Anti-pollution electrochemical immunosensor based on self-assembled gold nanoparticle-polypeptide hydrogel and preparation method and application thereof

Abstract

This invention relates to an antifouling electrochemical immunosensor based on a self-assembled gold nanoparticle-peptide hydrogel and its preparation method, belonging to the fields of electrochemical sensors and in vitro disease diagnostics. This invention discloses a method for constructing an antifouling electrochemical sensor with high sensing performance and detection accuracy by modifying electrodes with a self-assembled peptide hydrogel. Because the prepared self-assembled gold nanoparticle-peptide hydrogel modified interface has superior antifouling properties and biocompatibility, and accelerates electron and ion transport, it is expected to have wide applications in electrode biomaterials, flexible materials, or electrochemical sensing.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of electrochemical detection, and particularly relates to an anti-pollution electrochemical immunosensor based on self-assembled gold nanoparticles-polypeptide hydrogel, a preparation method thereof and application thereof in quantitative detection of biological small molecules. BACKGROUND

[0002] With the development of economy and the improvement of people's living standards, people pay more and more attention to physical health. Early diagnosis of common diseases has also attracted widespread attention, and early detection, early diagnosis and early treatment of diseases will effectively reduce disease mortality and the degree of pain of patients. Electrochemical sensors are widely used in early diagnosis of diseases due to their low cost and high sensitivity. However, in actual clinical application, the electrochemical sensor constructed must still have high selectivity in complex biological environment (such as human serum) to be able to resist the non-specific adsorption of interfering proteins in complex biological fluids. The common solution is to construct an anti-pollution sensing interface, so that the surface has high hydrophilicity and electrical neutrality to resist the non-specific adsorption of proteins. Polyethylene glycol and zwitterionic polymer are the most commonly used anti-pollution materials, but polyethylene glycol is easily oxidized and degraded under physiological conditions, limiting its long-term application stability. Zwitterionic polymers, such as polycarboxybetaine and polysulfobetaine polymers, have a relatively complex synthesis process. In addition, the current anti-pollution electrochemical sensing interface depends on the modification of non-conductive anti-pollution materials on the electrode surface, which is equivalent to passivating the electrode surface to obtain excellent anti-pollution ability, which inevitably loses the detection sensitivity.

[0003] Many mental and neurodegenerative diseases are directly related to abnormal neurotransmitter metabolism in the brain. Neurological diseases such as Parkinson's disease, Alzheimer's disease, schizophrenia and drug addiction are related to abnormal dopamine levels, and dopamine is a primary catecholamine neurotransmitter molecule. Similar to the effects of other neurotransmitters, the effect of dopamine depends on its overall concentration level. Therefore, sensitive and selective in vitro detection of dopamine is one of the most challenging tasks in neuroscience.

[0004] Polypeptide is a natural zwitterion, which can form polypeptide hydrogel with high water content through self-assembly. Polypeptide hydrogel has good biocompatibility and chemical stability, and is simple to prepare. Polypeptide hydrogel produces strong surface hydration through hydrogen bonding and ionic solvation, which creates an additional energy barrier for non-specific adsorption of interfering proteins and other molecules. And polypeptide as a natural zwitterion makes the overall charge neutral, the hydrophilicity and electrical neutrality of polypeptide hydrogel can effectively resist the hydrophobic interaction and electrostatic interaction of non-specific adsorption of interfering proteins, thereby having excellent anti-pollution performance. In addition, the three-dimensional network structure of hydrogel can accelerate electron transfer and ion transfer, and the supramolecular pi-pi stacking backbone formed by the aromatic residues of the self-designed polypeptide may provide effective intermolecular electron delocalization, which will promote charge transfer and increase electrical conductivity. Polypeptide and gold nanoparticles are blended and self-assembled into hydrogel, and the gold nanoparticles are connected with the cysteine of polypeptide through gold-sulfur bond, which ensures that the gold nanoparticles are uniformly dispersed on the polypeptide hydrogel, thereby effectively improving the detection sensitivity. The polypeptide hydrogel containing gold nanoparticles is drop-coated on the surface of the electrode for electrochemical detection of biological small molecules, which avoids the complex modification process and the layer-by-layer accumulation of various functional materials. Polypeptide is used to resist biological pollution, which has certain application in the field of electrochemical sensing, but there is no report on the application of self-assembled polypeptide hydrogel-gold nanoparticles for simultaneously improving detection sensitivity and accuracy. SUMMARY

[0005] Therefore, the application designs a preparation method of self-assembled gold nanoparticle-polypeptide hydrogel and an anti-pollution electrochemical sensor based on the same.

[0006] It should be noted that the commonly used solution is to construct an anti-pollution sensing interface, so that the surface has high hydrophilicity and electrical neutrality to resist non-specific adsorption of proteins. Polyethylene glycol and zwitterionic polymer are the most commonly used anti-pollution materials, but polyethylene glycol is easily oxidized and degraded under physiological conditions, which limits its long-term application stability. Zwitterionic polymers, such as polycarboxybetaine and polysulfobetaine polymers, have a complex synthesis process. In addition, the current anti-pollution electrochemical sensing interface relies on the modification of non-conductive anti-pollution materials on the electrode surface, which is equivalent to passivating the electrode surface to obtain excellent anti-pollution ability, which inevitably loses the detection sensitivity.

[0007] In order to achieve the above purpose, the application discloses an anti-pollution electrochemical sensor with high sensing performance and strong detection accuracy, which is constructed by modifying an electrode with self-assembled polypeptide hydrogel, and specifically adopts the following technical scheme:

[0008] An anti-pollution electrochemical immunosensor based on self-assembled gold nanoparticle-polypeptide hydrogel, the sensor is an anti-pollution electrochemical sensor constructed by modifying an electrode with self-assembled polypeptide hydrogel; and,

[0009] The polypeptide sequence is: Fmoc-FFCCEKEKEK;

[0010] The structural formula of the polypeptide hydrogel is:

[0011]

[0012] The second object of the present application is to provide a preparation method of the above-mentioned anti-pollution electrochemical immunosensor based on self-assembled gold nanoparticle-polypeptide hydrogel.

[0013] In order to achieve the above-mentioned object, the present application adopts the following technical solution:

[0014] A preparation method of an anti-pollution electrochemical immunosensor based on self-assembled gold nanoparticle-polypeptide hydrogel, comprising the following steps:

[0015] I. Synthesis of gold nanoparticle-polypeptide hydrogel: gold nanoparticles synthesized by sodium citrate reduction are purified by centrifugation and then re-dispersed in double-distilled water; polypeptide freeze-dried powder is added to a solution containing the gold nanoparticles, and a polypeptide hydrogel is prepared by rapid shaking; the self-assembled polypeptide hydrogel loaded with gold nanoparticles is obtained by placing at room temperature.

[0016] II. Preparation of gold nanoparticle-polypeptide hydrogel modified electrode: the gold nanoparticle-polypeptide hydrogel synthesized in step I is drop-coated on the working electrode of a screen-printed electrode, and the gold nanoparticle-polypeptide hydrogel is uniformly spread on the entire working electrode; the screen-printed electrode with drop-coated gold nanoparticle-polypeptide hydrogel is placed in an oven, and after drying, it is naturally cooled to room temperature to obtain a gold nanoparticle-polypeptide hydrogel modified electrode.

[0017] III. Assembly of sensor: the gold nanoparticle-polypeptide hydrogel synthesized in step I is drop-coated on the working electrode of a screen-printed electrode, and the carbon electrode on the screen-printed electrode is used as a counter electrode and the silver / silver chloride electrode is used as a reference electrode; then the three-electrode system is connected to an electrochemical workstation to form the anti-pollution electrochemical immunosensor.

[0018] Further, the specific method of step I is as follows:

[0019] 50 mL of a 0.01-0.1% mass fraction chloroauric acid solution HAuCl4 is heated to boiling, then 2 mL of a 0.1-1% mass fraction sodium citrate solution is added, and stirred rapidly; after the solution is kept boiling for 5 min, it is cooled to room temperature, and the obtained gold nanoparticles are purified by centrifugation and then re-dispersed in double-distilled water; polypeptide freeze-dried powder is added to a solution containing the gold nanoparticles, and a polypeptide hydrogel is prepared by rapid shaking for 30 s, and the concentration of the polypeptide hydrogel is 20 mg mL -1 , 10 mg mL-1 5 mg / mL -1 1 mg / mL -1 The polypeptide hydrogel is placed at room temperature to obtain a self-assembled polypeptide hydrogel loaded with gold nanoparticles.

[0020] Further, the specific method of step II is as follows:

[0021] 3-8 μL of the gold nanoparticle-polypeptide hydrogel prepared in step I is dropped on the working electrode of the screen-printed electrode, and the gold nanoparticle-polypeptide hydrogel is uniformly spread on the whole working electrode; the screen-printed electrode with the gold nanoparticle-polypeptide hydrogel dropped thereon is placed in an oven, and is dried at 50-100 ℃ for 1 h, and is naturally cooled to room temperature to obtain a gold nanoparticle-polypeptide hydrogel modified electrode.

[0022] A third object of the present application is to provide an application of the anti-pollution electrochemical immunosensor based on the self-assembled gold nanoparticle-polypeptide hydrogel in external diagnosis / detection.

[0023] Further, the application of the sensor is in detection of biological small molecules.

[0024] Further, the application includes quantitative detection of dopamine in human serum; and the specific operation steps of the quantitative detection are as follows:

[0025] First, the gold nanoparticle-polypeptide hydrogel modified electrode prepared is subjected to current signal detection on an electrochemical system, and then a screen-printed electrode with the gold nanoparticle-polypeptide hydrogel as a working electrode, a carbon electrode as a counter electrode and a silver / silver chloride as a reference electrode is immersed in an electrolytic cell, and a series of target dopamines with different concentrations are added to the electrolytic cell at a specific time to obtain test data by the instant voltage method; wherein the voltage applied for dopamine detection is 0.3-0.55 V, preferably 0.45 V.

[0026] Alternatively, the electrolytic cell contains 5 mL of a PBS solution, and the concentration of the PBS solution is 0.2 M, and the pH is 7.4.

[0027] It is worth noting that the detection of the target biological small molecule dopamine adopts an electrochemical catalytic reaction. The modified electrode prepared above is immersed in a near-neutral buffer solution, and a target dopamine with a determined concentration is continuously added, and the change of the current signal is measured by an electrochemical workstation; and the loading of the three-dimensional porous structure polypeptide hydrogel and gold nanoparticles realizes the anti-pollution of the electrochemical sensor and the high-sensitivity detection of the target dopamine.

[0028] Compared with the prior art, the present application has the following beneficial effects:

[0029] (1) This invention provides an efficient performance enhancement strategy for electrochemical sensors, thereby effectively improving the sensitivity and accuracy of detecting small molecule disease biomarkers.

[0030] (2) The sensing interface constructed by the present invention has a single component, a simple preparation process, low cost, and universality, and is easy to be applied and produced on a large scale.

[0031] In summary, the self-assembled gold nanoparticle-peptide hydrogel modified interface prepared in this invention exhibits superior antifouling properties and biocompatibility, and accelerates electron and ion transport. Therefore, it is expected to find wide applications in electrode biomaterials, flexible materials, or electrochemical sensing. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0033] Figure 1 This is a photograph of the gold nanoparticle-peptide hydrogel synthesized in Example 1.

[0034] Figure 2 The image shows a circular dichroism chromatogram of the self-assembled polypeptide hydrogel synthesized in Example 1.

[0035] Figure 3 The Fourier transform infrared spectrum is shown for the self-assembled polypeptide hydrogel synthesized in Example 1.

[0036] Figure 4 The image shows the dynamic light scattering spectrum of the gold nanoparticles synthesized in Example 1.

[0037] Figure 5 The potential of the gold nanoparticle-peptide hydrogel synthesized in Example 1 is given.

[0038] Figure 6 This is a scanning electron microscope image of the gold nanoparticle-peptide hydrogel modified electrode prepared in Example 2.

[0039] Figure 7 This is a transmission electron microscope image of the gold nanoparticle-peptide hydrogel synthesized in Example 2.

[0040] Figure 8 The image shows the UV-Vis absorption spectrum of the gold nanoparticle-peptide hydrogel synthesized in Example 2.

[0041] Figure 9The current response diagrams of the gold nanoparticle-peptide hydrogel modified electrode and the bare screen printed electrode to the target analyte and interfering protein are shown.

[0042] Figure 10 The current response diagrams for the analysis of target analytes are shown for gold nanoparticle-peptide hydrogel modified electrodes and bare screen-printed electrodes before and after immersion in undiluted human serum.

[0043] Figure 11 Fluorescence images showing the adsorption of gold nanoparticle-peptide hydrogel-modified indium zinc oxide electrode and bare indium zinc oxide electrode by FITC-BSA.

[0044] Figure 12 The current response of the gold nanoparticle-peptide hydrogel modified electrode to a 50 nM target material under different applied voltages is shown in the figure.

[0045] Figure 13 The current response of different gold nanoparticle-peptide hydrogel drop volumes to 50 nM dopamine.

[0046] Figure 14 The image shows the current signal of the gold nanoparticle-peptide hydrogel modified electrode for detecting different concentrations of the target analyte dopamine.

[0047] Figure 15 This is a standard curve for the detection of target dopamine by the self-assembled gold nanoparticle-peptide hydrogel modified electrode in Example 2. Detailed Implementation

[0048] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0049] The following are embodiments of certain implementations of the present invention, which are not intended to limit the scope of the present invention.

[0050] In this embodiment of the invention, the current signal was tested on an electrochemical system with a catalytic potential of 0.45V, and the current signal was recorded using an electrochemical workstation. A commercially available screen-printed electrode, purchased from Qingdao Botan Technology Co., Ltd., was used for detection. The electrode substrate material was PET, and the modified area was 0.13 cm². 2 The carbon electrode is used as the working electrode, the carbon electrode is used as the counter electrode, and the Ag / AgCl electrode is used as the reference electrode.

[0051] The polypeptide sequence used was designed in-house and synthesized and purified by Hefei Guotai Biotechnology Co., Ltd. All other raw materials were commercially available products.

[0052] Example 1

[0053] Synthesis of gold nanoparticle-peptide hydrogel:

[0054] Gold nanoparticles were synthesized via a sodium citrate reduction method. 50 mL of a 0.01% chloroauric acid solution (HAuCl4) was heated to boiling, followed by the addition of 2 mL of a 1% sodium citrate solution (containing 0.05% citric acid), and the mixture was stirred rapidly. The solution was kept boiling for 5 min and then cooled to room temperature. The obtained gold nanoparticles were purified by centrifugation and redispersed in deionized water. Lyophilized peptide powder was added to the solution containing the gold nanoparticles, and the mixture was rapidly shaken for 30 s to prepare a 20 mg / mL solution. -1 10mg mL -1 5mg mL -1 1 mg mL -1 The peptide hydrogel was placed at room temperature to obtain a self-assembled peptide hydrogel loaded with gold nanoparticles.

[0055] The attached image shows the gold nanoparticle-peptide hydrogel in a centrifuge tube. Figure 1 As shown, the inverted centrifuge tube indicates a hydrogel state rather than a fluid state, proving the successful synthesis of the hydrogel. A circular dichroism chromatogram is attached. Figure 2 As shown, the negative peak at 214 nm represents the presence of typical β-sheets in the peptide hydrogel. (See attached image.) Figure 3 The Fourier transform infrared spectrum of the synthesized hydrogel is shown, with peak values ​​at 1643 and 1690 cm⁻¹. -1 The peaks are characteristic of the β-sheet conformation, attributed to the incomplete stacking of the amide groups and the stacked carbamate groups in the Fmoc moiety, respectively, indicating the successful synthesis of the self-assembled peptide hydrogel.

[0056] Appendix Figure 4 This indicates that the hydration radius of the synthesized gold nanoparticles is 50-80 nm. The electrokinetic potential of the gold nanoparticle-peptide hydrogel is shown in the attached figure. Figure 5 As shown, the synthesized gold nanoparticle-peptide hydrogel is electrically neutral and can avoid electrostatic adsorption of charged proteins.

[0057] Example 2

[0058] Preparation of gold nanoparticle-peptide hydrogel modified electrodes:

[0059] Take 3-8 μL (preferably 5 μL) of the gold nanoparticle-peptide hydrogel synthesized in Example 1 and drop it onto the working electrode of the screen-printed electrode, spreading the gold nanoparticle-peptide hydrogel evenly over the entire working electrode. Place the screen-printed electrode with the hydrogel drop coating in an oven and dry it at 80°C for 1 hour, then allow it to cool naturally to room temperature to obtain the gold nanoparticle-peptide hydrogel modified electrode.

[0060] The scanning electron microscope image is attached. Figure 6 As shown, the peptide hydrogel-modified electrode has a three-dimensional network structure, and the enhanced porosity can promote electron transfer and ion transport.

[0061] Transmission electron microscope as attached Figure 7 And the UV-Vis absorption spectrum is attached. Figure 8 As shown, gold nanoparticles were successfully modified in situ onto the peptide hydrogel, with a particle size of approximately 20 nm.

[0062] In addition, to further verify the excellent performance of the anti-pollution electrochemical immunosensor prepared in this invention, the inventors also conducted the following experiments:

[0063] Experimental Example 1

[0064] Antifouling ability of gold nanoparticle-peptide hydrogel modified electrodes

[0065] The gold nanoparticle-peptide hydrogel electrode and the bare screen-printed electrode prepared in Example 2 were connected to an electrochemical workstation in a near-neutral buffer solution, and the current signals were measured under different conditions. At 100 s, 50 nM of the target dopamine was slowly injected into the buffer solution, and at 300 s, 1 mg / mL of [amount missing] was added. -1 Interfering protein bovine serum albumin (BSA) is rapidly dispersed by magnetic stirring.

[0066] As attached Figure 9 As shown, the results indicate that the modified electrode exhibits a greater current response to the target analyte. This is attributed to the fact that the three-dimensional network structure facilitates electron and ion transport processes, and the supramolecular π-π stacked backbone formed by electron-conducting aromatic residues may provide effective intermolecular electron delocalization, which promotes charge transport and increases conductivity. Furthermore, the current signal of the modified electrode remained essentially unchanged after the addition of a high concentration of interfering protein, while the current signal of the bare screen-printed electrode decreased significantly, indicating that the modified electrode possesses a strong resistance to non-specific protein adsorption.

[0067] The gold nanoparticle-peptide hydrogel electrode and bare screen-printed electrode prepared in Example 2 above were immersed in 50 μL of undiluted human serum for 2 h. The target analyte was then detected by current measurement using PBS and deionized water. At 50 s, 100 s, and 150 s, 50 nM of dopamine was added, and the target analyte was rapidly dispersed using magnetic stirring. The results are attached. Figure 10 As shown, the detection signal of the target analyte differs significantly before and after soaking in human serum, while the electrode modified with anti-fouling peptide hydrogel has little impact on the detection of the target molecule after soaking in human serum. This indicates that the modified electrode has excellent anti-fouling properties, which can effectively enhance sensing sensitivity and improve analytical detection accuracy, thus avoiding false positive results.

[0068] The gold nanoparticle-peptide hydrogel prepared in Example 2 above was used to modify an indium tin oxide (ITO) electrode. The gold nanoparticle-peptide hydrogel-modified ITO electrode and the bare ITO electrode were immersed in 2 mg / mL water. -1 The electrode surface was imaged using a confocal microscope with an excitation wavelength of 488 nm after 4 hours in fluorescein isothiocyanate-bovine serum albumin (FITC-BSA) and then thoroughly cleaned.

[0069] The results are attached. Figure 11 As shown, fluorescence intensity and significant protein adsorption were observed on the bare indium tin oxide (ITO) electrode, indicating severe biocontamination. However, no fluorescence intensity was observed on the gold nanoparticle-peptide hydrogel-modified ITO electrode, suggesting that the gold nanoparticle-peptide hydrogel-modified electrode exhibits excellent resistance to nonspecific adsorption of interfering proteins. This indicates its potential for detection of dopamine in complex biofluids.

[0070] Experimental Example 2

[0071] Detection of the target biomolecule dopamine based on a gold nanoparticle-peptide hydrogel modified electrode.

[0072] To obtain optimal sensing performance, the effects of catalytic potential and the drop volume of gold nanoparticle-peptide hydrogel on the sensing performance of the electrode surface were investigated; among them, the choice of catalytic potential directly reflects the specificity and sensitivity of the detection. The influence of the gold nanoparticle-peptide hydrogel modified interface on the catalytic performance of 50 nM dopamine under applied potentials of 0.3–0.55 V was also studied.

[0073] As attached Figure 12 As shown, a large current response (2.95 μA) was obtained when the catalytic potential was 0.45 V. Therefore, 0.45 V was chosen as the optimal dopamine catalytic potential for subsequent experiments.

[0074] Furthermore, as attached Figure 13As shown, a 5 μL gold nanoparticle-peptide hydrogel volume corresponds to a large catalytic current, indicating that the optimal dropping volume is 5 μL.

[0075] The gold nanoparticle-peptide hydrogel modified electrode prepared in Example 2 was used to detect current signals in an electrochemical system. The three-electrode system was immersed in a near-neutral buffer solution, and a fixed concentration of dopamine solution was added every 50 seconds under magnetic stirring, while monitoring changes in the current signal. (See attached...) Figure 14 As shown, the detection results indicate that the current signal gradually increases with the increase of the concentration of the target substance dopamine.

[0076] As attached Figure 15 As shown, in the dopamine concentration range of 0.2 nM to 1.9 μM, the current signal value is linearly related to the dopamine concentration, with a linear correlation coefficient of 0.9994 and a detection limit of 0.12 nM. This suggests the potential for ultrasensitive detection of dopamine in complex biological fluids (such as human serum).

[0077] Based on the analysis of the above experimental results, it can be seen that the preparation method described in this invention is simple and efficient, has a single interfacial component, low cost, strong practicality, and is easy to mass-produce.

[0078] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. An anti-fouling electrochemical immunosensor based on self-assembled gold nanoparticle-polypeptide hydrogel, characterized in that, The sensor is an anti-pollution electrochemical sensor constructed by modifying an electrode with a self-assembled polypeptide hydrogel; and the polypeptide sequence is Fmoc-FFCCEKEKEK. The structural formula of the polypeptide hydrogel is: In the sensor, gold nanoparticles are in-situ modified onto the polypeptide hydrogel.

2. A method for preparing a self-assembled gold nanoparticle-polypeptide hydrogel-based anti-fouling electrochemical immunosensor according to claim 1, characterized in that, The method comprises the following steps: I. Synthesis of gold nanoparticle-polypeptide hydrogel: gold nanoparticles synthesized by sodium citrate reduction are purified by centrifugation and then re-dispersed in double-distilled water; polypeptide lyophilized powder is added to the solution containing the gold nanoparticles, and a polypeptide hydrogel is prepared by rapid shaking; the polypeptide hydrogel is left at room temperature to obtain a self-assembled polypeptide hydrogel loaded with gold nanoparticles; II. Preparation of gold nanoparticle-polypeptide hydrogel modified electrode: gold nanoparticle-polypeptide hydrogel synthesized in step I is dropped onto the working electrode of a screen-printed electrode, and the gold nanoparticle-polypeptide hydrogel is uniformly spread on the entire working electrode; the screen-printed electrode with the dropped gold nanoparticle-polypeptide hydrogel is placed in an oven, and after drying, it is naturally cooled to room temperature to obtain a gold nanoparticle-polypeptide hydrogel modified electrode. III. Assembly of sensor: gold nanoparticle-polypeptide hydrogel synthesized in step I is dropped onto the working electrode of a screen-printed electrode, and the carbon electrode on the screen-printed electrode is used as a counter electrode, and a silver / silver chloride electrode is used as a reference electrode; Then, the three-electrode system is connected to an electrochemical workstation to form the anti-pollution electrochemical immunosensor.

3. The method for preparing a self-assembled gold nanoparticle-polypeptide hydrogel-based anti-fouling electrochemical immunosensor according to claim 2, characterized in that, The specific method of step I is: 50 mL of 0.01-0.1% mass fraction chloroauric acid solution HAuCl4 was heated to boiling, then 2 mL of 0.1-1% mass fraction sodium citrate solution was added and stirred quickly; after the solution was kept boiling for 5 min, it was cooled to room temperature, the obtained gold nanoparticles were purified by centrifugation and then redispersed in double-distilled water; polypeptide lyophilized powder was added to the solution containing gold nanoparticles, and quickly shaken for 30 s to prepare polypeptide hydrogel with a concentration of 20 mg mL -1 , 10 mg mL -1 , 5 mg mL -1 , 1 mg mL -1 , and placed at room temperature to obtain self-assembled polypeptide hydrogel loaded with gold nanoparticles.

4. The method for preparing a self-assembled gold nanoparticle-polypeptide hydrogel-based anti-fouling electrochemical immunosensor according to claim 2, characterized in that, The specific method of step II is: 3-8 μL of gold nanoparticle-polypeptide hydrogel synthesized in step I is dropped onto the working electrode of a screen-printed electrode, and the gold nanoparticle-polypeptide hydrogel is uniformly spread on the entire working electrode; the screen-printed electrode with the dropped gold nanoparticle-polypeptide hydrogel is placed in an oven, and dried at 50-100°C for 1 h, and then naturally cooled to room temperature to obtain a gold nanoparticle-polypeptide hydrogel modified electrode.

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