Electrochemical aptamer sensor as well as preparation method and application thereof

Abstract

The invention discloses an electrochemical aptamer sensor as well as 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 surface of the electrode contains polydopamine and a zwitterionic polymer; the nucleic acid aptamer is modified by sulfydryl; the nucleic acid aptamer is arranged on the surface of the electrode. According to the electrochemical sensor provided by the invention, the polydopamine and the zwitterionic polymer are introduced on the surface of the electrode, so that the electrochemical sensor is high in pollution resistance, high in sensitivity and good in stability, meanwhile, the nucleic acid aptamer is modified by sulfydryl, the stability of the electrochemical sensor is further improved, and the electrochemical sensor has good selectivity on a target detection object. The electrochemical sensor can solve the problem that the existing electrochemical sensor is easy to pollute in food detection, and is suitable for food safety detection, especially AFB1 detection.

Description

Technical Field

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

[0002] Aflatoxins usually come from Aspergillus flavus and Aspergillus parasiticus. Cereals with high starch content and their products are susceptible to mold invasion, resulting in excessive aflatoxin residues, which seriously affects food safety. Currently, 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 Agency for Research on Cancer. Therefore, it is of great significance to quickly and accurately detect AFB1 in food.

[0003] Among the numerous detection methods, sensor technology based on electrochemical aptamers has attracted extensive attention due to its advantages of 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 sensors, electrode surface modification strategies are usually studied to improve the sensitivity and accuracy of detection.

[0004] However, for existing electrochemical aptamer sensors, biofouling is one of the key factors affecting their application. Biofouling refers to nonspecific adhesion that causes negative effects, that is, the process in which macromolecules such as proteins and microorganisms reversibly or irreversibly adhere to the surface of the material substrate. This layer of adsorbed proteins can serve as a platform for microorganisms to attach and form biofilms, ultimately leading to sensor failure. The matrix in food is complex, and it is inevitable that the sensor surface will be contaminated during the detection process, affecting the specificity, stability and repeatability of the sensor. Therefore, giving the surface of the electrochemical aptamer sensor anti-fouling properties is crucial to improving sensor performance. Summary of the invention

[0005] The present invention aims to solve at least one of the above-mentioned technical problems existing in the prior art. To this end, the present invention aims to provide an electrochemical aptamer sensor with strong anti-pollution performance, high sensitivity, selectivity and good stability.

[0006] The second aspect of the present invention is to provide a method for preparing an electrochemical aptamer sensor.

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

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

[0009] The first aspect of the present invention is to provide an electrochemical aptamer sensor, comprising a nucleic acid aptamer and an electrode; the electrode surface contains polydopamine and a zwitterionic polymer; the nucleic acid aptamer is modified with thiol; and the nucleic acid aptamer is modified on the electrode surface.

[0010] It can be understood that, since the electrode surface contains polydopamine and zwitterionic polymer, modifying the aptamer on the electrode surface means fixing the aptamer on the polydopamine and / or zwitterionic polymer on the electrode surface.

[0011] Electrochemical aptamer sensor refers to a sensor based on electrochemical nucleic acid aptamers, in which the nucleic acid aptamer is fixed to the electrode surface, and is also called an electrochemical nucleic acid aptamer sensor. The zwitterionic polymer on the surface of the electrochemical aptamer sensor of the present invention contains zwitterionic groups, and the solvation and hydrogen bonding of the charged functional groups can form a hydration layer on the surface of the zwitterionic polymer. This hydration layer surface formed based on electrostatic action can effectively resist nonspecific adsorption, has high anti-fouling performance, and has high repeatability and stability under long-term storage. Dopamine has the structure of catechol and can adhere to the surface of various objects. Dopamine is modified on the electrode surface. After dopamine oxidative polymerization, the nucleic acid aptamer can be firmly fixed to the electrode surface, for example, by Michael addition fixation. In addition, polydopamine can bind water molecules through hydrogen bonds, thereby slightly improving the anti-fouling performance of the sensor.

[0012] The aptamer is modified with thiol and the anchoring effect of thiol is utilized to fix the modified functionalized probe and ensure the specificity of the electrochemical aptamer sensor.

[0013] The electrochemical aptamer sensor of the present invention has high sensitivity itself and does not need to be further enhanced for electrical signals.

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

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

[0016] In some embodiments of the present invention, the nucleic acid aptamer includes an AFB1 aptamer; the nucleotide sequence of the AFB1 aptamer is shown in SEO ID NO: 1; the sequence of the AFB1 aptamer after thiol modification is 5′-SH-(CH2)6-GCA TCA CTA CAG TCATTACGC ATC GGG TAAGCG GAACTG AGG AGT GGG AGG TAAATC GTGTGAAGT GCT GTC CC-3′.

[0017] The present invention further selects an AFB1 aptamer, which can improve the selectivity of the electrochemical aptamer sensor for AFB1 and is used in the rapid detection of AFB1 in food.

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

[0019] Michael addition has the characteristics of relatively mild reaction conditions, simple operation, high yield, and specific regional selection, which is simpler than other addition methods. For example, the method of activating the carboxyl group and then cross-linking it with the amino-containing aptamer requires the addition of activators 1-ethyl-(3-dimethylaminopropyl)carbamide (EDC) and N-hydroxysuccinimide (NHS), 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 cumbersome.

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

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

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

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

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

[0025] In some embodiments of the present invention, the polydopamine and the zwitterionic polymer are obtained by in situ polymerization of dopamine and a 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 dopamine and zwitterionic polymers undergo in situ polymerization to form polydopamine and zwitterionic polymers, as well as some copolymers of dopamine and zwitterionic polymers.

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

[0028] Dopamine and zwitterionic polymer are mixed and dissolved, applied to the electrode surface, and polymerized in situ; nucleic acid aptamers are modified on the electrode surface 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 invention, the mass ratio of the dopamine to the zwitterionic polymer is (1.5-3.5):5.

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

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

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

[0034] Adjusting the mass ratio of dopamine to zwitterionic polymer can also help improve the performance of electrochemical sensors.

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

[0036] The thiol-modified nucleic acid aptamer was mixed with TCEP and subjected to reduction reaction at room temperature to reduce the thiol groups with disulfide bonds.

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

[0038] In some embodiments of the present invention, the reduction reaction time is 25 to 35 minutes.

[0039] In some embodiments of the present invention, the method for preparing the zwitterionic polymer comprises the following steps:

[0040] The zwitterionic monomer compound is dissolved, an initiator is added, and the mixture is mixed in an organic solvent, and a polymerization reaction is carried out under an oxygen-free condition to obtain the zwitterionic polymer.

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

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

[0043] In some embodiments of the present invention, the polymerization reaction temperature is 65-75°C.

[0044] In some embodiments of the present invention, the polymerization reaction time is 23 to 25 hours.

[0045] In some embodiments of the present invention, the polymerization reaction further includes dialysis and freeze-drying steps; the dialysis is ultrapure water dialysis to remove unreacted monomers or oligomers.

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

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

[0048] In some embodiments of the present invention, the in-situ polymerization time is 3 to 5 hours.

[0049] In some embodiments of the present invention, the step of disposing the nucleic acid aptamer on the surface of the electrode comprises: placing the electrode in a solution of the nucleic acid aptamer and refrigerating to obtain the electrochemical sensor; the refrigeration temperature is 3-5°C.

[0050] In some embodiments of the present invention, the refrigeration method is to place in a refrigerator; the refrigeration time is 7 to 9 hours.

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

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

[0053] The third aspect of the present invention is to provide an application of the electrochemical aptamer sensor described in the first aspect of the present invention in food safety detection.

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

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

[0056] In some embodiments of the present invention, the electrochemical sensor is used to detect AFB1.

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

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

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

[0060] In some embodiments of the present invention, during the detection process, the spiked concentration of the AFB1 standard solution is 0.1-1 ng / mL, for example, 0.1 ng / mL, 0.5 ng / mL or 1 ng / mL.

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

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

[0063] Figure 1 The MS spectra of AEMA used in Examples and Comparative Examples are shown in FIG.

[0064] Figure 2 The MS spectra of CBMA used in Examples and Comparative Examples are shown in FIG.

[0065] Figure 3 This is the MS spectrum of pDA-pCBAE0 of Comparative Example 2.

[0066] Figure 4 This is the MS spectrum of pDA-pCBAE1 in Example 1.

[0067] Figure 5 This is the MS spectrum of pDA-pCBAE2 in Example 2.

[0068] Figure 6 It is the signal suppression rate of the electrochemical anti-fouling sensor in Test Example 1 in single protein solutions of different concentrations.

[0069] Figure 7 It is the signal suppression rate of the electrochemical anti-fouling sensor in Test Example 1 in different molecular weight sugar solutions with different concentrations.

[0070] Figure 8 It is the signal suppression rate of the electrochemical anti-fouling sensor in Test Example 1 in different food samples with different dilutions.

[0071] Fig. 9 The effects of different AFB1 aptamer concentrations and different protein solutions of different concentrations on the electrical signals of the electrochemical anti-fouling sensor in Test Example 2; wherein (a) is a graph showing changes in electrochemical signals at different concentrations of AFB1 aptamer; (b) to (d) respectively represent graphs showing changes in electrochemical signals of the electrochemical anti-fouling sensor pDA-pCBAE1 / GCE of Example 1 in different concentrations of BSA, LYS and Hb.

[0072] Fig.10 is a scanning electron microscope image of the glassy carbon electrode, the electrochemical sensor of Example 1 and Comparative Example 1; wherein (a 0～2 )~(c 0～2 ) represent the glassy carbon electrode, the electrochemical sensor of comparative example 1 and the electrochemical sensor of embodiment 1 respectively.

[0073] Fig.11 The figure is a graph showing the relationship between the AFB1 concentration and the test results of the electrochemical aptamer sensor in Test Example 2 and a standard curve; 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 the AFB1 concentration and the signal change rate, and (d) is the standard curve between the AFB1 concentration and the absorbance (enzyme-linked immunosorbent assay).

[0074] Fig.12 The results of the selectivity, stability and repeatability tests of the electrochemical aptamer sensor in Test Example 2 are shown in Figure 2; wherein (a) to (d) represent selectivity, storage stability, stability of electrodes from the same root, and stability of electrodes from the same batch, respectively.

[0075] Fig.13This is a graph showing the anti-pollution performance test results of the electrochemical aptamer sensor in Test Example 3.

[0076] Fig.14 Schematic diagram of the preparation process of the electrochemical aptamer sensor in the embodiment. DETAILED DESCRIPTION

[0077] The present invention is further described in detail below by specific examples. Unless otherwise specified, the raw materials, reagents or devices used in the examples and comparative examples can be obtained from conventional commercial sources or can be obtained by prior art methods. Unless otherwise specified, the experiments or test methods are conventional methods in the art.

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

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

[0080] Lysozyme (Lys) was purchased from Boster Biotechnology;

[0081] Potassium ferrocyanide trihydrate (K4Fe(CN)6·3H2O), potassium ferrocyanide (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 Sangon Biotechnology and modified with thiol groups, with the sequence of 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 Blue Season Technology Co., Ltd.;

[0084] 3-[[2-(Methacryloyloxy)ethyl]dimethylammonium]propionate (CBMA), N -methacryloylethylenediamine 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 Amaclin Biochemical Technology Co., Ltd.;

[0086] Ultrapure water (18.2MΩcm-1 ) was made in the laboratory.

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

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

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

[0090] Among them, the preparation method of PBS is: accurately weigh 35.8g of disodium hydrogen phosphate (Na2HPO4·12H2O) and 15.6g of sodium dihydrogen phosphate (NaH2PO4·2H2O), place them in a 500mL volumetric flask, and dilute to the scale with ultrapure water to obtain 0.2mol / LNa2HPO4 solution and 0.2mol / L NaH2PO4 solution; use a measuring cylinder to measure 324mL of 0.2mol / L Na2HPO4 solution and 76mL of 0.2mol / LNaH2PO4 solution, respectively, and then add 3.6g of solid sodium chloride (NaCl), mix well, and obtain a 0.2mol / L PBS solution with a pH of 7.4, place it at room temperature, and then dilute it according to the required concentration before use.

[0091] The preparation method of the electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]) is as follows: accurately weigh 0.8231 g of potassium ferrocyanide (K3[Fe(CN)6]) solid, 1.0560 g of potassium ferrocyanide (K4[Fe(CN)6]) and 7.4550 g of potassium chloride (KCl), then dissolve the above drugs with 0.01 mol / L PBS solution at pH 7.4, and finally make up to 500 mL volumetric flask to obtain a bright yellow solution, that is, a 5 mmol / L electrochemical probe (K3[Fe(CN)6] / K4[Fe(CN)6], containing 0.2 mol / L KCl) solution, wrap it with tin foil, and store it in a 4°C refrigerator for use.

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

[0093] In the following examples and comparative examples of the present invention, DPV was used to record the current response values ​​of the modified electrode before and after incubation (the electric signal before incubation was I0, and the electric signal after incubation was I), and the electric signal change value (△I=I0-I) and the electric signal suppression rate (Signal Suppression (%)=[(I0-I) / I0]×100) were calculated based on these values.

[0094] The preparation process of the electrochemical aptamer sensor in the embodiment of the present invention is as follows: Fig.14 As shown, the following is a detailed description in conjunction with specific embodiments.

[0095] Example 1

[0096] This embodiment provides an electrochemical aptamer sensor, including 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 disposed on the surface of the glassy carbon electrode;

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

[0098] Preparation of zwitterionic polymer: The amount of CBMA is fixed at 687.84 mg (3 mmol), 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) ACVA in DMSO, and then purging with nitrogen to fill the entire reaction environment with nitrogen to ensure that the reaction is in an oxygen-free state, raising the temperature to 70°C to initiate polymerization, and after reacting for 24 hours, dialyzing with ultrapure water to remove unreacted monomers or oligomers, and then freeze-drying to obtain a zwitterionic polymer;

[0099] Preparation of electrochemical antifouling sensor: dopamine (DA) was dissolved in 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, 10 μL of the solution containing 2 mg / mL DA and 5 mg / mL zwitterionic polymer was drop-coated on the surface of glassy carbon electrode, and placed at room temperature for 4 hours for in-situ polymerization. After the reaction was completed, it was rinsed with ultrapure water to obtain an electrochemical antifouling sensor (pDA-pCBAE1 / GCE), that is, a glassy carbon electrode containing polydopamine and zwitterionic polymer on the surface;

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

[0101] Among them, polydopamine and zwitterionic polymer are denoted as pDA-pCBAE1.

[0102] Example 2

[0103] Example 2 provides an electrochemical aptamer sensor, which is similar to 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] Among them, polydopamine and zwitterionic polymer are denoted as pDA-pCBAE2.

[0105] Example 3

[0106] Example 3 provides an electrochemical aptamer sensor, which is similar to 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 similar to 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 similar to 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 similar to 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 similar to 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 similar to 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 similar to 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 similar to 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 specifically the same as Example 1, except that the surface of the glassy carbon electrode only contains polydopamine, does not include zwitterionic polymers, and zwitterionic polymers are not introduced during the preparation process, that is, 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 similar to Example 1 except that AEMA is not added, that is, the molar amount of AEMA is 0% of the total amount of CBMA and AEMA.

[0125] Among them, polydopamine and zwitterionic polymer are denoted as pDA-pCBAE0.

[0126] The performance of the electrochemical aptamer sensors of the above-mentioned embodiments and comparative examples are tested and compared below.

[0127] Test Example 1

[0128] Test Example 1 The electrochemical anti-fouling sensors of Examples 1-2 and Comparative Examples 1-2 were tested.

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

[0130] 1. Immerse different electrochemical anti-fouling sensors and untreated glassy carbon electrodes (GCE) in 1.0 mg / mL, 5.0 mg / mL, 10.0 mg / mL bovine serum albumin, lysozyme, hemoglobin, glucose, lactose and starch solutions respectively and incubate at room temperature for 30 minutes. Immerse the electrochemical anti-fouling sensor to be tested in the electrochemical probe solution, scan with DPV in the potential range of -0.2V to 0.6V, record the current response value before and after incubation, and obtain the signal suppression rate, so as to more intuitively compare the anti-fouling ability of different electrochemical anti-fouling sensors. The results are as follows: Figures 6-7 As shown, according to Figure 6 and Figure 7 It can be concluded that the signal suppression rate of the electrochemical anti-fouling sensor of Example 1 is always the lowest, indicating that it has the best anti-fouling performance, which further illustrates that the electrochemical aptamer sensor of Example 1 has the best anti-fouling performance.

[0131] 2. Weigh 1mg, 5mg, 10mg, 20mg, and 30mg of pure milk powder (protein content of 43%) and dissolve them in 100mL of 20.0mmol / L PBS buffer solution at pH7.4; dissolve 10g of whole wheat flour in 100mL of methanol / PBS solution (volume ratio of 1:4), centrifuge at 6000r / min for 10min, take the supernatant and dilute it with the same PBS buffer solution (pH7.4, 20.0mmol / L) at a volume concentration of 1%, 5%, 10%, 20%, and 30%; immerse different electrochemical antifouling sensors and untreated glassy carbon electrodes (GCE) in the above solutions and incubate for 30min, scan with DPV in the potential range of -0.2V to 0.6V, and record the current response values ​​before and after incubation to obtain the signal inhibition rate. The results are as follows Figure 8 As shown, according to Figure 8 It can be concluded that the signal suppression rates of the electrochemical anti-fouling sensor of Example 1 in 1% whole wheat flour, pure milk powder and soy milk are as low as 1.2%, 4.1% and 1.4%, respectively. Even in 30% whole wheat flour, pure milk powder and soy milk, the signal suppression rates are only 13.92%, 6.27% and 9.31%, which are much lower than the signal suppression rates of GCE, which are 84.0%, 74.9% and 64.9%, respectively. This indicates that the electrochemical anti-fouling sensor of the present invention has good anti-pollution performance, and correspondingly, 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 to 6.

[0134] 1. Using the electrochemical anti-fouling sensor of Example 1 and Examples 3 to 6, 10 μL of 10 ng / mL AFB1 standard solution was added to the electrochemical anti-fouling sensor, and the unbound AFB1 aptamer was rinsed off with ultrapure water after incubation at 37°C for 30 min to form a working electrode to be detected; then the working electrode to be detected was immersed in an electrochemical probe solution, and scanned by differential pulse voltammetry (DPV) in the potential range of -0.2V to 0.6V. The change value of the electrical signal was calculated by recording the DPV signal before and after the detection (ΔI=I–I0, I0 is the peak value of the electrical signal before incubation of AFB1, and I is the peak value of the electrical signal after incubation). The change value of the electrical signal reflects the amount of AFB1 bound to the electrochemical anti-fouling sensor. The results are as follows: Fig. 9 As shown in (a), through Fig. 9 (a) It can be seen that when the concentration of AFB1 aptamer is 3 μmol / L, the electrical signal changes the most and tends to be stable, that is, when the concentration of AFB1 aptamer is 3 μmol / L, a more sensitive and stable electrochemical aptamer sensor can be obtained. Fig. 9 (b) to (d) show that when the concentration of dopamine solution remains unchanged, the concentration of zwitterionic polymer pCBAE1 solution changes, and the anti-pollution performance also changes, and it has a better anti-pollution effect when the concentration is 5 to 6 mg / mL; in the figure, BSA is bovine serum albumin, LYS is lysozyme, and Hb is hemoglobin.

[0135] 2. The electrochemical aptamer sensor of Example 1 was used, and 10 μL of AFB1 standard solutions with concentrations 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 were respectively applied on the surface of the sensor, and the unbound AFB1 was rinsed off with ultrapure water to form a working electrode to be detected. The working electrode to be detected was then immersed in an electrochemical probe solution, and DPV scanning was performed in the potential range of -0.2 V to 0.6 V to record the DPV response signals before and after detection (such as Fig.11 (a)). The electric signal change value was calculated by the response signal value, and the standard curve was obtained according to the electric signal change value and the logarithmic value of the AFB1 concentration: y=22.36x+7.25, (R2=0.997, S / N=3), and the detection limit of AFB1 was 0.19 pg / mL. Fig.11(b) is the signal change rate of the electrochemical aptamer sensor after incubation with different concentrations of AFB1. Fig.11 (c) is the standard curve between AFB1 concentration and signal change rate, Fig.11 (d) is the standard curve between AFB1 concentration and absorbance (enzyme-linked immunosorbent assay).

[0136] 3. 0.1 ng / mL AFB1 and 10 ng / mL aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone, vomitoxin and their mixtures were used as detection objects and the electrochemical aptamer sensor of Example 1 was used for detection. The results are shown in FIG. Fig.12 As shown in (a), the electrical signal changes of aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone and vomitoxin were very low, which were 8.5%, 7.2%, 6.6%, 5.7%, 2.9% and 1.4% respectively; while when AFB1 and their mixture were detected alone, the signal suppression rates were 30.7% and 23.1%, respectively, indicating that the electrochemical aptamer sensor has excellent selectivity. At the same time, combined with Fig.12 (b) to (d) show that the electrochemical aptamer sensor of the present invention has good storage stability ( Fig.12 (b)) and excellent repeatability stability ( Fig.12 (c)~(d)).

[0137] Test Example 3

[0138] Test Example 3 The electrochemical aptamer sensors of Example 1 and Examples 7 to 10 were tested.

[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 with the least interference from the pollutant, the better the anti-fouling effect. Fig.13 As shown by Fig.13 It can be seen that the best anti-fouling effect is achieved when the mass ratio of dopamine to zwitterionic polymer is 2:5.

[0140] Test Example 4

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

[0142] The soy milk was diluted 100 times by volume with a 20.0 mmol / L PBS buffer solution at a pH of 7.4; 1 g of pure milk powder was dissolved with 100 mL of a 20.0 mmol / L PBS buffer solution at a pH of 7.4; 10 g of whole wheat flour was fully dissolved with 100 mL of methanol / PBS (volume ratio of 1:4) solution, and then centrifuged at 6000 r / min for 10 min, the supernatant was taken and diluted 100 times by volume; different concentrations of AFB1 standard solutions were added to the three diluted food samples, and the spiked concentrations were 0.1 ng / mL, 0.5 ng / mL, and 1.0 ng / mL, respectively, to ensure that the solution contained 0.1 ng / mL, 0.5 ng / mL and 1.0 ng / mL of AFB1 after spiked, and then the anti-fouling electrochemical aptamer sensor prepared in Example 1 was used for detection to obtain the spiked recovery rate in the food samples. The spiked food samples were tested using an enzyme-linked immunosorbent assay kit (purchased from Shenzhen Finder Biotechnology Co., Ltd.) to verify the results obtained by the constructed electrochemical aptasensor 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 ELISA kit

[0145]

[0146] It can be seen from Table 1 that the electrochemical aptamer sensor prepared in the present invention can sensitively detect AFB1 in the sample to be tested and has broad application prospects in food matrices.

[0147] The above embodiments are preferred implementation modes of the present invention, but the implementation modes of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the protection scope of the present invention.

Claims

1. An electrochemical aptamer sensor, characterized in that: The invention comprises 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 thiol; and the nucleic acid aptamer is modified on the surface of the electrode.

2. The electrochemical aptamer sensor according to claim 1, characterized in that: The electrode comprises a glassy carbon electrode; And / or, the nucleic acid aptamer includes an AFB1 aptamer; the nucleotide sequence of the AFB1 aptamer is shown in SEO ID NO: 1; the sequence of the AFB1 aptamer after thiol modification is 5′-SH-(CH2)6-GCA TCA CTA CAGTCA TTACGC ATC GGG TAAGCG GAACTG AGG AGT GGG AGG TAA ATC GTG TGAAGT GCT GTC CC-3′.

3. The electrochemical aptamer sensor according to claim 1 or 2, characterized in that: The nucleic acid aptamer is immobilized on the polydopamine through Michael addition.

4. The electrochemical aptamer sensor according to claim 1, characterized in that: The zwitterionic polymer includes a copolymer of CBMA and AEMA; the molar ratio of the CBMA to the AEMA is 1:(0.05-3).

5. A method for preparing the electrochemical aptamer sensor according to any one of claims 1 to 4, characterized in that: The steps include: Dopamine and zwitterionic polymer are mixed and dissolved, applied to the electrode surface, and polymerized in situ; nucleic acid aptamers are modified on the electrode surface to obtain the electrochemical aptamer sensor.

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

7. The preparation method according to claim 5, characterized in that: The step of arranging the nucleic acid aptamer on the surface of the electrode comprises: placing the electrode in a solution of the nucleic acid aptamer and refrigerating to obtain the electrochemical sensor; The refrigeration temperature is 3-5°C.

8. The preparation method according to claim 7, characterized in that: The solution concentration of the nucleic acid aptamer is 1-5 μmol / L.

9. Use of the electrochemical aptamer sensor according to any one of claims 1 to 4 in food safety detection.

10. The use according to claim 9, characterized in that: The electrochemical aptamer sensor is used to detect AFB1.

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