Electrochemical aptamer sensor and preparation method and application thereof

Abstract

The application discloses an electrochemical aptamer sensor and a preparation method and application thereof, and relates to the technical field of sensors.The electrochemical sensor comprises a nucleic acid aptamer and an electrode; the electrode surface contains polydopamine and a zwitterionic polymer; the nucleic acid aptamer is modified by a mercapto group; and the nucleic acid aptamer is arranged on the electrode surface.The electrochemical sensor provided by the application introduces polydopamine and a zwitterionic polymer on the electrode surface, has strong anti-pollution performance, high sensitivity and good stability, and the stability of the electrochemical sensor is further improved by modifying the nucleic acid aptamer by a mercapto group, and the electrochemical sensor has good selectivity for a target detection object.The electrochemical sensor can solve the problem that existing electrochemical sensors are easily polluted in food detection, and is suitable for food safety detection, in particular, AFB1 detection.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of sensors, in particular to an electrochemical aptamer sensor and a preparation method and application thereof. BACKGROUND

[0002] Aflatoxins are usually derived from Aspergillus flavus and Aspergillus parasiticus, and grains with high starch content and their products are prone to be invaded by mold, resulting in excessive aflatoxin residues, which seriously affects food safety. More than 20 types of aflatoxins have been identified, such as B1, B2, M1, M2, G1 and G2. Among them, aflatoxin B1 (AFB1) shows the strongest toxicity and carcinogenicity, and has been classified as a class I carcinogen by the International Cancer Research Agency. Therefore, it is of great significance to rapidly and accurately detect AFB1 in food.

[0003] Among numerous detection methods, electrochemical aptamer-based sensor technology has attracted widespread attention due to its short detection time, high sensitivity and easy miniaturization, and has potential application value in on-site food safety detection. In order to improve the detection performance of the sensor, the modification strategy of the electrode surface is usually studied, so as to improve the sensitivity and accuracy of the detection.

[0004] However, for the existing electrochemical aptamer sensor, biofouling is one of the key factors affecting its application. Biofouling refers to the process of non-specific adhesion that causes negative effects, that is, the reversible or irreversible adhesion of macromolecules such as proteins and microorganisms to the surface of the material substrate. This layer of adsorbed proteins can serve as a platform for microorganisms to attach and form a biofilm, ultimately leading to sensor failure. The matrix in food is complex, and the sensor surface will inevitably be contaminated during the detection process, affecting the specificity, stability and repeatability of the sensor. Therefore, it is crucial to endow the electrochemical aptamer sensor surface with anti-fouling properties to improve the performance of the sensor. SUMMARY

[0005] The present application aims to at least solve one of the above-mentioned technical problems in the prior art. To this end, the purpose of the present application is to provide an electrochemical aptamer sensor with strong anti-fouling property and high sensitivity, selectivity and stability.

[0006] The second aspect of the present application is to provide a preparation method of an electrochemical aptamer sensor.

[0007] The third aspect of the present application is to provide an application of an electrochemical aptamer sensor.

[0008] In order to achieve the above-mentioned purposes, the technical solutions adopted by the present application are as follows:

[0009] The first aspect of the present application provides an electrochemical aptamer sensor, comprising a nucleic acid aptamer and an electrode; the surface of the electrode contains polydopamine and a zwitterionic polymer; the nucleic acid aptamer is modified by a thiol group; and the nucleic acid aptamer is modified on the surface of the electrode.

[0010] It can be understood that, since the surface of the electrode contains polydopamine and a zwitterionic polymer, the modification of the nucleic acid aptamer on the surface of the electrode means that the nucleic acid aptamer is fixed on the polydopamine and / or the zwitterionic polymer on the surface of the electrode.

[0011] The electrochemical aptamer sensor refers to a sensor based on electrochemical nucleic acid aptamer, and the nucleic acid aptamer is fixed on the surface of the electrode, also known as an electrochemical nucleic acid aptamer sensor. The zwitterionic polymer on the surface of the electrochemical aptamer sensor of the present application contains a zwitterionic group, and the solvation and hydrogen bonding of the charged functional groups can form a hydration layer on the surface of the zwitterionic polymer. The surface of the hydration layer formed based on electrostatic interaction can effectively resist non-specific adsorption, has high anti-pollution performance, and has high repeatability and stability during long-term storage. Dopamine has a catechol structure and can adhere to the surface of various objects. After the modification of dopamine on the surface of the electrode, the nucleic acid aptamer can be firmly fixed on the surface of the electrode, for example, by Michael addition. In addition, polydopamine can bind water molecules through hydrogen bonds to slightly improve the anti-pollution performance of the sensor.

[0012] The thiol group is used to modify the aptamer, and the anchoring effect of the thiol group is conducive to the fixation of the modified functional probe, thereby ensuring the specificity of the electrochemical aptamer sensor.

[0013] The electrochemical aptamer sensor of the present application itself has high sensitivity, and there is no need to perform electrical signal enhancement.

[0014] It should be noted that the nucleic acid aptamer is modified on the surface of the electrode containing polydopamine and a zwitterionic polymer, i.e., on the polydopamine and the zwitterionic polymer.

[0015] In some embodiments of the present application, the electrode comprises a glassy carbon electrode.

[0016] In some embodiments of the present application, the nucleic acid aptamer comprises an AFB1 aptamer; the nucleotide sequence of the AFB1 aptamer is as shown in SEQ ID NO: 1; and the sequence of the AFB1 aptamer after thiol modification is 5'-SH-(CH2)6-GCA TCA CTA CAG TCA TTA CGC ATC GGG TAA GCG GAA CTC GGG AGT GGG AGG TAA ATC GTG TGA AGT GCT GTC CC-3'.

[0017] The AFB1 aptamer is further selected, so that the selectivity of the electrochemical aptamer sensor for AFB1 is improved, and the electrochemical aptamer sensor is used for rapid detection of AFB1 in food.

[0018] In some embodiments of the present application, the nucleic acid aptamer is immobilized on the polydopamine through Michael addition.

[0019] Michael addition has the characteristics of relatively mild reaction conditions, simple operation, high yield, and regioselective specificity, and is more convenient than other addition methods. For example, after the activated carboxyl group is used to crosslink the aptamer containing an amino group, an activating agent 1-ethyl-(3-dimethylaminopropyl) carbonyl dihydrazine (EDC) and N-hydroxysuccinimide (NHS) need to be added, and the pH value needs to be adjusted twice to successfully connect the amino group and the carboxyl group. The experimental operation process is relatively complicated.

[0020] In some embodiments of the present application, the zwitterionic polymer comprises a copolymer of CBMA (carboxybetaine methyl methacrylate) and AEMA (N-(2-aminoethyl) methacrylamide hydrochloride); and the molar ratio of the CBMA to the AEMA is 1:(0.05-0.3).

[0021] In some embodiments of the present application, the molar ratio of the CBMA to the AEMA is 1:(0.09-0.25).

[0022] In some specific embodiments of the present application, the molar ratio of the CBMA to the AEMA is 1:(0.1-0.25).

[0023] In some examples of the present application, the molar ratio of the CBMA to the AEMA is 1:(0.1-0.12).

[0024] In the electrochemical aptamer sensor of the present application, by adjusting the molar ratio of the zwitterionic polymer monomer, the anti-pollution property, the sensitivity, the selectivity, the stability and other properties of the electrochemical aptamer sensor can be further adjusted.

[0025] In some embodiments of the present application, the polydopamine and the zwitterionic polymer are obtained by in-situ polymerization of dopamine and zwitterionic polymer, and the mass ratio of the dopamine to the zwitterionic polymer is (1.5-3.5):5, preferably (1.8-3.2):5, further preferably (2-3):5, and more preferably (2-2.5):5.

[0026] It should be understood that the dopamine and the zwitterionic polymer are polymerized in-situ to form the polydopamine and the zwitterionic polymer, and a copolymer of part of the dopamine and the zwitterionic polymer.

[0027] The second aspect of the present application is a method for preparing the electrochemical aptamer sensor of the first aspect of the present application, comprising the following steps:

[0028] mixing and dissolving dopamine and zwitterionic polymer, applying to the surface of the electrode, in-situ polymerization; modifying nucleic acid aptamer on the surface of the electrode to obtain the electrochemical aptamer sensor.

[0029] It should be understood that after in-situ polymerization, the dopamine forms polydopamine. The nucleic acid aptamer is modified on the surface of the electrode containing polydopamine and zwitterionic polymer.

[0030] In some embodiments of the present application, the mass ratio of the dopamine to the zwitterionic polymer is (1.5-3.5):5.

[0031] In some embodiments of the present application, the mass ratio of the dopamine to the zwitterionic polymer is (1.8-3.2):5.

[0032] In some specific embodiments of the present application, the mass ratio of the dopamine to the zwitterionic polymer is (2-3):5.

[0033] In some examples of the present application, the mass ratio of the dopamine to the zwitterionic polymer is (2-2.5):5.

[0034] By adjusting the mass ratio of dopamine to zwitterionic polymer, the performance of the electrochemical sensor can also be improved.

[0035] In some embodiments of the present application, the nucleic acid aptamer can be obtained by conventional commercial means, or prepared by a preparation method comprising the following steps:

[0036] mixing the thiol-modified nucleic acid aptamer with TCEP, and performing a reduction reaction at room temperature to reduce the thiol with disulfide bond.

[0037] wherein TCEP is tris(2-carboxyethyl)phosphine.

[0038] In some embodiments of the present application, the time of the reduction reaction is 25-35 min.

[0039] In some embodiments of the present application, the preparation method of the zwitterionic polymer comprises the following steps:

[0040] dissolving the zwitterionic monomer compound, adding an initiator, mixing in an organic solvent, and performing a polymerization reaction under anaerobic conditions to obtain the zwitterionic polymer.

[0041] In some embodiments of the present application, the initiator comprises 4,4'-azobis(4-cyanopentanoic acid) (ACVA).

[0042] In some embodiments of the present application, the organic solvent comprises dimethyl sulfoxide (DMSO).

[0043] In some embodiments of the present application, the temperature of the polymerization reaction is 65-75℃.

[0044] In some embodiments of the present application, the time of the polymerization reaction is 23-25h.

[0045] In some embodiments of the present application, the polymerization reaction further comprises a dialysis and a lyophilization step after the polymerization reaction; the dialysis is an ultra-pure water dialysis to remove unreacted monomers or oligomers.

[0046] In some embodiments of the present application, the zwitterionic polymer and dopamine are dissolved in a Tris-HCl buffer solution; the pH of the buffer solution is 8-9, and the concentration is 9-11mmol / L.

[0047] In some embodiments of the present application, after the dissolution, the concentration of the zwitterionic polymer is 4-6mg / L; and the concentration of the dopamine is 0.5-1.5mg / L.

[0048] In some embodiments of the present application, the time of the in-situ polymerization is 3-5h.

[0049] In some embodiments of the present application, the step of disposing the aptamer on the surface of the electrode comprises: placing the electrode in a solution of the aptamer, and obtaining the electrochemical sensor after refrigeration; the temperature of the refrigeration is 3-5℃.

[0050] In some embodiments of the present application, the refrigeration is performed by placing in a refrigerator; and the time of the refrigeration is 7-9h.

[0051] In some embodiments of the present application, the concentration of the solution of the aptamer is 1-5μmol / L.

[0052] In some specific embodiments of the present application, the concentration of the solution of the aptamer is 2-4μmol / L.

[0053] The third aspect of the present application provides an application of the electrochemical aptamer sensor of the first aspect of the present application in food safety detection.

[0054] The electrochemical aptamer sensor has high anti-pollution, high sensitivity, good selectivity, good stability and high repeatability, and can be used for detecting toxins in food, especially AFB1 (aflatoxin B1), and is suitable for food safety detection.

[0055] In some embodiments of the present application, the food sample includes whole wheat flour, pure milk powder or soy milk during the detection.

[0056] In some embodiments of the present application, the electrochemical sensor is used for detecting AFB1.

[0057] In some embodiments of the present application, the detection limit of AFB1 is 0.15-0.25 pg / mL during the detection.

[0058] In some embodiments of the present application, the process of detecting AFB1 includes the following steps:

[0059] The food sample is diluted, AFB1 standard solution is added, and the electrochemical aptamer sensor of the first aspect of the present application is used for detection; the concentration of the detected AFB1 can be obtained by bringing the electrical signals before and after the detection into the standard curve constructed.

[0060] In some embodiments of the present application, the standard addition concentration of the AFB1 standard solution is 0.1-1 ng / mL during the detection. For example, it can be 0.1 ng / mL, 0.5 ng / mL or 1 ng / mL.

[0061] Compared with the prior art, the present application has the following beneficial effects:

[0062] The electrochemical aptamer sensor provided by the present application introduces polydopamine and zwitterionic polymer on the surface of the electrode, has high anti-pollution, high sensitivity and good stability, and further improves the stability of the electrochemical aptamer sensor by modifying the nucleic acid aptamer with thiol, and has good selectivity for the target detection object. The electrochemical sensor can solve the problem that the existing electrochemical aptamer sensor is easily polluted in food detection, and is suitable for food safety detection, especially AFB1 detection. BRIEF DESCRIPTION OF DRAWINGS

[0063] Figure 1 MS spectrum of AEMA used for examples and comparative examples.

[0064] Figure 2 MS spectrum of CBMA used for examples and comparative examples.

[0065] Figure 3 MS spectrum of pDA-pCBAE0 of comparative example 2.

[0066] Figure 4 MS spectrum of pDA-pCBAE1 of Example 1.

[0067] Figure 5 MS spectrum of pDA-pCBAE2 of Example 2.

[0068] Figure 6 Signal inhibition rate of the electrochemical anti-fouling sensor in Example 1 in different concentrations of single protein solution.

[0069] Figure 7 Signal inhibition rate of the electrochemical anti-fouling sensor in Example 1 in different concentrations of different molecular weight sugar solution.

[0070] Figure 8 Signal inhibition rate of the electrochemical anti-fouling sensor in Example 1 in different dilutions of different food samples.

[0071] Figure 9 Effect of different AFB1 aptamer concentrations and different concentrations of different protein solutions on the electrical signal of the electrochemical anti-fouling sensor in Example 2; wherein (a) is the electrochemical signal change graph under different concentrations of AFB1 aptamer; (b)-(d) are the electrochemical signal change graphs of the electrochemical anti-fouling sensor pDA-pCBAE1 / GCE of Example 1 in different concentrations of BSA, LYS and Hb, respectively.

[0072] Figure 10 Scanning electron microscope images of the glassy carbon electrode, the electrochemical sensor of Example 1 and the electrochemical sensor of Comparative Example 1; wherein (a 0～2 )-(c 0～2 ) are the glassy carbon electrode, the electrochemical sensor of Comparative Example 1 and the electrochemical sensor of Example 1, respectively.

[0073] Figure 11 AFB1 concentration and electrochemical aptamer sensor test result change graph and standard curve in Example 2; wherein (a) is the DPV response signal of the electrochemical aptamer sensor combined with different concentrations of AFB1; (b) is the signal change rate of the electrochemical aptamer sensor after incubation with different concentrations of AFB1, (c) is the standard curve between AFB1 concentration and signal change rate, and (d) is the standard curve between AFB1 concentration and absorbance (enzyme-linked immunoassay).

[0074] Figure 12 Selectivity, stability and repeatability test result graphs of the electrochemical aptamer sensor in Example 2; wherein (a)-(d) are the selectivity, storage stability, same-root electrode stability and same-batch electrode stability, respectively.

[0075] Figure 13The test results of the anti-pollution performance test of the electrochemical aptamer sensor in Example 3 are shown in the figure.

[0076] Figure 14 The schematic diagram of the preparation process of the electrochemical aptamer sensor in the example is shown in the figure. DETAILED DESCRIPTION

[0077] The content of the present application is further described in detail below through specific examples. The raw materials, reagents or devices used in the examples and comparative examples are commercially available unless otherwise specified, or can be obtained by existing technical methods. Unless otherwise specified, the test or test method is a conventional method in the art.

[0078] Some of the raw materials used in the following examples and comparative examples of the present application are described as follows:

[0079] Bovine serum albumin (BSA) and hemoglobin (Hb) were purchased from Beijing Solabio Technology Co., Ltd.;

[0080] Lysozyme (Lys) was purchased from Dr. Deki Biological Technology Co., Ltd.

[0081] Potassium ferrocyanide trihydrate (K4Fe(CN)6·3H2O), potassium ferricyanide (K3Fe(CN)6), potassium chloride (KCl), sodium dihydrogen phosphate dihydrate (NaH2PO4·2H2O), disodium hydrogen phosphate dodecahydrate (Na2HPO4·12H2O), and sodium chloride (NaCl) were purchased from Sinopharm Group;

[0082] The AFB1 aptamer was purchased from Shanghai Shengong Biological Technology Co., Ltd. and was modified with thiol. The sequence was 5'-SH-(CH2)6-GCATCACTACAG TCATTACGC ATC GGG TAAGCG GAACTG AGG AGT GGG AGG TAAATC GTG TGA AGT GCTGTC CC-3';

[0083] D-(+)-glucose (Glu), α-lactose (Lac), and starch (Sta) were purchased from Shanghai Lanji Technology Co., Ltd.

[0084] 3-[[2-(methacryloyloxy)ethyl]dimethylammonium] propionate (CBMA), N-methyl acryloyl ethylenediamine hydrochloride (AEMA), dimethyl sulfoxide (DMSO), and 4,4'-azobis(4-cyanovaleric acid) (ACVA) were purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.

[0085] N,N-dimethylformamide (DMF) was purchased from Shanghai Amiclin Biochemical Technology Co., Ltd.

[0086] Ultrapure water (18.2 MΩcm-1 ) were made in the lab.

[0087] The electrode system used in the following examples and comparative examples of the present application is based on the traditional three-electrode system: glassy carbon electrode as working electrode, platinum electrode as counter electrode, and saturated calomel electrode as reference electrode.

[0088] The electrochemical experiments of the following examples and comparative examples of the present application were all carried out on CHI440c Shanghai Chenhua electrochemical workstation.

[0089] All electrochemical tests were carried out in the electrochemical probe solution. The electrochemical probe solution was PBS containing 5 mmol / L K3[Fe(CN)6] / K4[Fe(CN)6] and 0.2 mol / L KCl.

[0090] The preparation method of PBS is as follows: 35.8 g of sodium hydrogen phosphate (Na2HPO4·12H2O) and 15.6 g of sodium dihydrogen phosphate (NaH2PO4·2H2O) were accurately weighed and placed in a 500 mL volumetric flask, and then ultrapure water was added to the mark to obtain 0.2 mol / L Na2HPO4 solution and 0.2 mol / L NaH2PO4 solution; 324 mL of 0.2 mol / L Na2HPO4 solution and 76 mL of 0.2 mol / L NaH2PO4 solution were measured by a measuring cylinder, respectively, and then 3.6 g of sodium chloride (NaCl) solid was added, and after mixing, a 0.2 mol / L PBS solution with pH 7.4 was obtained, which was placed at room temperature, and then diluted according to the required concentration for subsequent use.

[0091] The preparation method of the electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]) is as follows: 0.8231 g of potassium ferricyanide (K3[Fe(CN)6]) solid, 1.0560 g of potassium ferrocyanide (K4[Fe(CN)6]) and 7.4550 g of potassium chloride (KCl) were accurately weighed, and then the above medicines were dissolved in 0.01 mol / L PBS solution with pH 7.4, and finally the volume was made up to 500 mL volumetric flask to obtain a bright yellow solution, i.e. 5 mmol / L electrochemical probe (K3[Fe(CN)6] / K4[Fe(CN)6] containing 0.2 mol / L KCl) solution, which was wrapped with tin foil paper and stored in a 4°C refrigerator for standby use.

[0092] The preparation method of the 10.0 mmol / L Tris-HCl buffer solution with pH 8.5 is as follows: 0.6057 g of Tris is weighed and placed in a 50 mL volumetric flask, and ultrapure water is used for constant volume to obtain a 0.1 mol / L Tris solution; the obtained Tris solution is mixed with 14.7 mL of a previously prepared HCl solution (0.1 mol / L), and the pH is adjusted to 8.5, and finally ultrapure water is used for constant volume to 100 mL, and ultrapure water is used for constant volume to 500 mL to obtain a 10.0 mmol / L Tris-HCl buffer solution with pH 8.5, which is stored at room temperature for standby use.

[0093] In the following examples and comparative examples of the present application, the current response values of the modified electrode before and after incubation are recorded by DPV (the electrical signal before incubation is I0, and the electrical signal after incubation is I), and the electrical signal change value (ΔI = I0-I) and the electrical signal suppression rate (Signal Suppression (%) = [(I0-I) / I0] x 100) are calculated by these values.

[0094] The preparation process of the electrochemical aptamer sensor in the embodiment of the present application is shown in Figure 14 The following will be described in detail in combination with specific examples.

[0095] Example 1

[0096] The present embodiment provides an electrochemical aptamer sensor, comprising an AFB1 aptamer and a glassy carbon electrode; the AFB1 aptamer is modified by a thiol group; the surface of the glassy carbon electrode contains polydopamine and a zwitterionic polymer; the AFB1 aptamer is arranged on the surface of the glassy carbon electrode;

[0097] The preparation method of the electrochemical aptamer sensor of the present embodiment comprises the following steps:

[0098] The zwitterionic polymer CBMA is prepared in an amount of 687.84 mg (3 mmol), and the molar amount of AEMA is 10% of the total amount of CBMA and AEMA (i.e. the molar ratio of CBMA to AEMA is 9:1, i.e. 1:0.11), and the formed copolymer is called pCBAE1; the specific steps include: dissolving CBMA and AEMA in ultrapure water, mixing with 5.0 mg (0.03 mmol) of ACVA in DMSO, then nitrogen blowing to make nitrogen fill the whole reaction environment to ensure the reaction in an anaerobic state, increasing the temperature to 70℃ to initiate polymerization, after 24 h of reaction, dialysis is performed with ultrapure water to remove unreacted monomers or oligomers, then freeze-drying to obtain the zwitterionic polymer;

[0099] Preparation of an electrochemical anti-fouling sensor: dissolve dopamine (DA) in a 10.0 mmol / L Tris-HCl buffer solution with a pH of 8.5 to prepare a solution containing 2 mg / mL DA and 5 mg / mL zwitterionic polymer, take 10 μL of the solution containing 2 mg / mL DA and 5 mg / mL zwitterionic polymer, drop coat the surface of a glassy carbon electrode, and place it at room temperature for 4 h for in-situ polymerization, then wash it with ultrapure water after the reaction is completed to obtain an electrochemical anti-fouling sensor (pDA-pCBAE1 / GCE), that is, a glassy carbon electrode containing polydopamine and zwitterionic polymer on the surface;

[0100] Preparation of an electrochemical aptamer sensor: the AFB1 aptamer solution is prepared using a 20.0 mmol / L PBS buffer solution with a pH of 7.4, the electrochemical anti-fouling sensor is placed in 100 μL of the AFB1 aptamer solution containing 3 μmol / L, and incubated at 4°C for 8 h in a refrigerator, and the AFB1 aptamer is assembled on the surface of the electrochemical anti-fouling sensor through Michael addition to obtain an electrochemical aptamer sensor (Apt / pDA-pCBAE1 / GCE).

[0101] wherein the polydopamine and zwitterionic polymer are denoted as pDA-pCBAE1.

[0102] Example 2

[0103] Example 2 provides an electrochemical aptamer sensor, which is the same as in Example 1, except that the molar ratio of CBMA to AEMA is 8:2 (1:0.25), that is, the molar amount of AEMA is 20% of the total amount of CBMA and AEMA.

[0104] wherein the polydopamine and zwitterionic polymer are denoted as pDA-pCBAE2.

[0105] Example 3

[0106] Example 3 provides an electrochemical aptamer sensor, which is the same as in Example 1, except that the concentration of the AFB1 aptamer solution is 1 μmol / L.

[0107] Example 4

[0108] Example 4 provides an electrochemical aptamer sensor, which is the same as in Example 1, except that the concentration of the AFB1 aptamer solution is 2 μmol / L.

[0109] Example 5

[0110] Example 5 provides an electrochemical aptamer sensor, which is the same as in Example 1, except that the concentration of the AFB1 aptamer solution is 4 μmol / L.

[0111] Example 6

[0112] Example 6 provides an electrochemical aptamer sensor, which is the same as Example 1 except that the concentration of the AFB1 aptamer solution is 5 μmol / L.

[0113] Example 7

[0114] Example 7 provides an electrochemical aptamer sensor, which is the same as Example 1 except that the mass concentration of dopamine in the Tris-HCl buffer solution is 1.5 mg / mL.

[0115] Example 8

[0116] Example 8 provides an electrochemical aptamer sensor, which is the same as Example 1 except that the mass concentration of dopamine in the Tris-HCl buffer solution is 2.5 mg / mL.

[0117] Example 9

[0118] Example 9 provides an electrochemical aptamer sensor, which is the same as Example 1 except that the mass concentration of dopamine in the Tris-HCl buffer solution is 3 mg / mL.

[0119] Example 10

[0120] Example 10 provides an electrochemical aptamer sensor, which is the same as Example 1 except that the mass concentration of dopamine in the Tris-HCl buffer solution is 1 mg / mL.

[0121] Comparative Example 1

[0122] Comparative Example 1 provides an electrochemical aptamer sensor, which is the same as Example 1 except that the glassy carbon electrode surface only contains polydopamine and does not include the zwitterionic polymer, and the zwitterionic polymer is not introduced during preparation, i.e., the electrochemical anti-fouling sensor of Comparative Example 1 is a glassy carbon electrode containing polydopamine on the surface.

[0123] Comparative Example 2

[0124] Comparative Example 2 provides an electrochemical aptamer sensor, which is the same as Example 1 except that no AEMA is added, i.e., the molar amount of AEMA is 0% of the total amount of CBMA and AEMA.

[0125] wherein the polydopamine and the zwitterionic polymer are denoted as pDA-pCBAE0.

[0126] The performances of the electrochemical aptamer sensors of the above examples and comparative examples are tested and compared below.

[0127] Test Example 1

[0128] Example 1 to 2 and Comparative Example 1 to 2 were tested.

[0129] It should be understood that although the electrochemical anti-fouling sensor is not directly mentioned in Example 1 to 2 and Comparative Example 1 to 2, it is the same as Example 1 except for the specifically stated, and therefore also includes the steps of preparing the electrochemical anti-fouling sensor.

[0130] 1. Different electrochemical anti-fouling sensors and untreated glassy carbon electrodes (GCE) were respectively immersed in 1.0 mg / mL, 5.0 mg / mL, 10.0 mg / mL bovine serum albumin, lysozyme, hemoglobin, glucose, lactose and starch solutions for incubation at room temperature for 30 min. The electrochemical probe solution was immersed in the electrochemical anti-fouling sensor to be tested, and was scanned by DPV in the potential range of -0.2 V to 0.6 V. The current response values before and after incubation were recorded to obtain the signal inhibition rate, and the anti-fouling ability of different electrochemical anti-fouling sensors was more directly compared. The results are shown in Table 1. Figures 6-7 As shown in Table 1, according to Figure 6 and Figure 7 It can be concluded that the signal inhibition rate of the electrochemical anti-fouling sensor of Example 1 is always the lowest, indicating that it has the best anti-pollution performance, and further indicating that the electrochemical aptamer sensor of Example 1 has the best anti-pollution performance.

[0131] 2. 1 mg, 5 mg, 10 mg, 20 mg and 30 mg of pure milk powder (protein content of 43%) were respectively dissolved in 100 mL of pH 7.4 20.0 mmol / L PBS buffer solution; 10 g of whole wheat flour was dissolved in 100 mL of methanol / PBS solution (volume ratio of 1:4), and after centrifugation at 6000 r / min for 10 min, the supernatant was taken and diluted with the same PBS buffer solution (pH 7.4, 20.0 mmol / L) to 1%, 5%, 10%, 20% and 30% by volume concentration; different electrochemical anti-fouling sensors and untreated glassy carbon electrodes (GCE) were respectively immersed in the above solutions for incubation for 30 min, and were scanned by DPV in the potential range of -0.2 V to 0.6 V. The current response values before and after incubation were recorded to obtain the signal inhibition rate. The results are shown in Table 2. Figure 8 As shown in Table 2, according to Figure 8 It can be concluded that in 1% whole wheat flour, pure milk powder and soy milk, the signal inhibition rate of the electrochemical anti-fouling sensor of Example 1 is as low as 1.2%, 4.1% and 1.4% respectively, and even in 30% whole wheat flour, pure milk powder and soy milk, the signal inhibition rate is only 13.92%, 6.27% and 9.31% respectively, which is much lower than that of GCE, which is 84.0%, 74.9% and 64.9% respectively, indicating that the electrochemical anti-fouling sensor has good anti-pollution performance, and accordingly, the electrochemical aptamer sensor also has good anti-pollution performance.

[0132] Test Example 2

[0133] Test Example 2 is a related test of the electrochemical anti-fouling sensor and the electrochemical aptamer sensor of Example 1 and Examples 3-6.

[0134] 1. Using the electrochemical anti-fouling sensor of Example 1 and Examples 3-6, 10 μL of 10 ng / mL AFB1 standard solution was dropped on the electrochemical anti-fouling sensor, and after incubation at 37°C for 30 min, the AFB1 aptamer that was not firmly bound was washed away with ultrapure water to form a working electrode to be tested. Then the working electrode to be tested was immersed in an electrochemical probe solution, and was scanned by differential pulse voltammetry (DPV) in a potential range of -0.2 V to 0.6 V. The change in the electrical signal (ΔI = I - I0, I0 is the electrical signal peak value before incubation with AFB1, and I is the electrical signal peak value after incubation) was calculated by recording the DPV signals before and after testing, and the amount of AFB1 binding to the electrochemical anti-fouling sensor was reflected by the change in the electrical signal. The results are shown in Figure 9 (a). It can be seen from Figure 9 (a) that when the AFB1 aptamer concentration is 3 μmol / L, the electrical signal change is the highest and tends to be stable, i.e., when the AFB1 aptamer concentration is 3 μmol / L, a more sensitive and stable electrochemical aptamer sensor can be obtained. Meanwhile, it can be seen from Figure 9 (b)-(d) that when the dopamine solution concentration is unchanged, the anti-fouling performance also changes with the change in the concentration of the zwitterionic polymer pCBAE1 solution, and the anti-fouling effect is better when the concentration is 5-6 mg / mL. In the figure, BSA is bovine serum albumin, LYS is lysozyme, and Hb is hemoglobin.

[0135] 2. Using the electrochemical aptamer sensor of Example 1, 10 μL of AFB1 standard solution with a concentration of 0.001 ng / mL, 0.005 ng / mL, 0.01 ng / mL, 0.05 ng / mL, 0.1 ng / mL, 0.5 ng / mL, 1 ng / mL, 0.5 ng / mL, and 10 ng / mL was dropped on the surface of the electrochemical aptamer sensor, respectively, and after incubation at 37°C for 30 min, the AFB1 that was not firmly bound was washed away with ultrapure water to form a working electrode to be tested. Then the working electrode to be tested was immersed in an electrochemical probe solution, and was scanned by DPV in a potential range of -0.2 V to 0.6 V. The DPV response signals before and after testing were recorded (as shown in Figure 11 (a)). The change in the electrical signal was calculated by the response signal value, and the standard curve was obtained according to the change in the electrical signal and the logarithmic value of the AFB1 concentration, i.e., y = 22.36x + 7.25 (R2= 0.997, S / N = 3), and the detection limit of AFB1 was 0.19 pg / mL. Figure 11(b) signal change rate of the electrochemical aptamer sensor after incubation with AFB1 at different concentrations, Figure 11 (c) standard curve between AFB1 concentration and signal change rate, Figure 11 (d) standard curve between AFB1 concentration and absorbance (enzyme-linked immunoassay).

[0136] 3. AFB1 at 0.1 ng / mL and aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone, vomitoxin and their mixture at 10 ng / mL were detected by the electrochemical aptamer sensor of Example 1. The results are shown in Figure 12 (a) The signal change values of the electrochemical aptamer sensor for aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone and vomitoxin were very low, being 8.5%, 7.2%, 6.6%, 5.7%, 2.9% and 1.4%, respectively; while the signal inhibition rates for AFB1 and their mixture were 30.7% and 23.1%, respectively, indicating that the electrochemical aptamer sensor had excellent selectivity. Meanwhile, it can be seen from Figure 12 (b)-(d) that the electrochemical aptamer sensor of the present application had good storage stability Figure 12 (b)) and excellent repeatability stability Figure 12 (c)-(d)).

[0137] Test Example 3

[0138] The electrochemical aptamer sensors of Examples 1, 7-10 were tested in Test Example 3.

[0139] After the electrochemical aptamer sensor was incubated in a 10 mg / mL BSA solution at room temperature for 0.5 h, the change in the electrical signal was calculated. The smaller the change in the electrical signal, the better the anti-pollution effect. The results are shown in Figure 13 It can be seen from Figure 13 that the anti-pollution effect was best when the mass ratio of dopamine to zwitterionic polymer was 2:5.

[0140] Test Example 4

[0141] Whole wheat flour, pure milk powder (protein content 43%) and soy milk were selected as the samples to be detected, and the electrochemical aptamer sensor of Example 1 of the present application was used to verify the actual application performance in detecting AFB1 in food by a standard addition recovery experiment.

[0142] The soy milk is diluted by 100 times of volume with 20.0 mmol / L PBS buffer solution with pH of 7.4; 1 g of pure milk powder is dissolved in 100 mL of 20.0 mmol / L PBS buffer solution with pH of 7.4; 10 g of whole wheat flour is dissolved in 100 mL of methanol / PBS (volume ratio of 1:4) solution, and then centrifuged at 6000 r / min for 10 min, and the supernatant is taken and diluted by 100 times of volume; different concentrations of AFB1 standard solution are added in the diluted three kinds of food samples, and the adding concentration is 0.1 ng / mL, 0.5 ng / mL and 1.0 ng / mL respectively, so as to ensure that the solution after adding contains 0.1 ng / mL, 0.5 ng / mL and 1.0 ng / mL of AFB1, and then the anti-pollution electrochemical aptamer sensor prepared in Example 1 is used for detection, so as to obtain the recovery rate of the food sample after adding. The food sample after adding is detected by using an enzyme-linked immunoassay kit (purchased from Shenzhen Fender Biotechnology Co., Ltd.), and the result obtained by the constructed electrochemical aptamer sensor is verified, so as to ensure the accuracy of the analysis.

[0143] The specific results are shown in Table 1.

[0144] Table 1: Detection results of food samples by electrochemical aptamer sensor and enzyme-linked reaction kit

[0145]

[0146] As can be seen from Table 1, the electrochemical aptamer sensor prepared in the application can sensitively detect AFB1 in the sample to be detected, and has a broad application prospect in food matrix.

[0147] The above examples are the preferred embodiments of the application, but the embodiments of the application are not limited by the above examples, and any changes, modifications, substitutions, combinations and simplifications made without departing from the spirit and principles of the application shall be equivalent replacement modes, and all shall be included in the protection scope of the application.

Claims

1. An electrochemical aptamer sensor, characterized in that, The device includes a nucleic acid aptamer and an electrode; the electrode surface contains polydopamine and a zwitterionic polymer; the nucleic acid aptamer is modified with thiol groups; the nucleic acid aptamer is modified on the electrode surface; the electrode includes a glassy carbon electrode; the nucleic acid aptamer includes an AFB1 aptamer; the nucleotide sequence of the AFB1 aptamer is shown in SEO ID NO:1; the thiol-modified sequence of the AFB1 aptamer is 5′-SH-(CH2)6-GCATCA CTA CAG TCA TTA CGC ATC GGG TAA GCG GAA CTG AGG AGT GGG AGG TAA ATC GTGTGA AGT GCT GTC CC-3′; the nucleic acid aptamer is immobilized on the polydopamine by Michael addition; the zwitterionic polymer includes a copolymer of CBMA and AEMA; the molar ratio of CBMA to AEMA is 1:(0.05~3).

2. A method for preparing the electrochemical aptamer sensor according to claim 1, characterized in that, Includes the following steps: Dopamine and a zwitterionic polymer are mixed and dissolved, applied to the electrode surface, and polymerized in situ; a nucleic acid aptamer is then modified onto the electrode surface to obtain the electrochemical aptamer sensor.

3. The preparation method according to claim 2, characterized in that, The mass ratio of dopamine to the zwitterionic polymer is (1.5~3.5):5; And / or, after dissolution, the concentration of the zwitterionic polymer is 4~6 mg / L; the concentration of dopamine is 0.5~1.5 mg / L.

4. The preparation method according to claim 2, characterized in that, The step of placing the nucleic acid aptamer on the electrode surface includes: placing the electrode in a solution of the nucleic acid aptamer and refrigerating it to obtain an electrochemical sensor; the refrigeration temperature is 3~5℃.

5. The preparation method according to claim 4, characterized in that, The concentration of the nucleic acid aptamer solution is 1~5 μmol / L.

6. The application of the electrochemical aptamer sensor according to claim 1 in food safety detection.

7. The application according to claim 6, characterized in that, The electrochemical aptamer sensor is used to detect AFB1.

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