A polypeptide-based anti-fouling electrochemical sensor, its construction method and application

Abstract

The application discloses a polypeptide-based anti-fouling electrochemical sensor and a construction method and application thereof. In the application, cysteine is used as an anchoring end, an alpha-helix structure is formed by inserting polyproline to give a supporting effect to the whole peptide, then angiotensin converting enzyme inhibitory peptide (KSRE) and alpha-glucosidase inhibitory peptide (EKDER) are used as branches to prepare the anti-fouling polypeptide. The anti-fouling polypeptide has a better anti-fouling effect than other anti-fouling polypeptides, and the working electrode is modified by the anti-fouling polypeptide to prepare an anti-fouling electrochemical aptamer sensor for detecting AFB1, so that the anti-pollutant interference effect of the sensor can be greatly improved, the accuracy and sensitivity of AFB1 detection can be improved, and the anti-fouling electrochemical aptamer sensor has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of anti-fouling electrochemical sensor technology, in particular to a polypeptide-based anti-fouling electrochemical sensor and a construction method and application thereof. BACKGROUND

[0002] Aflatoxin is a kind of double furan ring toxin produced by Aspergillus flavus and parasitic Aspergillus strains. Due to the double bond at the end of the double furan ring, it is easy to be epoxidized to form an epoxide that destroys nucleic acid molecules, and has the hazards of carcinogenesis, teratogenesis and mutagenesis. There are about 20 derivatives of aflatoxin, among which aflatoxin B1 (AFB1) has the strongest toxicity and the strongest carcinogenicity. Since the pollution of AFB1 is widespread and the contaminated food will cause irreversible harm to animals and humans, qualitative and quantitative detection of AFB1 has always been the focus of research in the field of food safety.

[0003] In related technologies, 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, etc. Among them, electrochemical analysis technology, as a new technology, has great application prospects. It is to use the electrochemical properties of the measured substance or the specific reaction between the sensing electrode and the measured substance to generate corresponding electrochemical signals for rapid detection of the measured substance. This method has the advantages of simple operation, high sensitivity and fast detection speed. However, in the process of electrochemical detection, due to the complexity of the actual sample to be detected, non-target components in the food matrix will be non-specifically adsorbed on the sensing interface, which will seriously affect the sensitivity, accuracy and practicability of the sensor. Therefore, introducing anti-fouling materials on the surface of the sensor to improve the anti-fouling performance of the electrochemical sensor is the key to realizing the anti-matrix interference. At present, common anti-fouling materials include polyethylene glycol and its derivatives, polysaccharides, zwitterionic polymers and polypeptides, etc. Among them, polypeptide anti-fouling materials have attracted more and more attention due to their good biocompatibility, sequence adjustability and commercial synthesis, and polypeptides with different sequences and structures have been designed and used for modification of anti-fouling sensing interface. However, conventional polypeptide anti-fouling materials have low anti-fouling ability and are difficult to achieve ideal anti-fouling effect in the detection of AFB1.

[0004] Therefore, the present application designs a new type of polypeptide modified anti-fouling electrochemical aptamer sensor with the advantages of low detection limit, good selectivity and high sensitivity, which can greatly improve the detection efficiency when applied to the detection of AFB1. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application provides an anti-fouling polypeptide. The present application takes cysteine as an anchor end, gives the whole peptide a supporting effect by inserting polyproline to form an alpha-helix structure, and then prepares an anti-fouling sequence by taking angiotensin converting enzyme inhibitor peptide and alpha-glucosidase inhibitor peptide as branch sequences. Based on the steric hindrance effect, the anti-fouling polypeptide has a more optimal anti-fouling effect than other anti-fouling polypeptides. Moreover, the anti-fouling polypeptide is used to modify a 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 to improve the accuracy and sensitivity of AFB1 detection.

[0006] The present application also provides an anti-fouling polypeptide for use in anti-fouling materials.

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

[0008] The present application also provides a preparation method of a polypeptide-based anti-fouling electrode.

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

[0010] The present application also provides a construction method of a polypeptide-based anti-fouling electrochemical aptamer sensor.

[0011] The present application also provides an electrochemical detection method of aflatoxin B1.

[0012] In a first aspect of the present application, an anti-fouling polypeptide is provided, and the amino acid sequence of the anti-fouling polypeptide is as follows:

[0013] CPPPPEK(KSRE)DER.

[0014] According to the anti-fouling polypeptide of the embodiments of the present application, at least the following beneficial effects are achieved:

[0015] The present application takes cysteine as an anchor end, gives the whole peptide a supporting effect by inserting polyproline to form an alpha-helix structure, and then prepares an anti-fouling sequence by taking angiotensin converting enzyme inhibitor peptide and alpha-glucosidase inhibitor peptide as branch sequences. The anti-fouling polypeptide of the present application has a more optimal anti-fouling effect than other anti-fouling polypeptides.

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

[0017] In some embodiments of the present application, the chemical structure of the anti-fouling polypeptide is as follows:

[0018]

[0019] In a second aspect of the present application, the anti-fouling polypeptide of the first aspect is provided for use in an anti-fouling material.

[0020] In a third aspect of the present application, a polypeptide-based anti-fouling electrode is provided, wherein the electrode is surface-modified with the anti-fouling polypeptide of the first aspect.

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

[0022] In some embodiments of the present application, the material of the electrode is 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 application, 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, with a platinum electrode as a counter electrode and a saturated calomel electrode as a reference electrode, for constant potential deposition; then after washing, it is placed in an H2SO4 solution for etching treatment, and after taking out, it is washed to obtain the electrode.

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

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

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

[0029] In some embodiments of the present application, the time for constant potential deposition is 100-300 s.

[0030] In some embodiments of the present application, the constant potential for etching treatment is 0.8-1.5 V.

[0031] In some embodiments of the present application, the time for etching treatment is 200-600 s.

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

[0033] In some embodiments of the present application, the method for modification comprises connecting the thiol group on the cysteine of the anti-fouling polypeptide to the Au of the electrode through self-assembly.

[0034] In a fourth aspect of the present application, a method for preparing an anti-fouling polypeptide-based electrode is provided, comprising the following steps:

[0035] The anti-fouling polypeptide of 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 the electrode is obtained after incubation.

[0036] In some embodiments of the present application, 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 application, the buffer is phosphate buffer.

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

[0039] In some preferred embodiments of the present application, the concentration of the anti-fouling polypeptide solution is 0.2-0.5 mg mL -1 .

[0040] In some embodiments of the present application, 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 application, 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, a platinum electrode is used as a counter electrode, and a saturated calomel electrode is used as a reference electrode to perform constant potential deposition; then the electrode is placed in an H2SO4 solution for etching treatment after washing, and the electrode is obtained after washing.

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

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

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

[0047] In some embodiments of the present application, the time of the potentiostatic deposition is 100-300 s.

[0048] In some embodiments of the present application, the potential of the etching treatment is 0.8-1.5 V.

[0049] In some embodiments of the present application, the time of the etching treatment is 200-600 s.

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

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

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

[0053] In some embodiments of the present application, the time of the incubation is 6-12 h.

[0054] In a fifth aspect of the present application, a polypeptide-based anti-fouling electrochemical aptamer sensor is provided, comprising:

[0055] a working electrode, wherein the working electrode is fixed with an aptamer and an anti-fouling polypeptide according to 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 embodiments of the present application has at least the following beneficial effects: the polypeptide-based anti-fouling electrochemical aptamer sensor of the present application has excellent anti-fouling effect.

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

[0060] In some embodiments of the present application, the material of the working electrode is 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 application, the aptamer is a nucleic acid aptamer.

[0063] In some embodiments of the present application, the aptamer includes but is not limited to an 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 application, the nucleotide sequence of the AFB1 aptamer is as follows:

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

[0066] In a sixth aspect of the present application, a method for constructing a polypeptide-based anti-fouling electrochemical aptamer sensor is provided, and the method comprises the following steps:

[0067] S1, mixing the aptamer and the anti-fouling polypeptide in a solvent to prepare an aptamer-polypeptide solution, then contacting an electrode with the aptamer-polypeptide solution, and incubating to obtain a working electrode;

[0068] S2, electrically connecting the working electrode with a counter electrode and a reference electrode, and the method is completed.

[0069] The polypeptide-based anti-fouling electrochemical aptamer sensor construction method according to the embodiments of the present application has at least the following beneficial effects:

[0070] The polypeptide-based anti-fouling electrochemical aptamer sensor construction method according to the embodiments of the present application has at least the following beneficial effects: -1 -1 -1 -1 -1

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

[0072] In some embodiments of the present application, the concentration of the aptamer in the aptamer-polypeptide solution is 0.1-5 μmol / L. -1

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

[0074] ​​​In some embodiments of the present application, the concentration of the anti-fouling polypeptide in the aptamer-polypeptide solution is 0.05-1 mg / mL -1 .

[0075] In some preferred embodiments of the present application, the concentration of the anti-fouling polypeptide is 0.1-0.5 mg / mL -1 .

[0076] In some embodiments of the present application, the temperature of the incubation is 2-10℃.

[0077] In some preferred embodiments of the present application, the temperature of the incubation is 2-5℃.

[0078] In some embodiments of the present application, the time of the incubation is 6-12 h.

[0079] In a seventh aspect of the present application, an electrochemical detection method of AFB1 is provided, which comprises 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 embodiments of the present application has at least the following beneficial effects: the AFB1 electrochemical detection method of the present application is simple and easy to miniaturize, wherein the linear range of the target AFB1 is 0.001 ng / mL -1 -10 ng / mL -1 , the detection limit is 0.26 pg / mL -1 , and the method has excellent selectivity, sensitivity and accuracy.

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

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

[0083] In some embodiments of the present application, 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 application will be described in the following description. BRIEF DESCRIPTION OF DRAWINGS

[0085] The present application will be further described below in conjunction with the accompanying drawings and examples, in which:

[0086] Figure 1 Results of the anti-fouling ability detection of the four anti-fouling polypeptide-modified electrodes in bovine serum albumin solution;

[0087] Figure 2 The anti-fouling performance detection results of the electrode modified by the anti-fouling polypeptide of the application in real food samples with different dilution times;

[0088] Figure 3 The detection results of the influence of different concentrations of aptamers on signal inhibition rates;

[0089] Figure 4 The sensitivity detection results of the anti-fouling electrochemical aptamer sensor of the application, 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 concentration of AFB1 and the signal inhibition rate;

[0090] Figure 5 The specificity detection results of the anti-fouling electrochemical aptamer sensor of the embodiment of the application. DETAILED DESCRIPTION

[0091] The concept and the technical effects generated by the application will be described below in combination with embodiments, so as to fully understand the purpose, features and effects of the application. Obviously, the described embodiments are only part of the embodiments of the application, but not all the embodiments. Based on the embodiments of the application, other embodiments obtained by those skilled in the art without creative labor are within the protection scope of the application.

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

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

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

[0095] In the description of the application, the description of the term "one embodiment", "some embodiments", "exemplary embodiment", "example", "specific example", or "some examples" means that the specific features, structures, materials, or characteristics described in connection with the embodiment or example are included in at least one embodiment or example of the application. In this specification, the exemplary description of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0096] In the description of the application, the term "amino acid" refers to the basic unit that constitutes a protein, which gives the protein a specific molecular structure form, so that its molecule has biochemical activity. For example, the common "amino acid" includes 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 application, the polypeptide-based anti-fouling sensing interface is to assemble active polypeptides on the surface of the electrode, and to block pollutants by using the charge carried by the active polypeptides and / or the steric hindrance of the nanostructure formed.

[0098] In the embodiment of the application, all electrochemical experiments are carried out in a VersaSTAT3 electrochemical workstation. All electrochemical tests are carried out in an electrochemical probe solution, which is a PBS containing 5 mmol L -1 K3[Fe(CN)6] / K4[Fe(CN)6] and 0.2 mol L -1 KCl PBS. Wherein:

[0099] The preparation method of PBS includes: accurately weighing 35.8 g of sodium 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. Respectively, 0.2 mol L -1Na2HPO4 solution 324 mL and 0.2 mol / L - 1 NaH2PO4 solution 76 mL, then add sodium chloride solid 3.6 g, after stirring well, 0.2 mol / L -1 pH 7.4 PBS solution 400 mL, room temperature.

[0100] The preparation method of the electrochemical probe solution (K3[Fe(CN)6] / K4[Fe(CN)6]) comprises the following steps: accurately weighing potassium ferricyanide (K3[Fe(CN)6]) solid 164.62 mg, potassium ferrocyanide (K4[Fe(CN)6]) 184.17 mg and potassium chloride (KCl) 1491.02 mg. 0.2 mol / L -1 pH 7.4 PBS solution is diluted to 0.01 mol / L -1 then 0.01 mol / L -1 pH 7.4 PBS solution is used as a solvent to dissolve the above-mentioned drugs, and finally the volume is made up to 100 mL in a volumetric flask to obtain a bright yellow solution, that is, 5 mmol / L -1 The electrochemical probe (K3[Fe(CN)6] / K4[Fe(CN)6], containing 0.2 mol / L -1 KCl) solution, wrapped with tin foil paper, stored in a 4℃ refrigerator for standby use.

[0101] In the present application, the differential pulse voltammetry (DPV) is used to record the current response values before and after the incubation of the modified electrode (the electrical signal before incubation is I0, and the electrical signal after incubation is I), and the signal suppression rate (Signal Suppression (%) = [(I0-I) / I0]x100) is calculated through these values.

[0102] The specific conditions not mentioned in the examples are carried out according to the conventional conditions or the conditions recommended by the manufacturer. The reagents or instruments used are not marked with the manufacturer, which are all conventional products that can be purchased on the market.

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

[0104] 1. Construction of polypeptide-based anti-fouling sensing interface

[0105] (1) Preparation of anti-fouling polypeptide Pep4 modified working electrode:

[0106] The application takes angiotensin converting enzyme inhibitory peptides in cheese and alpha-glucosidase inhibitory peptides in egg yolk protein as branch sequences of anti-fouling polypeptides respectively, and designs an anti-fouling polypeptide Pep4 with an amino acid sequence of CPPPPEK(KSRE)DER and a chemical structural formula as follows by taking cysteine (C) as an anchoring end and polyproline (PPPP) as a support:

[0107]

[0108] Further, 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 a well-polished glassy carbon electrode (GCE) in 4 mL of a mixed solution containing 1 mmol L - 1 HAuCl4 and 1 mmol L -1 CuSO4, perform constant potential deposition of gold-copper alloy by adopting a constant potential technology, set the constant potential to be 0.15 V, and the deposition time is 200 s; after the deposition is completed, take out the working electrode; then immerse the working electrode in 2 mL of a 0.5 mol L -1 H2SO4 solution for copper etching, set the constant potential to be 1.0 V, and the deposition time is 400 s; after the deposition is completed, take out the working electrode, wash it with ultrapure water, and obtain NPG / GCE;

[0110] Step S2, modification of anti-fouling polypeptide: entrust Suzhou Modiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep4 containing a mercapto group, and dissolve it in 0.02 mol L -1 of PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, incubate overnight at 4°C, obtain a working electrode modified by the anti-fouling polypeptide Pep4 (hereinafter referred to as Pep4 / NPG / GCE), and the construction of the polypeptide-based anti-fouling sensing interface is completed.

[0111] (2) Preparation of a working electrode modified by the anti-fouling polypeptide Pep1:

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

[0113] Step S1, refer to the above-mentioned modification method of nanoporous gold to prepare NPG / GCE;

[0114] Step S2, entrust Suzhou Merdiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep1 containing sulfhydryl, and dissolve it in 0.02 mol / L PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, and incubate at 4°C overnight to obtain a working electrode modified by the anti-fouling polypeptide Pep1 (hereinafter referred to as Pep1 / NPG / GCE). -1 Step S2, entrust Suzhou Merdiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep1 containing sulfhydryl, and dissolve it in 0.02 mol / L PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, and incubate at 4°C overnight to obtain a working electrode modified by the anti-fouling polypeptide Pep1 (hereinafter referred to as Pep1 / NPG / GCE).

[0115] (3) Preparation of working electrode modified by anti-fouling polypeptide Pep2:

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

[0117] Step S1, refer to the above-mentioned modification method of nano-porous gold to prepare NPG / GCE;

[0118] Step S2, entrust Suzhou Merdiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep2 containing sulfhydryl, and dissolve it in 0.02 mol / L PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, and incubate at 4°C overnight to obtain a working electrode modified by the anti-fouling polypeptide Pep2 (hereinafter referred to as Pep2 / NPG / GCE). -1 Step S2, entrust Suzhou Merdiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep2 containing sulfhydryl, and dissolve it in 0.02 mol / L PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, and incubate at 4°C overnight to obtain a working electrode modified by the anti-fouling polypeptide Pep2 (hereinafter referred to as Pep2 / NPG / GCE).

[0119] (4) Preparation of working electrode modified by anti-fouling polypeptide Pep3:

[0120] As a control group, the most reported polypeptide at present, i.e., the anti-fouling polypeptide Pep3 (NH2-CPPPPEKEKEKE, SEQ ID No. 3) is used as an anti-fouling polypeptide to further construct another polypeptide-based anti-fouling sensing interface, and the specific method is as follows:

[0121] Step S1, refer to the above-mentioned modification method of nano-porous gold to prepare NPG / GCE;

[0122] Step S2, entrust Suzhou Merdiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep3 containing sulfhydryl, and dissolve it in 0.02 mol / L PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, and incubate at 4°C overnight to obtain a working electrode modified by the anti-fouling polypeptide Pep3 (hereinafter referred to as Pep3 / NPG / GCE). -1 Step S2, entrust Suzhou Merdiv Biological Technology Co., Ltd. to synthesize the anti-fouling polypeptide Pep3 containing sulfhydryl, and dissolve it in 0.02 mol / L PBS buffer solution to prepare an anti-fouling polypeptide solution; then immerse the prepared NPG / GCE in 150 μL of the anti-fouling polypeptide solution, and incubate at 4°C overnight to obtain a working electrode modified by the anti-fouling polypeptide Pep3 (hereinafter referred to as Pep3 / NPG / GCE).

[0123] 2. Antifouling performance test

[0124] To more intuitively compare the antifouling capabilities of the four antifouling peptides, this embodiment refers to the above-mentioned method for constructing peptide-based antifouling sensing interfaces, and dissolves the four antifouling peptides (Pep1, Pep2, Pep3, and Pep4) containing thiol groups in 0.02 mol / L solutions. -1 Different concentration gradients (0.05 mg / mL) were prepared in PBS buffer solution. -1 0.1 mg mL -1 0.2 mg / mL -1 0.3 mg mL -1 0.4 mg mL -1 The antifouling peptide solution was prepared, and then NPG / GCE was immersed in 150 μL of the antifouling peptide solution and incubated overnight at 4 °C to obtain the corresponding working electrodes modified with different concentrations of antifouling peptides.

[0125] Furthermore, the working electrodes modified with the different antifouling peptides obtained above were immersed in 1 mg mL of water. -1 Bovine serum albumin was incubated for 30 min. The working electrode to be detected was immersed in the electrochemical probe solution, and DPV scanning was performed within a 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 obtained by recording the current response value before and after incubation, which allowed for a more intuitive comparison of the antifouling ability of these four peptides.

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

[0127] Table 1: Comparison of antifouling capabilities 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), Pep4 / NPG / GCE consistently exhibited the lowest signal inhibition rate at different concentrations, indicating its superior antifouling ability. The optimal concentration range was 0.2–0.4 mg / mL. -1 At that time, the signal inhibition rate was as low as 3.8%. Considering factors such as overall cost, 0.2 mg / mL was used. -1 The concentration is used for the subsequent construction of the antifouling sensing interface.

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

[0131] This part takes real samples as examples to detect the signal inhibition rate of Pep4 / NPG / GCE in milk, bean curd and rice powder. The specific detection method is as follows:

[0132] Dilute milk with 0.2 mol L -1 of PBS by 1%, 2.5%, 5%, 10% and 25%; dissolve bean curd into paste, and then dissolve it with methanol / PBS solution (volume ratio 1:4), after 20 min, take the supernatant and dilute it by 1%, 2.5%, 5%, 10% and 25%; dissolve rice powder with methanol / PBS solution, then centrifuge at 6000 r / min for 10 min, take the supernatant and dilute it by 1%, 2.5%, 5%, 10% and 25%. Then immerse Pep4 / NPG / GCE in the above solutions for 30 min, and immerse NPG / GCE as a control group for the same incubation. Scan with DPV in the potential range of-0.2V-0.6V, and record the current response values before and after incubation to obtain the signal inhibition rate.

[0133] The detection results are shown in Table 2 and Figure 2

[0134] Table 2: Signal inhibition rate detection results based on real samples

[0135]

[0136]

[0137] The results show that after modification by the anti-fouling polypeptide Pep4, the signal inhibition rate of the working electrode in real samples is significantly reduced, and the signal inhibition rate of Pep4 / NPG / GCE in 1% milk, bean curd and rice powder is as low as 1.97%, 1.37% and 1.17% respectively. In addition, even in 25% milk, bean curd and rice powder, the signal inhibition rate is only 13.92%, 6.27% and 9.31%, which is much lower than that of NPG / GCE (87.59%, 69.04% and 71.99%).

[0138] Example 2: Construction of anti-fouling electrochemical aptamer sensor for AFB1 detection and performance detection thereof

[0139] 1. Construction of anti-fouling electrochemical aptamer sensor

[0140] (1) Modification of nano-porous gold:

[0141] Prepare NPG / GCE according to the method of Example 1 above, which specifically includes immersing the polished GCE in 4 mL of 1 mmol L -1 of HAuCl4 and 1 mmol L -1 ​CuSO4 mixed solution, constant potential technology was used for constant potential deposition of gold-copper alloy, constant potential was set as 0.15V, deposition time was 200s, and the working electrode was taken out after deposition; the working electrode was rinsed with ultrapure water and then immersed into 2mL of 0.5mol / L -1 H2SO4 solution for copper etching, constant potential was set as 1.0V, deposition time was 400s, the working electrode was taken out after deposition and rinsed with ultrapure water, and NPG / GCE was obtained.

[0142] (2) Co-modification of aptamer and polypeptide:

[0143] The aptamer (such as AFB1 aptamer: 5'-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3', SEQ ID No. 4) modified with thiol at the 5' end was prepared into aptamer-polypeptide solution with anti-fouling polypeptide solution, the NPG / GCE was immersed into 150μL of the aptamer-polypeptide solution for overnight incubation at 4℃, and the working electrode co-modified with aptamer and polypeptide (Apt-Pep4 / NPG / GCE) was obtained.

[0144] (3) Preparation of electrochemical sensor:

[0145] The above working electrode co-modified with aptamer and polypeptide was used as working electrode, platinum electrode was used as counter electrode, and saturated calomel electrode was used as reference electrode, to jointly constitute an anti-fouling electrochemical aptamer sensor.

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

[0147] NPG / GCE was prepared according to the above method, and then the above AFB1 aptamer (5'-SH-(CH2)6-GTTGGGCACGTGTTGTCTCTCTGTGTCTCGTGCCCTTCGCTAGGCCCACA-3', SEQ ID No. 4) modified with thiol at the 5' end was prepared into aptamer-polypeptide solution with different concentration gradients (0.5μmol / L -1 , 1.0μmol / L -1 , 1.5μmol / L -1 , 2.0μmol / L -1 or 2.5μmol / L -1 ) of anti-fouling polypeptide solution. The NPG / GCE was immersed into 150μL of the above different aptamer-polypeptide solutions for overnight incubation at 4℃, and the corresponding working electrode co-modified with aptamer and polypeptide (Apt-Pep4 / NPG / GCE) was obtained.

[0148] Next, add 10 μL to 10 ng mL. -1 AFB1 standard solution was added dropwise to Apt-Pep4 / NPG / GCE and incubated for 60 min. Unbound AFB1 was then rinsed off with ultrapure water to form the working electrode for detection. The working electrode was then immersed in an electrochemical probe solution, and DPV scanning was performed within a potential range of -0.2 V to 0.6 V at 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 detection. The signal inhibition rate reflects the amount of AFB1 binding to Apt.

[0149] Test results as follows Figure 3 As shown, the aptamer concentration is 1.5 μmol L. -1 The signal suppression rate was highest and tended to stabilize at this time. Subsequently, 1.5 μmol L⁻¹ was selected. -1 Used in the construction of antifouling electrochemical aptamer sensors.

[0150] 3. Sensitivity Detection

[0151] The sensitivity of the constructed antifouling electrochemical aptamer sensor was tested using the following method:

[0152] Take the antifouling electrochemical aptamer sensor constructed in the above steps, and modify its surface with 10 μL of 0.001 ng / mL solution. -1 0.01 ng mL -1 0.1 ng mL -1 1 ng mL -1 and 10 ng mL -1 The AFB1 standard solution was incubated at room temperature for 60 min, and then the loosely bound AFB1 was rinsed off with ultrapure water to form the working electrode to be detected. The working electrode was then immersed in the electrochemical probe solution, and DPV scanning was performed within a potential range of -0.2 V to 0.6 V at 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 suppression rate.

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

[0154] 4. Specific detection

[0155] The specificity of the constructed antifouling electrochemical aptamer sensor was tested using the following method:

[0156] At 10 ng mL -1 AFB1 and 100 ng mL -1 Aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone, vomitoxin, and mixtures thereof were used as detection targets, and the antifouling electrochemical aptamer sensor prepared above was used for detection respectively.

[0157] The results are as follows Figure 5 As shown, the signal inhibition rate was highest when detecting aflatoxin B1, indicating that the binding amount of aflatoxin B1 was the highest. In contrast, the signal inhibition rates for detecting aflatoxin B2, aflatoxin G1, aflatoxin G2, ochratoxin A, zearalenone, and vomitoxin were very low, at 3.11%, 2.49%, 3.41%, 2.83%, 4.11%, and 3.79%, respectively, indicating that the binding amounts of these components were very small. This demonstrates that the antifouling electrochemical aptamer sensor of this invention exhibits excellent selectivity for aflatoxin B1.

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

[0159] In this embodiment, milk, tofu, and rice flour were selected as test samples. A spiked recovery experiment was used to verify the practical application performance of the constructed antifouling electrochemical aptamer sensor for detecting AFB1 in the test samples. The specific method is as follows:

[0160] Use 0.2 mol L -1 Milk was diluted 1% with PBS; tofu was mashed into a paste, dissolved in methanol / PBS solution (volume ratio 1:4), allowed to stand for 20 min, and then the supernatant was taken and diluted 1%; rice flour was thoroughly dissolved in methanol / PBS solution, centrifuged at 6000 r / min for 10 min, and the supernatant was taken and diluted 1%. Different concentrations (0.5 ng / mL) of PBS were added to the three diluted food samples. -1 2.5 ng mL -1 and 5.0 ng mL -1 AFB1 standard solution was prepared and then detected using the antifouling electrochemical aptamer sensor constructed above for AFB1 detection. The spiked recovery rate in food samples was obtained. Simultaneously, the spiked food samples were tested using an enzyme-linked immunosorbent assay (ELISA) kit to verify the results obtained by the constructed sensor and ensure the accuracy of the analysis.

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

[0162] Table 3: The results of the recovery rate of AFB1 in three food samples

[0163]

[0164] The results show that the anti-fouling electrochemical aptamer sensor prepared by the method can be used for sensitive detection of AFB1 in the sample, and has good detection accuracy, and the method has wide application prospect in food matrix detection.

[0165] To sum up, the application provides a polypeptide-based anti-fouling electrochemical sensor and a construction method and application thereof. The anti-fouling polypeptide is inspired by angiotensin converting enzyme inhibitory peptides in cheese and alpha-glucosidase inhibitory peptides in egg yolk proteins. Specifically, the application uses cysteine as an anchor end, forms an alpha-helix structure by inserting polyproline to give the whole peptide a supporting effect, and then prepares an anti-fouling sequence by using angiotensin converting enzyme inhibitory peptides and alpha-glucosidase inhibitory peptides as branch sequences. The anti-fouling polypeptide has a better anti-fouling effect than other anti-fouling polypeptides. The working electrode is modified by the anti-fouling polypeptide 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 to improve the accuracy and sensitivity of AFB1 detection. In addition, by changing the type of aptamer in the sensor, detection of a plurality of target substances can be realized.

[0166] The above has described the embodiments of the application in detail, but the application is not limited to the above embodiments. Within the scope of knowledge possessed by those skilled in the art, various changes can be made without departing from the purpose of the application. In addition, the embodiments of the application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A soil- resistant polypeptide, characterized in that, The chemical structural formula of the antifouling polypeptide is as follows: 。 2. Use of the antifouling polypeptide of claim 1 in the preparation of an antifouling material.

3. A polypeptide-based antifouling electrode, characterized in that, The electrode is modified with the antifouling polypeptide of claim 1.

4. A method for preparing a polypeptide-based antifouling electrode, characterized by, The method comprises the following steps: The antifouling polypeptide of claim 1 is dissolved in a buffer solution to obtain an antifouling polypeptide solution, and then the electrode is contacted with the antifouling polypeptide solution, and after incubation, the electrode is obtained.

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

6. A polypeptide-based anti-fouling electrochemical aptamer sensor, characterized in that, It comprises: A working electrode, the working electrode is fixed with an aptamer and the antifouling polypeptide of claim 1; A counter electrode, the counter electrode is selected from a platinum electrode; A reference electrode, the reference electrode is selected from a saturated calomel electrode.

7. The polypeptide-based anti-fouling electrochemical aptamer sensor of claim 6, wherein, The aptamer is a nucleic acid aptamer.

8. The polypeptide-based anti-fouling electrochemical aptamer sensor of claim 7, wherein, The aptamer is an aflatoxin B1 aptamer.

9. The method of constructing a polypeptide-based antifouling electrochemical aptamer sensor according to any one of claims 6 to 8, wherein, The method comprises the following steps: S1, in a solvent, the aptamer is mixed with the antifouling polypeptide to prepare an aptamer-polypeptide solution, then the electrode is contacted with the aptamer-polypeptide solution, and after incubation, a working electrode is obtained; S2, the working electrode is electrically connected with the counter electrode and the reference electrode, and the electrode is obtained.

10. An electrochemical method for detecting aflatoxin B1 for non-disease diagnostic purposes, characterized by, The detection comprises cyclic voltammetry detection and / or differential pulse voltammetry detection.

11. The electrochemical detection method according to claim 10, wherein, The detection comprises cyclic voltammetry detection and / or differential pulse voltammetry detection.

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