITO, incubate in the dark for 30 minutes at room temperature, and then rinse with distilled water. After stirring in an ethanol solution containing 0.1M hydrochloric acid for 5 minutes and removing the SM from the silica nanochannel, an aldehyde-functionalized silica nanochannel array film modified electrode was obtained, named GA / NH2-SNF / ITO.

[0084] Then, 50 μL of 0.5 μM cDNA solution was drop-coated and incubated at 4°C in a refrigerator for 60 min, and then rinsed with PBS solution to obtain cDNA / GA / NH2-SNF / ITO. Then, 20 μL of MEA was drop-coated on the electrode and reacted for 0.5 h to block the remaining nonspecific binding sites to obtain MEA / cDNA / GA / NH2-SNF / ITO. Finally, 50 μL of 0.5 μM OTA aptamer solution was drop-coated, incubated at 4°C for 45 min, and rinsed with PBS to obtain MEA / Apt-cDNA / GA / NH2-SNF / ITO.

[0085] Application Example 1

[0086] The process of detecting ochratoxin A in wheat by electrochemical method using MEA / Apt-cDNA / GA / NH2-SNF / ITO in Example 1:

[0087] The wheat sample was crushed, 1.0 g of the powdered sample was accurately weighed and placed in 5 mL of methanol extract (70%), vortexed for 5 minutes, ultrasonicated for 30 minutes, centrifuged at 6000 rpm for 10 minutes, the supernatant was collected, and filtered with a 0.22 μm filter membrane. The wheat extract was diluted 10 times with a PBS (pH 7.4) solution with a concentration of 0.01 mol / L, and a certain concentration of ochratoxin A was added, and the ochratoxin A concentrations in the wheat samples were finally obtained to be 100 fg / mL, 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL and 100 ng / mL.

[0088] Wheat samples containing 20U Exo I and different concentrations of ochratoxin A were dripped onto the MEA / Apt-cDNA / GA / NH2-SNF / ITO electrode, incubated at 37°C for 45 minutes, and then rinsed with 0.01mol / L PBS (pH 7.4) solution to obtain OTA-Exo I / MEA / Apt-cDNA / GA / NH2-SNF / ITO, which was then subjected to differential pulse voltammetry in a PBS solution (0.01mol / L, pH 7.4) containing 1mM K3[Fe(CN)6]. A simple three-electrode system was used, with a platinum electrode and a silver / silver chloride electrode as the counter electrode and reference electrode, respectively.

[0089] Performance observation test

[0090] 1. Electrode preparation and detection principle

[0091] The preparation flow chart of MEA / Apt-cDNA / GA / NH2-SNF / ITO electrode in Example 1 and the electrochemical detection principle of ochratoxin A (OTA) are as follows Figure 1 As shown. First, the EASA method was used to prepare an amino-functionalized silica nanochannel array film on the surface of the ITO electrode in one step, named NH2-SNF / ITO. Using GA as a cross-linker and MEA as a blocking agent, cDNA was covalently fixed on the outer surface of NH2-SNF / ITO to obtain MEA / cDNA / GA / NH2-SNF / ITO. Then, the OTA aptamer (Apt) was further combined to form an Apt-DNA double-stranded structure on the outer surface of the silica nanochannel array film to obtain the MEA / Apt-cDNA / GA / NH2-SNF / ITO aptamer sensor.

[0092] The size of the Apt-DNA double-stranded complex creates a steric hindrance that hinders the electrochemical probe Fe(CN)6 3-The aptamer enters the silica nanochannel and reaches the electrode surface, obtaining a lower electrochemical signal. When OTA is present, OTA can compete with cDNA, form an OTA-aptamer complex with the OTA aptamer, and fall off from the electrode surface, significantly reducing the steric hindrance effect. 3- At the same time, ExoⅠ can specifically cut the DNA single strand from 3' to 5', that is, the cDNA exposed on the electrode surface and the single-stranded aptamer on the OTA-aptamer complex, releasing the target OTA, which continues to participate in the reaction on the electrode surface, achieving the effect of signal cyclic amplification.

[0093] 2. Morphology observation

[0094] The morphological characterization results of the amino-functionalized silica nanochannel array film modified ITO electrode SM@NH2-SNF / ITO obtained in step (1) of Example 1 are as follows: Figure 2 shown. Figure 2 A is the TEM top view and cross-sectional view of NH2-SNF. The top view shows that the surface of the peeled NH2-SNF film is flat, without obvious cracks and defects, and the pore size of the nanochannel is 2-3nm. The TEM cross-sectional view shows that the nanochannels of NH2-SNF are long-range ordered and oriented perpendicular to the substrate, with a thickness of about 90nm. Figure 2 B is the SEM cross-sectional image of NH2-SNF, showing the obvious layered structure of NH2-SNF / ITO, which is NH2-SNF, ITO and glass from top to bottom. The thickness of NH2-SNF film is about 90nm, which is consistent with the results of TEM cross-sectional image.

[0095] 3. Electrochemical characterization of electrodes

[0096] Figure 3 The cyclic voltammetry curves of bare ITO electrode (ITO), ITO electrode modified with amino-functionalized silica nanochannel array film with micelles retained in the pores (SM@NH2-SNF / ITO), and ITO electrode modified with amino-functionalized silica nanochannel array film (NH2-SNF / ITO) were tested in Ru(NH3)6Cl3 solution (0.2mM). The electrolyte solution was potassium hydrogen phthalate (0.05mol / L).

[0097] It can be found that on the SM@NH2-SNF / ITO electrode, the positively charged electrochemical probe Ru(NH3)6 3+ No Faraday current was generated, only charging current was presented, indicating that the surfactant micelles inside the pores prevented the probe from entering the pores and reaching the electrode surface, and also proved that the film grown on the electrode surface was intact. NH2-SNF / ITO electrode has open nanochannels, Ru(NH3)63+ It can enter the nanochannel and reach the electrode surface to generate electrochemical signals. In addition, due to the protonation of the amino groups of NH2-SNF / ITO, the channel surface is positively charged and can enrich the positively charged Ru(NH3)6 3+ , so compared with the bare ITO electrode, Ru(NH3)6 3+ The electrochemical signal on the NH2-SNF / ITO electrode increased. The above results prove that the NH2-SNF / ITO electrode has charge selectivity and can enrich negatively charged species.

[0098] 4. Construction and characterization of label-free electrochemical aptamer sensors

[0099] Figure 4 The verified pulse voltammetry curves of NH2-SNF / ITO, GA / NH2-SNF / ITO, cDNA / GA / NH2-SNF / ITO, MEA / cDNA / GA / NH2-SNF / ITO, MEA / Apt-cDNA / GA / NH2-SNF / ITO and 1ng / mL OTA-Exo I / MEA / Apt-cDNA / GA / NH2-SNF / ITO (ochratoxin A in wheat sample is 1ng / mL) in K3[Fe(CN)6] are shown.

[0100] Since NH2-SNF is negatively charged probe Fe(CN)6 3- The electrode showed a higher oxidation peak current after sequential modification of GA, cDNA and MEA. 3- The mass transfer was hindered, and the oxidation peak current signal gradually decreased. The obtained MEA / cDNA / GA / NH2-SNF / ITO was further combined with the OTA aptamer to form a rigid double-stranded structure on the outer surface of the silica nanochannel array film. The resulting steric hindrance effect and self-electronegativity hindered the Fe(CN)6 3- After entering the nanochannel, the oxidation peak current further decreased. When the target OTA was present, due to the strong affinity effects of electrostatics, hydrogen bonds and spatial matching effects, OTA and OTA aptamers were more inclined to form OTA-aptamer complexes rather than aptamer-cDNA duplexes.

[0101] Therefore, the OTA aptamer on the double-stranded structure falls off, and the steric hindrance effect on the electrode surface is reduced, resulting in the Fe(CN)6 3- The oxidation peak current increases. At the same time, ExoⅠ can digest the decomposed cDNA and aptamer single strand from the 3' end, causing OTA to be released from the OTA-aptamer complex and continue to participate in the reaction on the electrode surface, achieving the effect of signal amplification. The above data prove the successful construction of the label-free electrochemical aptamer sensor for the detection of ochratoxin A.

[0102] 5. Exonuclease cleavage cycle amplification

[0103] Exonuclease I (ExoⅠ) is a biological enzyme that is highly selective for denatured or single-stranded deoxyribonucleotides. ExoⅠ can attack from the 3'hydroxyl end of single-stranded deoxyribonucleotides, and then gradually release deoxyribonucleoside 5'-monophosphate, but the terminal dinucleotide will remain intact and has no activity against double-stranded DNA or RNA.

[0104] In order to verify the effect of ExoⅠ-induced target cyclic amplification, the cyclic voltammetry curves of NH2-SNF / ITO in 0.01M PBS (pH=7.4) without and with 5μM cDNA, 20U Exo I, and 5μM cDNA and 20U Exo I were compared by electrochemical method. Figure 5 As shown in the figure, it can be found that in the PBS solution containing only ExoⅠ and single-stranded DNA, no obvious oxidation peak is observed for the NH2-SNF / ITO electrode, and only charging current is presented. When ExoⅠ and single-stranded DNA are present at the same time and react for a period of time, an oxidation peak can be observed, indicating that in the presence of ExoⅠ, the oligonucleotides of single-stranded DNA are sheared into small fragments and reach the electrode surface through the pores, and an oxidation reaction occurs, which proves that ExoⅠ is effective in shearing single-stranded DNA.

[0105] Application Example 2: Optimization of detection conditions

[0106] In order to give full play to the advantages of electrochemical sensing and obtain the best electrochemical signal, Fe(CN)6 3- The concentration parameters of the signal probe were optimized. Firstly, differential pulse voltammetry was used to detect the electrochemical properties of the three electrodes at different concentrations of Fe(CN)6 3- The signal intensity in the signal probe was calculated, and the signal change rate of the sensor in the probe with different concentrations after adding the same concentration of OTA was calculated. The results are shown in Figure 6 As shown. a ,I b and I c They are MEA / Apt-cDNA / GA / NH2-SNF / ITO, GA / NH2-SNF / ITO and OTA-Exo I / MEA / Apt-cDNA / GA / NH2-SNF / ITO. Figure c shows the incubation of Apt-cDNA / GA / NH2-SNF / ITO (same as a) with 10 pg / mL OTA and 20 U EXO I at different concentrations of Fe(CN)6 3- The DPV signal intensity measured in the quenching rate is calculated as follows:

[0107]

[0108] As Fe(CN)6 3- With the increase of concentration, the DPV signal increases significantly. 3- Provides higher absolute signal values, but the quenching rate decreases with Fe(CN)6 3- As the increase of Fe(CN)6 3- The optimal concentration is 1 mM.

[0109] Application Example 3: Electrochemical Detection of Ochratoxin A

[0110] Figure 7 The differential pulse voltammetry curves of MEA / Apt-cDNA / GA / NH2-SNF / ITO electrode for detecting different concentrations of ochratoxin A in wheat samples under the optimal experimental conditions. The concentrations of ochratoxin A were 100fg / mL, 1pg / mL, 10pg / mL, 100pg / mL, 1ng / mL, 10ng / mL and 50ng / mL. The detection solution contained 0.1mol / L K3[Fe(CN)6] and KCl. As the concentration of ochratoxin A increased, the DPV signal increased; with the current intensity as the ordinate and the concentration of ochratoxin A (C OTA ) logarithm (logC OTA ) as the horizontal axis, such as Figure 7 (B) The DPV signal intensity and the logarithmic concentration of ochratoxin A showed a good linear relationship in the range of 100 fg / mL to 50 ng / mL, and the detection limit was 77 fg / mL, indicating that it had good sensitivity.

[0111] Application Example 4: Selectivity of Label-free Electrochemical Aptamer Sensors

[0112] Figure 8 The selectivity and anti-interference data of the constructed label-free electrochemical aptamer sensor. Aflatoxin B1, okadaic acid and zearalenone are potential co-existing interfering substances. The constructed Apt-cDNA / GA / NH2-SNF / ITO electrode was used to detect the above interfering substances (concentration of 1.0 ng / mL) and the coexistence of the above substances (concentration of 1.0 ng / mL) and ochratoxin A (concentration of 0.1 ng / mL), and the obtained electrochemical signals were compared with the signals for detecting ochratoxin A.

[0113] The results showed that higher concentrations of interfering substances had little effect on the detection of ochratoxin A, and only ochratoxin A could produce an obvious electrochemical response. The above data show that the proposed sensor has good selectivity and anti-interference properties, and can easily distinguish ochratoxin A from other toxins.

[0114] Fig. 9 The DPV detection signals of Apt-cDNA / GA / NH2-SNF / ITO to different concentrations of OTA were compared without (left) and with (right) 20U EXO I. The results showed that in both cases, the DPV current response increased with the increase of OTA concentration, but at the same OTA concentration, the DPV signal increased significantly when 20U EXO I was present in the detection solution. This satisfactory sensitivity improvement is attributed to the fact that ExoⅠ can specifically shear the detached aptamer and cDNA on the electrode surface, release OTA, and continue to participate in the reaction on the electrode surface to achieve signal amplification.

[0115] The above analysis results demonstrate that the combination of the anti-pollution effect of the silica nanochannel array film and the electrostatic enrichment effect on the electrochemical probe, the specificity of the ochratoxin A aptamer, and the signal amplification effect of nuclease I can achieve highly sensitive and selective detection of ochratoxin A in wheat samples.

Claims

1. A label-free electrochemical aptamer sensor for detecting ochratoxin A, characterized in that: The label-free electrochemical aptamer sensor comprises a three-electrode system of a modified working electrode, a reference electrode and a counter electrode, and the electrolyte contains exonuclease I and potassium ferrocyanide; The modified working electrode is a VMSF modified electrode obtained by sequentially covalently fixing cDNA and ochratoxin A aptamer.

2. The label-free electrochemical aptasensor for detecting ochratoxin A according to claim 1, characterized in that: The preparation process of the modified working electrode comprises the following steps: Step 1, drop-coating glutaraldehyde solution on the surface of the micelle NH2-VMSF modified electrode, and incubating in the dark to obtain a surface aldehyde functionalized VMSF modified electrode; Step 2, after removing the micelle template from the aldehyde-functionalized VMSF-modified electrode, a cDNA buffer solution is drop-coated on the surface for incubation; The blocking agent solution is then dropped onto the electrode to block the remaining aldehyde sites, and then the buffer solution of the ochratoxin A aptamer is dropped and incubated to obtain the modified working electrode.

3. The label-free electrochemical aptasensor for detecting ochratoxin A according to claim 2, characterized in that: The amino group in the micelle-containing NH2-VMSF modified electrode is aminosilane modified on the surface of the micelle-containing VMSF modified electrode by a one-step modification method or a post-modification method; The micelle VMSF modified electrode is prepared by growing a nanometer-sized silica porous membrane on the electrode surface, and the growth method includes an electrochemical assisted method, Any one of solution growth method, two-phase layered growth method, evaporation-induced self-assembly method, π-π interaction induced method, epitaxial growth method, strong magnetic field method, electric field method, and organic solvent-induced self-assembly method; The electrode in the micellar NH2-VMSF modified electrode includes any one of an indium tin oxide electrode, a glassy carbon electrode, a fluorine-doped tin oxide electrode, a gold electrode, a screen-printed electrode, a graphite electrode, and a carbon fiber electrode; The aminosilane includes 3-aminopropyltriethoxysilane, 3-aminopropyltrimethoxysilane or 11-aminoundecyltrimethoxysilane; The blocking agent includes one or more of ethanolamine, diethanolamine, acetamide, propionamide, mercaptoethanol, and mercaptopropionic acid.

4. The label-free electrochemical aptasensor for detecting ochratoxin A according to claim 2, characterized in that: The solvent of the glutaraldehyde solution in step 1 includes any one or more of ethanol, water, and ether, wherein the volume proportion of glutaraldehyde is 0.02% to 10%; And / or, in step 1, the incubation time is 0.1 to 2 hours and the temperature is 4-40°C.

5. The label-free electrochemical aptasensor for detecting ochratoxin A according to claim 2, characterized in that: The molar concentration of the ochratoxin A aptamer in the buffer solution of the ochratoxin A aptamer is 0.02 to 2 μM; The molar concentration of cDNA in the cDNA buffer solution is 0.02 to 2 μM; The molar concentration of the blocking agent in the blocking agent solution is 0.02 to 2 mM; And / or, the buffer solution comprises any one or more of a sodium chloride aqueous solution, a sodium sulfate aqueous solution, a potassium chloride aqueous solution or a phosphate buffer solution; And / or, in step 2, each incubation time is independently 0.1 to 2 hours, and the incubation temperature is 0-5°C.

6. The label-free electrochemical aptasensor for detecting ochratoxin A according to claim 2, characterized in that: The ochratoxin A aptamer sequence is 5'-GATCGGGTGTGGGTGGCGTAAAGGGAGCATCGGACA-3'; The cDNA is an amino-modified single-stranded DNA chain, and its sequence is 5'-NH2-TGTCCGATGCTCCCTTTACGCCACCCACACCCGATC-3'.

7. The label-free electrochemical aptasensor for detecting ochratoxin A according to claim 1, characterized in that: The molar concentration of exonuclease I in the test solution is 2 to 50 U; the molar concentration of potassium ferrocyanide is 0.1 to 5 mM; And / or, the pH value of the electrolyte is 6-8.

8. The method for constructing a label-free electrochemical aptasensor for detecting ochratoxin A according to any one of claims 1 to 7, characterized in that: include: Step 1, drop-coating glutaraldehyde solution on the surface of the micelle NH2-VMSF modified electrode, and incubating in the dark to obtain a surface aldehyde functionalized VMSF modified electrode; Step 2, after removing the micelle template from the aldehyde-functionalized VMSF-modified electrode, a cDNA buffer solution is drop-coated on the surface for incubation; Then, a blocking agent solution is dripped onto the electrode to block the remaining aldehyde sites, and then a buffer solution of ochratoxin A aptamer is dripped and incubated to obtain the modified working electrode; Step 3, using the three-electrode system formed by the modified working electrode, the reference electrode and the counter electrode, and using a solution containing exonuclease I and potassium ferrocyanide as an electrolyte, to construct a label-free electrochemical aptamer sensor for detecting ochratoxin A.

9. Use of the label-free electrochemical aptasensor according to any one of claims 1 to 7 in detecting ochratoxin A, characterized in that: Includes steps: The exonuclease I and the test solution are dripped on the working electrode and incubated to obtain the test electrode; the test electrode, the reference electrode and the counter electrode form a three-electrode system, and the electrochemical luminescence signal is detected in a potassium ferrocyanide buffer solution.

10. Use of the label-free electrochemical aptasensor according to claim 9 in detecting ochratoxin A, characterized in that: The incubation time is 35-38°C for 0.5-2h.

Images