Polypeptide-based anti-fouling electrochemical sensor as well as construction method and application thereof

Abstract

The invention discloses a polypeptide-based anti-fouling electrochemical sensor as well as a construction method and application thereof. Cysteine is used as an anchoring end, polyproline is inserted to form an alpha-helical structure to provide a supporting effect for the whole peptide, and then angiotensin converting enzyme inhibitory peptide (KSRE) and alpha-glucosidase inhibitory peptide (EKDER) are used as branches to prepare the anti-fouling polypeptide. Compared with other anti-pollution polypeptides, the anti-pollution polypeptide has a better anti-pollution effect, the anti-pollution polypeptide is used for modifying a working electrode and preparing an anti-pollution electrochemical aptamer sensor for detecting AFB1, the anti-pollution interference effect of the sensor can be greatly improved, the accuracy and sensitivity of AFB1 detection can be improved, and the application prospect is wide. Wide application prospects are realized.

Description

Technical Field

[0001] The present invention relates to the technical field of anti-fouling electrochemical sensors, and in particular to a polypeptide-based anti-fouling electrochemical sensor and a construction method and application thereof. Background Art

[0002] Aflatoxin is a type of bifuranoid toxin produced by strains such as Aspergillus flavus and Aspergillus parasiticus. The double bond at the end of the bifuranoid ring is easily epoxidized to form epoxides that damage nucleic acid molecules, which are carcinogenic, teratogenic and mutagenic. There are about 20 derivatives of aflatoxin, among which aflatoxin B1 (AFB1) is the most toxic and carcinogenic. Given the widespread presence of AFB1 contamination and the irreversible harm that contaminated food can cause to animals and humans, the qualitative and quantitative detection of AFB1 has always been the focus of research in the field of food safety.

[0003] In the related technology, the detection methods of AFB1 include gas chromatography, high performance liquid chromatography, liquid chromatography-mass spectrometry detection, electrochemical analysis technology, colorimetric analysis technology and fluorescence analysis technology. Among them, electrochemical analysis technology, as an emerging technology, has great application prospects. It is to quickly detect the object to be tested by utilizing the electrochemical properties of the object to be tested or the specific reaction of the sensing electrode and the object to be tested to generate the corresponding electrochemical signal. This method has the advantages of simple operation, high sensitivity and fast detection speed. However, in the electrochemical detection process, due to the complex composition of the actual sample to be tested, the non-target components in the food matrix will be non-specifically adsorbed at the sensing interface, which seriously affects the sensitivity, accuracy and practicality of the sensor. Therefore, introducing anti-fouling materials on the sensor surface and improving the anti-fouling performance of the electrochemical sensor itself are the key to achieving anti-matrix interference. At present, common antifouling materials include polyethylene glycol and its derivatives, polysaccharides, zwitterionic polymers and peptides, among which peptide antifouling materials have attracted more and more attention due to their good biocompatibility, sequence adjustability and commercial synthesis. Peptides with different sequences and structures have been gradually designed and used to modify antifouling sensing interfaces. However, conventional peptide antifouling materials are difficult to achieve ideal antifouling effects in the AFB1 detection process due to their low antifouling ability.

[0004] Based on this, the present invention designs a novel polypeptide-modified anti-fouling electrochemical aptamer sensor with the advantages of low detection limit, good selectivity and high sensitivity. Applying it to the detection of AFB1 can greatly improve the detection efficiency. Summary of the invention

[0005] The present invention aims to solve at least one of the technical problems existing in the prior art. To this end, the present invention proposes an anti-fouling polypeptide. The present invention uses cysteine ​​as the anchor end, inserts polyproline to form an α-helical structure to give the entire peptide a supporting effect, and then uses angiotensin converting enzyme inhibitory peptide and α-glucosidase inhibitory peptide as branch sequences to prepare anti-fouling sequences. Based on the steric hindrance effect, the anti-fouling polypeptide has a better anti-fouling effect than other anti-fouling polypeptides, and the anti-fouling polypeptide is used to modify the working electrode and prepare an anti-fouling electrochemical aptamer sensor for detecting AFB1, which can greatly improve the anti-pollutant interference effect of the sensor and help improve the accuracy and sensitivity of AFB1 detection.

[0006] The invention also proposes the application of an anti-fouling polypeptide in anti-fouling materials.

[0007] The invention also provides a polypeptide-based anti-fouling electrode.

[0008] The invention also provides a method for preparing a polypeptide-based anti-fouling electrode.

[0009] The invention also provides a polypeptide-based anti-fouling electrochemical aptamer sensor.

[0010] The invention also proposes a method for constructing a polypeptide-based anti-fouling electrochemical aptamer sensor.

[0011] The invention also proposes an electrochemical detection method for aflatoxin B1.

[0012] The first aspect of the present invention provides an anti-fouling polypeptide, wherein the amino acid sequence of the anti-fouling polypeptide is:

[0013] CPPPPEK(KSRE)DER.

[0014] The anti-fouling polypeptide according to the embodiments of the present invention has at least the following beneficial effects:

[0015] The present invention uses cysteine ​​as the anchor end, inserts polyproline to form an α-helical structure to give the entire peptide a supporting effect, and then uses angiotensin converting enzyme inhibitory peptide and α-glucosidase inhibitory peptide as branch sequences to prepare an anti-fouling sequence. The anti-fouling polypeptide of the present invention has a better anti-fouling effect than other anti-fouling polypeptides.

[0016] In some embodiments of the invention, the anti-fouling polypeptide comprises a KSRE branch sequence and an EKDER branch sequence.

[0017] In some embodiments of the present invention, the chemical structural formula of the anti-fouling polypeptide is as follows:

[0018]

[0019] The second aspect of the present invention provides the use of the anti-fouling polypeptide described in the first aspect in an anti-fouling material.

[0020] The third aspect of the present invention provides a polypeptide-based antifouling electrode, wherein the surface of the electrode is modified with the antifouling polypeptide as described in the first aspect.

[0021] The polypeptide-based antifouling electrode according to the embodiment of the present invention has at least the following beneficial effects: the polypeptide-based antifouling electrode of the present invention has excellent antifouling performance, and the signal suppression rate in some real samples is as low as 1.17%.

[0022] In some embodiments of the present invention, the electrode is made of gold, silver, platinum or glassy carbon.

[0023] Preferably, the electrode is a nanoporous gold-modified electrode; more preferably, the electrode is a nanoporous gold-modified glassy carbon electrode.

[0024] In some embodiments of the present invention, the method for preparing the nanoporous gold modified electrode comprises the following steps:

[0025] The polished working electrode is immersed in a mixed solution of HAuCl4 and CuSO4, and a platinum electrode is used as a counter electrode and a saturated calomel electrode is used as a reference electrode for constant potential deposition; then, after washing, it is placed in a H2SO4 solution for etching, and then taken out and washed.

[0026] In some embodiments of the present invention, the concentration of HAuCl4 in the mixed solution of HAuCl4 and CuSO4 is 0.5 to 1.5 mmol L -1 , and / or, the concentration of CuSO4 is 0.5 to 1.5 mmol L -1 .

[0027] In some embodiments of the present invention, the concentration of the H2SO4 solution is 0.2 to 1 mol L -1 .

[0028] In some embodiments of the present invention, the potential of the constant potential deposition is 0.1-0.2V.

[0029] In some embodiments of the present invention, the constant potential deposition time is 100 to 300 seconds.

[0030] In some embodiments of the present invention, the constant potential of the etching process is 0.8-1.5V.

[0031] In some embodiments of the present invention, the etching process lasts for 200 to 600 seconds.

[0032] In some embodiments of the present invention, the washing solution is ultrapure water.

[0033] In some embodiments of the present invention, the modification method comprises utilizing the thiol group on the cysteine ​​of the anti-fouling polypeptide to connect with Au of the electrode through self-assembly.

[0034] A fourth aspect of the present invention provides a method for preparing a polypeptide-based antifouling electrode, comprising the following steps:

[0035] The anti-fouling polypeptide described in the first aspect is dissolved in a buffer to obtain an anti-fouling polypeptide solution, and then the electrode is contacted with the anti-fouling polypeptide solution and incubated to obtain the anti-fouling polypeptide solution.

[0036] In some embodiments of the present invention, the buffer is selected from any one of phosphate buffer, Tris-HCl buffer, sodium chloride buffer, and HEPES buffer.

[0037] In some preferred embodiments of the present invention, the buffer is a phosphate buffer.

[0038] In some embodiments of the present invention, the concentration of the antifouling polypeptide solution is 0.05-1 mg mL -1 .

[0039] In some preferred embodiments of the present invention, the concentration of the antifouling polypeptide solution is 0.2-0.5 mg / mL -1 .

[0040] In some embodiments of the present invention, the electrode is made of gold, silver, platinum or glassy carbon.

[0041] Preferably, the electrode is a nanoporous gold-modified electrode; more preferably, the electrode is a nanoporous gold-modified glassy carbon electrode.

[0042] In some embodiments of the present invention, the method for preparing the nanoporous gold modified electrode comprises the following steps:

[0043] The polished working electrode is immersed in a mixed solution of HAuCl4 and CuSO4, and a platinum electrode is used as a counter electrode and a saturated calomel electrode is used as a reference electrode for constant potential deposition; then, after washing, it is placed in a H2SO4 solution for etching, and then taken out and washed.

[0044] In some embodiments of the present invention, the concentration of HAuCl4 in the mixed solution of HAuCl4 and CuSO4 is 0.5 to 1.5 mmol L -1 , and / or, the concentration of CuSO4 is 0.5 to 1.5 mmol L -1 .

[0045] In some embodiments of the present invention, the concentration of the H2SO4 solution is 0.2 to 1 mol L -1 .

[0046] In some embodiments of the present invention, the potential of the constant potential deposition is 0.1-0.2V.

[0047] In some embodiments of the present invention, the constant potential deposition time is 100 to 300 seconds.

[0048] In some embodiments of the present invention, the constant potential of the etching process is 0.8-1.5V.

[0049] In some embodiments of the present invention, the etching process lasts for 200 to 600 seconds.

[0050] In some embodiments of the present invention, the washing solution is ultrapure water.

[0051] In some embodiments of the present invention, the incubation temperature is 2-10°C.

[0052] In some preferred embodiments of the present invention, the incubation temperature is 2-5°C.

[0053] In some embodiments of the present invention, the incubation time is 6 to 12 hours.

[0054] The fifth aspect of the present invention provides a polypeptide-based anti-fouling electrochemical aptamer sensor, comprising:

[0055] A working electrode, wherein the working electrode is immobilized with an aptamer and the anti-fouling polypeptide as described in the first aspect;

[0056] a counter electrode, wherein the counter electrode is selected from a platinum electrode;

[0057] A reference electrode, wherein the reference electrode is selected from a saturated calomel electrode.

[0058] The polypeptide-based anti-fouling electrochemical aptamer sensor according to the embodiment of the present invention has at least the following beneficial effects: the polypeptide-based anti-fouling electrochemical aptamer sensor of the present invention has excellent anti-fouling effect.

[0059] In some embodiments of the present invention, the working electrode is electrically connected to the counter electrode and the reference electrode.

[0060] In some embodiments of the present invention, the working electrode is made of gold, silver, platinum or glassy carbon.

[0061] Preferably, the working electrode is a nanoporous gold-modified electrode; more preferably, the electrode is a nanoporous gold-modified glassy carbon electrode.

[0062] In some embodiments of the present invention, the aptamer is a nucleic acid aptamer.

[0063] In some embodiments of the present invention, the aptamer includes but is not limited to the AFB1 aptamer. It can be understood that the type of the aptamer can be selected according to the type of the substance to be detected.

[0064] In some embodiments of the present invention, the nucleotide sequence of the AFB1 aptamer is as follows:

[0065] 5'-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCA CA-3' (SEQ ID No. 4).

[0066] The sixth aspect of the present invention provides a method for constructing the polypeptide-based anti-fouling electrochemical aptamer sensor as described in the fifth aspect, comprising the following steps:

[0067] S1. In a solvent, the aptamer and the anti-fouling polypeptide are mixed to prepare an aptamer-polypeptide solution, and then an electrode is contacted with the aptamer-polypeptide solution, and after incubation, a working electrode is obtained;

[0068] S2. The working electrode is electrically connected to the counter electrode and the reference electrode.

[0069] The method for constructing a polypeptide-based anti-fouling electrochemical aptamer sensor according to an embodiment of the present invention has at least the following beneficial effects:

[0070] The polypeptide-based antifouling electrochemical aptamer sensor of the present invention has a simple construction process and is easy to miniaturize, wherein the linear range of detection of the target AFB1 is 0.001 ng mL -1 ~10ng mL -1 , the detection limit was 0.26 pg mL -1 , with excellent selectivity, sensitivity and accuracy.

[0071] In some embodiments of the present invention, the solvent is phosphate buffer.

[0072] In some embodiments of the present invention, the concentration of the aptamer in the aptamer-peptide solution is 0.1 to 5 μmol L -1 .

[0073] In some preferred embodiments of the present invention, the concentration of the aptamer is 1-2 μmol L -1 .

[0074] In some embodiments of the present invention, the concentration of the antifouling polypeptide in the aptamer-polypeptide solution is 0.05-1 mg mL -1 .

[0075] In some preferred embodiments of the present invention, the concentration of the antifouling polypeptide is 0.1-0.5 mg mL -1 .

[0076] In some embodiments of the present invention, the incubation temperature is 2-10°C.

[0077] In some preferred embodiments of the present invention, the incubation temperature is 2-5°C.

[0078] In some embodiments of the present invention, the incubation time is 6 to 12 hours.

[0079] The seventh aspect of the present invention provides an electrochemical detection method for AFB1, comprising using the polypeptide-based anti-fouling electrochemical aptamer sensor as described in the sixth aspect for detection, wherein the aptamer is an AFB1 aptamer.

[0080] The electrochemical detection method according to the embodiment of the present invention has at least the following beneficial effects: the electrochemical detection method of AFB1 of the present invention is simple and easy to miniaturize, wherein the linear range of the detection of the target AFB1 is 0.001 ng mL -1 ~10ngmL -1 , the detection limit was 0.26 pg mL -1 , with excellent selectivity, sensitivity and accuracy.

[0081] In some embodiments of the present invention, the detection comprises cyclic voltammetry detection and / or differential pulse voltammetry detection.

[0082] In some embodiments of the present invention, the potential range of the differential pulse voltammetry detection is -0.2V to 0.6V.

[0083] In some embodiments of the present invention, the amplitude of the differential pulse voltammetry detection is 40-60 mV, and the scan rate is 8-12 mV S -1 .

[0084] Other features and advantages of the present invention will be set forth in the description which follows. BRIEF DESCRIPTION OF THE DRAWINGS

[0085] The present invention will be further described below with reference to the accompanying drawings and embodiments, wherein:

[0086] Figure 1 The anti-fouling ability test results of the electrodes modified with the four anti-fouling polypeptides of the present invention in bovine serum albumin solution;

[0087] Figure 2 The anti-fouling performance test results of the electrode modified with the anti-fouling polypeptide of the present invention in real food samples at different dilution multiples;

[0088] Figure 3 The results of the detection of the effects of different concentrations of aptamers on the signal inhibition rate of the present invention;

[0089] Figure 4 The sensitivity test results of the anti-fouling electrochemical aptamer sensor of the present invention are shown in Figure 1, wherein A is the DPV response signal of the anti-fouling electrochemical aptamer sensor combined with different concentrations of AFB1; B is the relationship curve between the AFB1 concentration and the signal inhibition rate;

[0090] Figure 5 This is the specific detection result of the anti-fouling electrochemical aptamer sensor in the embodiment of the present invention. DETAILED DESCRIPTION

[0091] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0092] The words "preferably", "more preferably" and the like in the present invention refer to embodiments of the present invention that may provide certain beneficial effects in certain circumstances. However, other embodiments may also be preferred under the same circumstances or other circumstances. In addition, the description of one or more preferred embodiments does not imply that other embodiments are not applicable, nor is it intended to exclude other embodiments from the scope of the present invention.

[0093] When a numerical range is disclosed herein, the above range is considered to be continuous and includes the minimum and maximum values ​​of the range, as well as every value between such minimum and maximum values. Further, when a range refers to an integer, every integer between the minimum and maximum values ​​of the range is included. In addition, when multiple ranges are provided to describe features or characteristics, the ranges can be combined. In other words, unless otherwise indicated, all ranges disclosed herein should be understood to include any and all subranges included therein.

[0094] In the description of the present invention, the reference term "and / or" includes all and any combinations of one or more of the associated listed items.

[0095] In the description of the present invention, the description with reference to the terms "one embodiment", "some embodiments", "illustrative embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0096] In the description of the present invention, the reference term "amino acid" refers to the basic unit constituting protein, which gives the protein a specific molecular structure and makes its molecule have biochemical activity. For example, common "amino acids" include the following 20 natural amino acids: alanine (Ala or A), glycine (Gly or G), isoleucine (Ile or I), asparagine (Asn or N), arginine (Arg or R), lysine (Lys or K), cysteine ​​(Cys or C), aspartic acid (Asp or D), glutamic acid (Glu or E), glutamine (Gln or Q), histidine (His or H), leucine (Leu or L), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), valine (Val or V) and tyrosine (Tyr or Y).

[0097] In the description of the present invention, the polypeptide-based anti-fouling sensing interface is to assemble active polypeptides on the electrode surface, and utilize the charge carried by the active polypeptides and / or the steric hindrance of the formed nanostructures to block pollutants.

[0098] In the embodiment of the present invention, all electrochemical experiments were performed in a VersaSTAT3 electrochemical workstation. All electrochemical tests were performed in an electrochemical probe solution containing 5 mmol L -1 K3[Fe(CN)6] / K4[Fe(CN)6] and 0.2mol L -1 KCl in PBS. Where:

[0099] The preparation method of PBS includes: accurately weighing 35.8 g of disodium hydrogen phosphate (Na2HPO4·12H2O) and 15.6 g of sodium dihydrogen phosphate (NaH2PO4·2H2O), placing them in a 500 mL volumetric flask, and diluting to the mark with ultrapure water to obtain 0.2 mol L -1 Na2HPO4 solution and 0.2 mol L -1 NaH2PO4 solution. Use a measuring cylinder to measure 0.2 mol L -1324 mL of Na2HPO4 solution and 0.2 mol L - 1 76 mL of NaH2PO4 solution, then add 3.6 g of sodium chloride solid, stir and mix thoroughly to obtain 0.2 mol L -1 400 mL of PBS solution at pH 7.4 was placed at room temperature.

[0100] The preparation method of the electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]) includes: accurately weighing 164.62 mg of potassium ferrocyanide (K3[Fe(CN)6]) solid, 184.17 mg of potassium ferrocyanide (K4[Fe(CN)6]) and 1491.02 mg of potassium chloride (KCl). -1 PBS solution at pH 7.4 was diluted to 0.01 mol L -1 Then, 0.01 mol L -1 The above drugs were dissolved in PBS solution with pH 7.4 as solvent, and the volume was finally adjusted to 100 mL in a volumetric flask to obtain a bright yellow solution, that is, 5 mmol L -1 Electrochemical probe (K3[Fe(CN)6] / K4[Fe(CN)6], containing 0.2 mol L -1 Wrap the solution with tin foil and store it in a refrigerator at 4°C until use.

[0101] In the present invention, differential pulse voltammetry (DPV) was used to record the current response values ​​of the modified electrode before and after incubation (the electrical signal before incubation was I0, and the electrical signal after incubation was I), and the signal suppression rate (Signal Suppression (%) = [(I0-I) / I0] × 100) was calculated based on these values.

[0102] If the specific conditions are not specified in the examples, the experiments were carried out under conventional conditions or conditions recommended by the manufacturer. If the manufacturers of the reagents or instruments are not specified, they are all conventional products that can be purchased commercially.

[0103] Example 1: Construction of polypeptide-based antifouling sensing interface and verification of antifouling effect

[0104] 1. Construction of peptide-based antifouling sensing interface

[0105] (1) Preparation of antifouling peptide Pep4 modified working electrode:

[0106] The present invention uses angiotensin converting enzyme inhibitory peptide in cheese and α-glucosidase inhibitory peptide in egg yolk protein as branch sequences of anti-fouling polypeptides, and then uses cysteine ​​(C) as an anchor end and polyproline (PPPP) as a support to design an anti-fouling polypeptide Pep4 with an amino acid sequence of CPPPPEK(KSRE)DER). Its chemical structure is as follows:

[0107]

[0108] Furthermore, the anti-fouling polypeptide Pep4 is used as a modified sensing interface material to construct a polypeptide-based anti-fouling sensing interface, and the specific method is as follows:

[0109] Step S1, modification of nanoporous gold: immerse the polished glassy carbon electrode (GCE) in 4 mL of 1 mmol L - 1 HAuCl4 and 1mmol L -1 In the CuSO4 mixed solution, constant potential technology was used to carry out constant potential deposition of gold-copper alloy, in which the constant potential was set to 0.15V and the deposition time was 200s. After the deposition was completed, the working electrode was taken out; then the working electrode was rinsed with ultrapure water and immersed in 2mL of 0.5mol L -1 Copper was etched in H2SO4 solution, the constant potential was set to 1.0 V, the deposition time was 400 s, and after the deposition was completed, the working electrode was taken out and rinsed with ultrapure water to obtain NPG / GCE;

[0110] Step S2, modification of antifouling peptide: Suzhou Modifu Biotechnology Co., Ltd. was commissioned to synthesize the antifouling peptide Pep4 containing thiol groups and dissolve it in 0.02 mol L -1 The anti-fouling polypeptide solution was prepared by diluting the NPG / GCE prepared above into 150 μL of the anti-fouling polypeptide solution and incubating the solution at 4°C overnight to obtain a working electrode modified with the anti-fouling polypeptide Pep4 (hereinafter referred to as Pep4 / NPG / GCE), thereby completing the construction of the polypeptide-based anti-fouling sensing interface.

[0111] (2) Preparation of antifouling peptide Pep1 modified working electrode:

[0112] As a control group, this example further constructed another polypeptide-based anti-fouling sensing interface based on one branch of the polypeptide sequence Pep4, namely the anti-fouling polypeptide Pep1 (CPPPPKSRE-Ac, SEQ ID No. 1) as an anti-fouling polypeptide, and the specific method is as follows:

[0113] Step S1, preparing NPG / GCE by referring to the modification method of nanoporous gold;

[0114] Step S2: Suzhou Modifu Biotechnology Co., Ltd. was commissioned to synthesize the antifouling peptide Pep1 containing thiol groups, and the peptide was dissolved in 0.02 mol L -1 The antifouling polypeptide solution was prepared by dipping the NPG / GCE prepared above into 150 μL of the antifouling polypeptide solution and incubating the mixture at 4°C overnight to obtain a working electrode modified with the antifouling polypeptide Pep1 (hereinafter referred to as Pep1 / NPG / GCE).

[0115] (3) Preparation of antifouling polypeptide Pep2 modified working electrode:

[0116] As a control group, this example further constructed another polypeptide-based anti-fouling sensing interface based on another branch of the polypeptide sequence Pep4, namely the anti-fouling polypeptide Pep2 (NH2-CPPPPEKDER, SEQ ID No. 2) as an anti-fouling polypeptide, and the specific method is as follows:

[0117] Step S1, preparing NPG / GCE by referring to the modification method of nanoporous gold;

[0118] Step S2: entrust Suzhou Modifu Biotechnology Co., Ltd. to synthesize the antifouling polypeptide Pep2 containing thiol groups, and dissolve it in 0.02 mol L -1 The anti-fouling polypeptide solution was prepared by adding 150 μL of PBS buffer solution; the NPG / GCE prepared above was then immersed in 150 μL of the anti-fouling polypeptide solution and incubated overnight at 4°C to obtain a working electrode modified with the anti-fouling polypeptide Pep2 (hereinafter referred to as Pep2 / NPG / GCE).

[0119] (4) Preparation of antifouling polypeptide Pep3 modified working electrode:

[0120] As a control group, this example uses the most reported polypeptide, the anti-fouling polypeptide Pep3 (NH2-CPPPPEKEKEKE, SEQ ID No. 3) as the anti-fouling polypeptide, and further constructs another polypeptide-based anti-fouling sensing interface, and the specific method is as follows:

[0121] Step S1, preparing NPG / GCE by referring to the modification method of nanoporous gold;

[0122] Step S2: entrust Suzhou Modifu Biotechnology Co., Ltd. to synthesize the antifouling peptide Pep3 containing thiol groups, and dissolve it in 0.02 mol L -1 The anti-fouling polypeptide solution was prepared by adding PBS buffer solution; then the NPG / GCE prepared above was immersed in 150 μL of the anti-fouling polypeptide solution and incubated at 4°C overnight to obtain a working electrode modified with the anti-fouling polypeptide Pep3 (hereinafter referred to as Pep3 / NPG / GCE).

[0123] 2. Anti-fouling performance test

[0124] In order to more intuitively compare the anti-fouling ability of the above four anti-fouling peptides, this example refers to the construction method of the above peptide-based anti-fouling sensing interface, and the above four anti-fouling peptides containing thiol groups (Pep1, Pep2, Pep3 and Pep4) are dissolved in 0.02 mol L -1 PBS buffer solution was prepared into different concentration gradients (0.05 mg mL -1 , 0.1mg mL -1 , 0.2mgmL -1 , 0.3mg mL -1 , 0.4mg mL -1 ) anti-fouling polypeptide solution, and then immersed the NPG / GCE in 150 μL of the anti-fouling polypeptide solution and incubated overnight at 4°C to obtain the corresponding working electrodes modified with different concentrations of anti-fouling polypeptides.

[0125] Furthermore, the working electrodes modified with the different antifouling peptides obtained above were immersed in 1 mg mL -1 The working electrode to be tested was immersed in the electrochemical probe solution and scanned by DPV in the potential range of -0.2V to 0.6V with an amplitude of 50mV and a scan rate of 10mV S -1 , the current response values ​​before and after incubation were recorded to obtain the signal inhibition rate, and then the anti-fouling ability of these four peptides was more intuitively compared.

[0126] The test results are shown in Table 1 and Figure 1 As shown:

[0127] Table 1: Comparison of the antifouling ability of four antifouling peptides at different concentrations

[0128]

[0129] The results showed that compared with other antifouling peptide-modified working electrodes (such as Pep1 / NPG / GCE, Pep2 / NPG / GCE or Pep3 / NPG / GCE), the signal inhibition rate of Pep4 / NPG / GCE at different concentrations was always the lowest, indicating that it had the best antifouling ability, with a concentration of 0.2-0.4 mg mL -1 When the signal suppression rate was as low as 3.8%, considering the comprehensive cost and other factors, 0.2 mg mL -1 The concentration is used for the subsequent construction of antifouling sensing interface.

[0130] 3. Comparative analysis of signal suppression rate based on real samples

[0131] This section uses real samples as an example to detect the signal inhibition rate of Pep4 / NPG / GCE in milk, tofu and rice flour. The specific detection method is as follows:

[0132] Use 0.2 mol L -1 The milk was diluted to 1%, 2.5%, 5%, 10% and 25% with PBS; the tofu was mashed into a paste and dissolved with methanol / PBS solution (volume ratio of 1:4). After standing for 20 minutes, the supernatant was taken and diluted to 1%, 2.5%, 5%, 10% and 25%; after the rice flour was fully dissolved with methanol / PBS solution, centrifuged at 6000r / min for 10 minutes, the supernatant was taken and diluted to 1%, 2.5%, 5%, 10% and 25%. Then Pep4 / NPG / GCE were immersed in the above solutions and incubated for 30 minutes, and NPG / GCE was used as the control group for the same incubation. DPV scanning was used in the potential range of -0.2V to 0.6V, and the current response values ​​before and after incubation were recorded to obtain the signal inhibition rate.

[0133] The test results are shown in Table 2 and Figure 2 As shown:

[0134] Table 2: Signal suppression rate test results based on real samples

[0135]

[0136]

[0137] The results showed that after modification with the antifouling peptide Pep4, the signal inhibition rate of the working electrode in real samples was significantly reduced, with the signal inhibition rates of Pep4 / NPG / GCE in 1% milk, tofu and rice flour as low as 1.97%, 1.37% and 1.17%, respectively. In addition, even in 25% milk, tofu and rice flour, the signal inhibition rates were only 13.92%, 6.27% and 9.31%, which were much lower than the signal inhibition rates of NPG / GCE (87.59%, 69.04% and 71.99%).

[0138] Example 2: Construction of an antifouling electrochemical aptasensor for AFB1 detection and its performance testing

[0139] 1. Construction of anti-fouling electrochemical aptasensor

[0140] (1) Modification of nanoporous gold:

[0141] NPG / GCE was prepared by referring to the method of Example 1, specifically comprising immersing the polished GCE in 4 mL of 1 mmol L -1 HAuCl4 and 1mmol L -1In the CuSO4 mixed solution, constant potential technology was used to perform constant potential deposition of gold-copper alloy. The constant potential was set to 0.15 V and the deposition time was 200 s. After the deposition was completed, the working electrode was taken out; after rinsing with ultrapure water, the working electrode was immersed in 2 mL of 0.5 mol L -1 Copper was etched in H2SO4 solution, the constant potential was set to 1.0 V, and the deposition time was 400 s. After the deposition was completed, the working electrode was taken out and rinsed with ultrapure water to obtain NPG / GCE.

[0142] (2) Co-modification of aptamers and peptides:

[0143] The aptamer modified with a thiol group at the 5' end (such as AFB1 aptamer: 5'-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3', SEQ ID No. 4, which can also be replaced by other aptamers according to actual needs) was prepared into an aptamer-peptide solution using an anti-fouling polypeptide solution, and NPG / GCE was immersed in 150 μL of the aptamer-peptide solution and incubated overnight at 4°C to obtain a working electrode modified with an aptamer and a polypeptide (Apt-Pep4 / NPG / GCE).

[0144] (3) Preparation of electrochemical sensor:

[0145] The working electrode modified by the aptamer and the polypeptide prepared above is used as the working electrode, the platinum electrode is used as the counter electrode, and the saturated calomel electrode is used as the reference electrode to form an anti-fouling electrochemical aptamer sensor.

[0146] 2. Effect of aptamer concentration on signal inhibition rate

[0147] NPG / GCE was prepared by referring to the above method, and then the AFB1 aptamer modified with thiol at the 5' end (5'-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3', SEQ ID No. 4) was prepared into different concentration gradients (0.5 μmol L -1 , 1.0 μmol L -1 , 1.5 μmol L -1 , 2.0 μmol L -1 or 2.5 μmol L -1 ) aptamer-peptide solution. NPG / GCE was immersed in 150 μL of the above different aptamer-peptide solutions and incubated at 4°C overnight to obtain the corresponding working electrode (Apt-Pep4 / NPG / GCE) modified by aptamer and peptide.

[0148] Then 10 μL of 10 ng mL -1 The AFB1 standard solution was added dropwise onto Apt-Pep4 / NPG / GCE, and after incubation for 60 min, the unbound AFB1 was washed away with ultrapure water to form the working electrode to be detected. The working electrode to be detected was then immersed in the electrochemical probe solution and scanned by DPV in the potential range of -0.2 V to 0.6 V with an amplitude of 50 mV and a scan rate of 10 mV S -1 The signal inhibition rate was calculated by recording the DPV signals before and after the detection. The signal inhibition rate reflects the amount of AFB1 binding to Apt.

[0149] Test results such as Figure 3 As shown, the aptamer concentration was 1.5 μmol L -1 The signal inhibition rate was the highest and tended to be stable when 1.5 μmol L -1 Used as an antifouling electrochemical aptasensor for construction.

[0150] 3. Sensitivity test

[0151] The sensitivity of the anti-fouling electrochemical aptamer sensor constructed above was tested, and the specific method was as follows:

[0152] Take the antifouling electrochemical aptamer sensor constructed in the above steps and modify 10 μL of 0.001 ng mL -1 , 0.01ng mL -1 , 0.1ng mL -1 , 1ng mL -1 , and 10 ng mL -1 The AFB1 standard solution was incubated at room temperature for 60 min, and then the unbound AFB1 was washed away with ultrapure water to form the working electrode to be detected. The working electrode to be detected was then immersed in the electrochemical probe solution and scanned by DPV in the potential range of -0.2 V to 0.6 V with an amplitude of 50 mV and a scan rate of 10 mV S -1 , record the DPV response signals before and after detection, and calculate the signal inhibition rate.

[0153] Detection Figure 4 As shown, the standard curve obtained based on the signal inhibition rate and the logarithmic value of AFB1 concentration is y=4.84x+20.85 (R 2 =0.997, S / N=3), indicating that its detection limit is 0.26 pg mL -1 .

[0154] 4. Specificity detection

[0155] The specificity of the anti-fouling electrochemical aptamer sensor constructed above was detected, and the specific method was as follows:

[0156] 10 ng mL -1 of AFB1 and 100 ng mL -1 Aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone, vomitoxin and their mixtures were taken as detection objects and detected respectively using the anti-fouling electrochemical aptamer sensor prepared as above.

[0157] The results are as follows Figure 5 As shown, the signal inhibition rate is the highest when detecting aflatoxin B1, indicating that the binding amount of aflatoxin B1 is the highest; while the signal inhibition rates are very low when detecting aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone and vomitoxin, which are 3.11%, 2.49%, 3.41%, 2.83%, 4.11% and 3.79% respectively, indicating that the binding amount of these components is very small. It can be seen that the anti-fouling electrochemical aptamer sensor of the present invention has excellent selectivity for aflatoxin B1.

[0158] Example 3: Peptide-based antifouling electrochemical sensor for AFB1 detection in real samples

[0159] In this example, milk, tofu and rice flour were selected as samples to be tested, and the actual application performance of the anti-fouling electrochemical aptamer sensor for detecting AFB1 constructed above was verified in the samples to be tested by using a spike recovery experiment. The specific method is as follows:

[0160] Use 0.2 mol L -1 The milk was diluted to 1% with PBS; the tofu was mashed into a paste and dissolved with methanol / PBS solution (volume ratio of 1:4), and after standing for 20 minutes, the supernatant was taken and diluted to 1%; after the rice flour was fully dissolved with methanol / PBS solution, centrifuged at 6000r / min for 10 minutes, the supernatant was taken and diluted to 1%. Different concentrations (0.5ng mL -1 , 2.5ng mL -1 and 5.0 ng mL -1 ) of the AFB1 standard solution, and then use the anti-fouling electrochemical aptamer sensor for detecting AFB1 constructed above to detect it, and obtain the spike recovery rate in the food sample. At the same time, the spiked food sample is tested with an enzyme-linked immunosorbent assay kit to verify the results obtained by the constructed sensor to ensure the accuracy of the analysis.

[0161] The test results are shown in Table 3.

[0162] Table 3: AFB1 spike recovery results in three food samples

[0163]

[0164] The results show that the anti-fouling electrochemical aptamer sensor prepared by the present invention can sensitively detect AFB1 in the sample to be tested and has good detection accuracy. This method has broad application prospects in food matrix detection.

[0165] In summary, the present invention proposes a polypeptide-based anti-fouling electrochemical sensor and its construction method and application. The anti-fouling polypeptide designed by the present invention is inspired by the angiotensin converting enzyme inhibitory peptide in cheese and the α-glucosidase inhibitory peptide in egg yolk protein. Specifically, the present invention uses cysteine ​​as the anchor end, inserts polyproline to form an α-helical structure to give the entire peptide a supporting effect, and then uses the angiotensin converting enzyme inhibitory peptide and the α-glucosidase inhibitory peptide as branch sequences to prepare an anti-fouling sequence. The anti-fouling polypeptide designed by the present invention has a better anti-fouling effect than other anti-fouling polypeptides. The anti-fouling polypeptide is used to modify the working electrode and is used to prepare an anti-fouling electrochemical aptamer sensor for detecting AFB1, which can greatly improve the anti-pollutant interference effect of the sensor and help improve the accuracy and sensitivity of AFB1 detection. In addition, by changing the type of aptamer in the sensor, the detection of multiple targets can also be achieved.

[0166] The above is a detailed description of the embodiments of the present invention, but the present invention is not limited to the above embodiments. Various changes can be made within the knowledge of ordinary technicians in the relevant technical field without departing from the purpose of the present invention. In addition, the embodiments of the present invention and the features in the embodiments can be combined with each other without conflict.

Claims

1. An anti-fouling polypeptide, characterized in that The amino acid sequence of the antifouling polypeptide is: CPPPPEK(KSRE)DER.

2. Use of the antifouling polypeptide as claimed in claim 1 in antifouling materials.

3. A polypeptide-based antifouling electrode, characterized in that: The electrode surface is modified with the anti-fouling polypeptide as claimed in claim 1.

4. A method for preparing a polypeptide-based antifouling electrode, characterized in that: The following steps are involved: The anti-fouling polypeptide according to claim 1 is dissolved in a buffer to obtain an anti-fouling polypeptide solution, and then the electrode is contacted with the anti-fouling polypeptide solution and incubated to obtain the anti-fouling polypeptide solution.

5. The preparation method according to claim 4, characterized in that: The buffer is selected from any one of phosphate buffer, Tris-HCl buffer, sodium chloride buffer, and HEPES buffer; And / or, the concentration of the antifouling polypeptide solution is 0.05-1 mg mL -1 ; And / or, the material of the electrode is any one of gold, silver, platinum or glassy carbon; And / or, the incubation temperature is 2 to 10°C; And / or, the incubation time is 6 to 12 hours.

6. A polypeptide-based anti-fouling electrochemical aptamer sensor, characterized in that: include: A working electrode, wherein the working electrode is immobilized with an aptamer and the anti-fouling polypeptide according to claim 1; a counter electrode, wherein the counter electrode is selected from a platinum electrode; A reference electrode, wherein the reference electrode is selected from a saturated calomel electrode.

7. The polypeptide-based anti-fouling electrochemical aptamer sensor according to claim 6, characterized in that: The aptamer is a nucleic acid aptamer; Preferably, the aptamer is an aflatoxin B1 aptamer.

8. The method for constructing a polypeptide-based anti-fouling electrochemical aptamer sensor according to claim 6 or 7, characterized in that: The following steps are involved: S1. In a solvent, the aptamer and the anti-fouling polypeptide are mixed to prepare an aptamer-polypeptide solution, and then an electrode is contacted with the aptamer-polypeptide solution, and after incubation, a working electrode is obtained; S2. The working electrode is electrically connected to the counter electrode and the reference electrode.

9. An electrochemical detection method for aflatoxin B1, characterized in that: The method comprises using the polypeptide-based anti-fouling electrochemical aptamer sensor as described in claim 6 or 7 for detection, wherein the aptamer is an aflatoxin B1 aptamer.

10. The electrochemical detection method according to claim 9, characterized in that: The detection includes cyclic voltammetry detection and / or differential pulse voltammetry detection.

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