An antifouling hybrid peptide and its application in the construction of electrochemical biosensors

Abstract

This invention belongs to the field of electrochemical technology and relates to an antifouling hybrid peptide and its application in the construction of electrochemical biosensors. The sequence of the antifouling hybrid peptide is cpppp(NE-NK)4; where cpppp represents a sequence composed of D-type amino acids, c is the anchoring region sequence, and pppp is the linker region sequence; (NE-NK)4 represents the antifouling peptide sequence, and its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing. This invention also provides an antifouling interface comprising the aforementioned antifouling hybrid peptide and their application in the construction of electrochemical sensors. The antifouling hybrid peptide, antifouling interface, and electrochemical biosensor based on the hybrid peptide provided by this invention all exhibit good stability and antifouling performance, demonstrating great potential for development and application in the field of medical detection.

Description

Technical Field

[0001] This invention belongs to the field of electrochemical technology and relates to an antifouling hybrid peptide and its application in the construction of electrochemical biosensors. Background Technology

[0002] In most non-sterile environments, organisms tend to form biofilms on material surfaces, particularly in the healthcare industry, water treatment, and marine facilities. The global biofouling problem caused by biofilms not only leads to serious environmental issues but also exacerbates medical problems such as implanted device failure and false positive test results. Exploring effective and stable antifouling materials is crucial for addressing or preventing biofouling caused by biofilms. However, the complexity of the biological fluid environment and the diversity of interfering substances make the development of effective antifouling materials and technologies extremely difficult. Therefore, developing advanced antifouling materials to solve the contamination problem has become a key breakthrough.

[0003] Therefore, materials with antifouling properties have been extensively studied in various fields, with polyethylene glycol (PEG) and zwitterionic polymers being commonly used. However, PEG is easily damaged by oxidation and exhibits immunogenic reactions, while zwitterionic polymers have complex synthesis processes, greatly limiting the further development and application of these two types of materials. In recent years, peptides have been favored by researchers due to their tunable structure, ease of synthesis, and good biocompatibility. However, during development and use, it has been found that natural peptides are easily hydrolyzed by proteases, leading to a sharp decline in the antifouling effect of sensors constructed based on natural peptides. This, in turn, affects the sensor's detection results or causes sensor failure, severely limiting the further development and application of peptides and peptide-based sensors. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides a stable antifouling hybrid peptide, an antifouling interface constructed based on the antifouling hybrid peptide, and its application in the construction of electrochemical biosensors.

[0005] The present invention provides an antifouling hybrid peptide with the sequence cpppp(NE-NK)4; wherein cpppp represents a sequence composed of D-type amino acids, c is the anchoring region sequence, and pppp is the linker region sequence; (NE-NK)4 represents an antifouling peptide sequence, the amino acid sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0006] The present invention also provides an antifouling interface, the antifouling interface comprising the above-mentioned antifouling mixed peptide.

[0007] Furthermore, the antifouling interface is obtained by immobilizing an antifouling mixed peptide on a glassy carbon electrode; the glassy carbon electrode is obtained by sequentially modifying a conductive polymer (such as 3,4-ethylenedioxythiophene) and gold nanoparticles on a glassy carbon electrode.

[0008] The present invention further provides an electrochemical biosensor, wherein the electrochemical biosensor includes the above-mentioned antifouling hybrid peptide or antifouling interface.

[0009] Furthermore, the electrochemical biosensor is obtained by immobilizing an antifouling mixed peptide and an aptamer on a glassy carbon electrode; the glassy carbon electrode is obtained by sequentially modifying a conductive polymer (such as 3,4-ethylenedioxythiophene) and gold nanoparticles on a glassy carbon electrode.

[0010] The present invention further provides a method for preparing the antifouling interface, which specifically includes the following steps: 1) Electrode treatment: The glassy carbon electrode was polished with 0.3μm and 0.05μm alumina powder in sequence, and then ultrasonically cleaned with secondary water, anhydrous ethanol and secondary water in sequence.

[0011] 2) Electrode modification: The pretreated glassy carbon electrode (working electrode), saturated calomel electrode (reference electrode), and platinum wire electrode (counter electrode) were first placed in an aqueous solution containing conductive polymers with negatively charged groups (such as 3,4-ethylenedioxythiophene) doped with sodium citrate or sodium alginate for electrochemical polymerization using the it method. The potential was set to 1.0 V and the running time was 10-30 s to obtain the modified electrode. The obtained conductive nanomaterial modified electrode (working electrode), saturated calomel electrode (reference electrode), and platinum wire electrode (counter electrode) were placed in a chloroauric acid solution and modified with gold nanoparticles by cyclic voltammetry (segments: 2-8) to complete the final modification of the electrode.

[0012] 3) Preparation of antifouling interface: The mixed peptide solution was dropped onto the interface of the gold nanoparticle modified electrode and incubated at room temperature for 8-12 hours to obtain the antifouling interface.

[0013] Preferably, step 2) electrode modification is achieved by ultrasonically dispersing an aqueous solution of sodium citrate at a concentration of 4.0~8.0 mg / mL or sodium alginate at a concentration of 2.0~5.0 mg / mL and 1.1~2.7 mg / mL of 3,4-ethylenedioxythiophene, and then running it on a pretreated bare glassy carbon electrode at 1.0V for 20 s using the IT method.

[0014] More preferably, in step 2), electrode modification: 3.0 mg / mL sodium alginate is selected as the dopant in the aqueous solution of 3,4-ethylenedioxythiophene, and the aqueous solution of 1.1 mg / mL 3,4-ethylenedioxythiophene is ultrasonically dispersed for electrode modification.

[0015] The present invention further provides a method for preparing the electrochemical sensor, which specifically includes the following steps: 1) Electrode treatment: The glassy carbon electrode was polished with 0.3μm and 0.05μm alumina powder in sequence, and then ultrasonically cleaned with secondary water, anhydrous ethanol and secondary water in sequence.

[0016] 2) Electrode modification: The pretreated glassy carbon electrode (working electrode), saturated calomel electrode (reference electrode), and platinum wire electrode (counter electrode) were first placed in an aqueous solution containing conductive polymers with negatively charged groups (such as 3,4-ethylenedioxythiophene) doped with sodium citrate or sodium alginate for electrochemical polymerization using the it method. The potential was set to 1.0 V and the running time was 10-30 s to obtain the modified electrode. The obtained conductive nanomaterial modified electrode (working electrode), saturated calomel electrode (reference electrode), and platinum wire electrode (counter electrode) were placed in a chloroauric acid solution and modified with gold nanoparticles by cyclic voltammetry (segments: 2-8) to complete the final modification of the electrode.

[0017] 3) Preparation of mixed peptide and aptamer solution: Take the same volume of 0.2 ~ 0.6 mg / mL mixed peptide and 10 ~ 30 μM aptamer and mix them.

[0018] 4) Preparation of electrochemical biosensor: The mixed solution of the above-mentioned mixed peptide and aptamer is dropped onto the electrode interface modified with gold nanoparticles prepared in step 2), and incubated at room temperature for 8-12 hours to obtain the electrochemical biosensor.

[0019] Preferably, in step 2), electrode modification involves ultrasonically dispersing an aqueous solution of sodium citrate (4.0–8.0 mg / mL) or sodium alginate (2.0–5.0 mg / mL) and 3,4-ethylenedioxythiophene (1.1–2.7 mg / mL), and then performing an iterative process (it) on a pretreated bare glassy carbon electrode at 1.0 V for 20 s. The gold nanoparticles are then electrodeposited using a CV method with parameters set to a voltage range of -1.5–0.5 V, a scan rate of 0.05 V / s, and 6 scan segments.

[0020] Preferably, in step 3), the concentration of the mixed peptide is 0.4 mg / mL and the concentration of the aptamer is 20 μM.

[0021] More preferably, in step 2), electrode modification: 3.0 mg / mL sodium alginate is selected as the dopant in the aqueous solution of 3,4-ethylenedioxythiophene, and the aqueous solution of 1.1 mg / mL 3,4-ethylenedioxythiophene is ultrasonically dispersed for electrode modification.

[0022] Compared with the prior art, the present invention has the following beneficial effects: The antifouling hybrid peptide provided by this invention integrates multiple functions, comprising three functional regions: anchoring, linking, and antifouling, with corresponding sequences of D-Cys, (D-Pro)4, and (Nglu NLys)4, respectively. The anchoring and linking regions employ a D-configuration amino acid design to enhance stability against protease hydrolysis. The antifouling region adopts a peptide-like structure design. In addition to containing a large number of hydrophilic groups such as carboxyl and amino groups, similar to traditional peptide (GluLys)4, exhibiting good hydrophilicity, it also overcomes the weakness of traditional peptide (GluLys)4 being easily hydrolyzed by proteases. This allows the modified interface to form a dense hydration layer, providing not only excellent antifouling effects but also enhanced stability against protease hydrolysis.

[0023] The interface modification method using mixed peptides in this invention significantly enhances the stability and antifouling effect of protease hydrolysis. Furthermore, the electrochemical biosensor based on this mixed peptide can achieve highly sensitive and reliable detection of NSE, with a linear detection range of 0.001-500 ng / mL, demonstrating excellent selectivity, antifouling properties, and reproducibility.

[0024] The electrochemical biosensor of this invention exhibits excellent reliability and sensitivity in NSE analysis.

[0025] In summary, this invention has developed an antifouling hybrid peptide, an antifouling interface, and an electrochemical biosensor based on the hybrid peptide, all of which exhibit good stability and antifouling performance, demonstrating great potential for development and application in the field of medical testing. Attached Figure Description

[0026] Figure 1 The following is a characterization of the antifouling mixed peptide provided by the present invention; wherein, A. structural diagram; B. high performance liquid chromatography (HPLC) chromatogram; Figure 2 This is the mass spectrum of the antifouling mixed peptide provided by the present invention; Figure 3 The graph shows the theoretical simulation results of the antifouling mixed peptide and the contrasting peptide provided by this invention; where A is the free energy landscape; B is the root mean square deviation curve; and C is the radial distribution function. Figure 4 This is a test diagram of the antifouling interface's resistance to protease hydrolysis provided by the present invention; Figure 5 This is a test diagram of the long-term anti-fouling performance of the anti-fouling interface provided by the present invention; Figure 6 This is a schematic diagram illustrating the construction and detection of an electrochemical sensor based on antifouling hybrid peptides provided by the present invention; Figure 7 These are the experimental results of optimizing the preparation conditions of the electrochemical sensor provided by this invention; wherein, A. optimization of the effect of peptide concentration on current response; B. optimization of the effect of aptamer concentration on current response; Figure 8 These are characterization diagrams of the electrochemical sensor construction process provided by this invention; wherein, A. Differential pulse voltammetry (DPV) curve; B. Cyclic voltammetry (CV) curve; Figure 9 This is a comparison of the antifouling effects in serum at different concentrations; where A. bare glassy carbon electrode; B. Au / PEDOT / GCE modified electrode; C. antifouling interface; D. electrochemical biosensor; Figure 10 This invention provides a performance test of the electrochemical biosensor for detecting protein NSE; wherein, A. DPV current; B. linear fitting graph; Figure 11 This is a selectivity diagram of the electrochemical biosensor provided by the present invention; Figure 12 This is a stability diagram of the electrochemical biosensor provided by the present invention. Detailed Implementation

[0027] To make the objectives, technical solutions, and advantages of this invention clearer, the following embodiments are provided to further illustrate the invention. It should be understood that the embodiments described herein are not all embodiments, and the embodiments in this invention are only used to explain the invention and do not constitute any limitation on the invention.

[0028] It should be noted that, unless otherwise specified, the materials used in the embodiments described below can be obtained commercially or synthesized by known methods. Furthermore, unless otherwise specified, the instruments and methods used in the embodiments described below are conventional methods and instruments in this technical field.

[0029] Example 1: Preparation of antifouling hybrid peptides and antifouling interfaces (1) Preparation of antifouling mixed peptides The synthesized antifouling hybrid peptide sequence is cpppp(NE-NK)4, and its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing. The anchoring region sequence c and the linker region sequence pppp are designed with D-configuration amino acids to enhance stability during protease hydrolysis. The antifouling region (NE-NK)4 adopts a peptide-like structure (side chain on backbone N), and its molecular structure is designed as follows: Figure 1 As shown in Figure A, the synthesized mixed peptides, with a purity ≥95%, were characterized using high-performance liquid chromatography and mass spectrometry, and the results are as follows. Figure 1 China B and Figure 2 As shown.

[0030] (2) Preparation of antifouling interface Electrode treatment: The glassy carbon electrode (diameter Φ = 3 mm) was polished with 0.3 μm and 0.05 μm alumina powder in sequence, and then ultrasonically cleaned with secondary water, anhydrous ethanol and secondary water for 1 minute each in sequence.

[0031] Preparation of electrodeposition solution: The electrodeposition aqueous solution (1) includes sodium citrate 4.0~8.0 mg / mL or sodium alginate 2.0~5.0 mg / mL and 3,4-ethylenedioxythiophene 1.1~2.7 mg / mL; the electrodeposition solution (2) includes chloroauric acid and potassium nitrate.

[0032] Preparation of Au / PEDOT / GCE modified electrode: The pretreated glassy carbon electrode (working electrode), saturated calomel electrode (reference electrode), and platinum wire electrode (counter electrode) were sequentially placed in the electrodeposition solution (1) described above. The electrode was prepared by the it method on the pretreated bare glassy carbon electrode at a voltage of 1.0 V for 20 s. In the above deposition solution (2), electrodeposition was performed by the CV method: the parameters were set to a voltage range of -1.5 ~ 0.5 V, a scan rate of 0.05 V / s, and a scan segment of 6 to obtain the Au / PEDOT / GCE modified electrode.

[0033] Preparation of antifouling interface: The 0.2 mg / mL mixed peptide synthesized in this invention is fixed on the Au / PEDOT / GCE modified electrode interface through gold-sulfur bonding, thereby completing the assembly of the antifouling interface.

[0034] Example 2: Theoretical simulation calculation of antifouling hybrid peptides like Figure 3 As shown, all molecular dynamics (MD) simulations were performed using the GROMACS 2020.6 software package. The peptide structure was constructed using PyMOL, then dissolved in a cubic box containing TIP3P water molecules, and a three-dimensional periodic boundary was applied. To neutralize the system, Na was added to the simulation system. + and Cl - Neutralization was performed. Atomic interaction parameters were assigned based on the AMBER14SB force field. Energy minimization was achieved using the conjugate gradient algorithm. The system was equilibrated sequentially under NVT and NPT, reaching the target temperature of 298 K and the target pressure of 1 bar, respectively. Production MD simulations were then performed with a time step of 2 fs. The cutoff distance for nonbonded interactions was 1.2 nm.

[0035] Example 3: Antifouling interface stability against protease hydrolysis test To characterize the proteolytic stability of the mixed peptides, this embodiment selected site-specific trypsin and non-site-specific alkaline protease for enzymatic stability testing. First, an antifouling interface was prepared based on the mixed peptides. This interface was then treated with 2.0 mg / mL trypsin and 2.0 mg / mL alkaline protease for 2 hours, respectively. The interfaces before and after proteolytic treatment (S1 before hydrolysis, S2 after trypsin hydrolysis, and S3 after alkaline protease hydrolysis) were tested for antifouling in 100% human serum. The results are shown in [Figure number missing]. Figure 4 The antifouling interface constructed in this invention exhibits essentially no change in antifouling effect in either site-specific trypsin or non-site-specific alkaline proteases. In contrast, the control group, with its antifouling interface constructed from traditional peptides, showed a significant increase in signal inhibition rate after protease treatment, indicating a marked deterioration in antifouling effect. This demonstrates that the antifouling interface based on mixed peptides possesses excellent protease hydrolytic stability.

[0036] Example 4: Long-term antifouling experiment of antifouling interface To verify the long-term antifouling capability of the constructed antifouling interface, this invention conducted long-term antifouling experiments on the bare glassy carbon electrode and the antifouling interface separately in undiluted serum. The results are as follows: Figure 5 As shown in the figure. The antifouling effect is represented by the signal inhibition rate; the lower the signal inhibition rate, the better the antifouling effect, and vice versa. Before the electrode was immersed in serum, the interfacial current signal was measured in a 5.0 mM potassium ferricyanide / potassium ferrocyanide solution and recorded as I1. After the electrode was immersed in serum for different times, the interfacial current signal was measured in a 5.0 mM potassium ferricyanide / potassium ferrocyanide solution and recorded as I2. The signal inhibition rate (%) is calculated as [(I1-I2) / I1]×100%. The results show that compared with the bare electrode, the prepared antifouling interface still maintains a very low signal inhibition rate after 27 days, indicating that the antifouling interface has a very good antifouling effect within 27 days.

[0037] Example 5: Schematic diagram of the preparation and detection of an electrochemical sensor based on mixed peptides. Given that methods of directly modifying bare glassy carbon electrodes with biomaterials for sensor construction suffer from signal instability, this invention modifies the bare glassy carbon electrode using a conductive nanocomposite material before modifying the biomaterial. Specifically, electrodeposition is performed sequentially in an aqueous solution of sodium alginate-doped conductive polymer 3,4-ethylenedioxythiophene and a chloroauric acid solution to obtain the modified glassy carbon electrode. A mixed peptide and DNA aptamer are then co-incubated at the interface of the modified electrode in a single step to construct an electrochemical sensor based on the mixed peptide. The prepared electrochemical sensor is then directly immersed in NSE protein solutions of different concentrations or in undiluted clinical serum samples for detection, enabling accurate detection of the disease biomarker NSE. Figure 6 As shown.

[0038] Example 6: Optimization of electrochemical sensor conditions and characterization of its preparation process based on hybrid peptides To improve the performance of the constructed electrochemical biosensor, key influencing factors were first optimized during the sensor construction process. For example... Figure 7 As shown, the optimal concentrations of peptide (A) and DNA aptamer (B) during the construction process were optimized. The results showed that when the peptide concentration reached 0.2 mg / mL and the aptamer concentration reached 10 μM, the current change essentially reached equilibrium, indicating that the interfacial incubation volume had reached saturation, which is the optimal concentration. Therefore, in the sensor construction process, we first mixed equal volumes of 0.4 mg / mL peptide and 20 μM DNA aptamer, then co-incubated them in one step to complete the sensor construction.

[0039] To demonstrate the successful fabrication of the electrochemical sensor, such as Figure 8 As shown, we performed DPV and CV tests on each step of the assembly process. Figure 8 As shown in Figure A, after electrodepositing conductive nanocomposite materials on a bare glassy carbon electrode, the current signal increases, mainly due to the excellent conductivity of the conductive polymer, which promotes interfacial electron transfer. Following further electrodeposition of gold nanoparticles, the current signal increases again due to the excellent conductivity and increased specific surface area of ​​the gold nanoparticles. Subsequently, after incubating the peptide and aptamer, the current signal decreases because the electrochemical inertness of the peptide and aptamer hinders interfacial electron transfer. Finally, after specific recognition of the NSE protein, the electrochemical inertness of the protein continues to impede electron transfer, resulting in a decrease in the current signal. Similarly, cyclic voltammetry (CV) was used for characterization to further illustrate this.

[0040] Example 7: Antifouling interface based on hybrid peptides and antifouling performance of electrochemical sensors To verify the antifouling effect of the interface, a series of human serum samples with different concentrations (V / V) were prepared: 5%, 25%, 50%, 75%, and 100%. First, the different modified electrodes were immersed in a 5.0 mM potassium ferricyanide / potassium ferrocyanide solution, and the current signal was recorded. Then, the different modified electrodes were immersed in human serum solutions of different concentrations for 30 minutes, and the DPV current signal was measured in the 5.0 mM potassium ferricyanide / potassium ferrocyanide solution. The results are as follows: Figure 9As shown, the worse the antifouling effect of the electrode interface, the more severe the non-specific adsorption, and the more significant the current drop. The electrode interfaces represented by AD, in order, are: bare glassy carbon electrode, substrate electrode modified with conductive polymer and gold nanoparticles, antifouling interface based on mixed peptides, and electrochemical sensor based on mixed peptides. The results show that the antifouling effect of the antifouling interface constructed from mixed peptides and the electrochemical sensor is significantly better than that of the interfaces without mixed peptide modification (bare glassy carbon electrode, substrate electrode modified with conductive polymer and gold nanoparticles). (Comparison) Figure 9 As can be seen in A and B, the antifouling interface has a better antifouling effect than the electrochemical sensor. However, this is because a DNA probe is used as a recognition element during the sensor assembly process. The DNA probe is negatively charged and will inevitably adsorb some positively charged proteins in the serum, which will result in a slight decrease in the antifouling performance of the antifouling interface.

[0041] Example 8: Detection of specific enolase (NSE) using an electrochemical sensor based on mixed peptides Figure 10 In the diagram, A, a to i represent the NSE protein concentrations as follows: 1 pg / mL, 10 pg / mL, 100 pg / mL, 1 ng / mL, 10 ng / mL, 100 ng / mL, 200 ng / mL, and 500 ng / mL. The prepared hybrid peptide-based electrochemical sensor was immersed in the above-mentioned NSE protein solutions of different concentrations for 40 minutes, and the DPV current was recorded. Figure 9 A. Plotting the change in current (ΔI) before and after immersion on the ordinate and the logarithm of NSE concentration (LgC) on the abscissa, the fitted straight line is shown in Figure 1. Figure 10 The detection range of the electrochemical sensor of this invention is 0.001~500 ng / mL, and the linear correlation coefficient is R. 2 =0.9983.

[0042] Example 9: Selectivity of Electrochemical Sensors Based on Mixed Peptides like Figure 11 As shown, 1-6 represent: 5.0 μg / mL interfering proteins AFP, IgG, CRP, CEA, ANXA1, and 10.0 ng / mL target protein NSE, respectively. These five proteins were detected using an electrochemical biosensor based on a mixed peptide, and the changes in the electrochemical sensor's response current were recorded. The results show that even at a concentration of 500 times that of the target protein, the response signal of the electrochemical sensor to the interfering protein is not significant. This demonstrates that the electrochemical sensor based on the mixed peptide of this invention exhibits strong specificity for the target protein, i.e., excellent selectivity.

[0043] Example 10: Antifouling interface and stability of electrochemical sensor based on hybrid peptides To verify the stability of the antifouling interface and the electrochemical sensor, different modified electrodes were first prepared: a bare glassy carbon electrode, an antifouling interface, and an electrochemical sensor based on a mixed peptide. The three modified interfaces were then scanned seven times consecutively in a 5.0 mM potassium ferrocyanide / potassium ferrocyanide solution, and the DPV current values ​​were recorded. The results are as follows: Figure 12 As shown in the figure. The results show that the antifouling interface prepared in this invention and the DPV current of the electrochemical sensor based on mixed peptides remain basically unchanged, indicating good stability; conversely, the DPV current of the bare glassy carbon electrode changes significantly, indicating poor stability.

[0044] The descriptions of the disclosed embodiments are detailed and specific, intended to enable those skilled in the art to implement or use the present invention, and are not intended to limit the scope of the present invention. It should be clearly stated that any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A type of antifouling mixed peptide, characterized in that, The sequence of the antifouling hybrid peptide is cpppp(NE-NK)4; where cpppp represents a sequence composed of D-type amino acids, c is the anchoring region sequence, and pppp is the linker region sequence; (NE-NK)4 represents the antifouling region peptide sequence, and its amino acid sequence is shown in SEQ ID NO.1 of the sequence listing.

2. A stain-resistant interface, characterized in that, The antifouling interface contains the antifouling mixed peptide as described in claim 1.

3. The anti-fouling interface according to claim 2, characterized in that, The antifouling interface is obtained by incubating the antifouling mixed peptide of claim 1 on a glassy carbon electrode; the glassy carbon electrode is obtained by electrochemically polymerizing a conductive nanocomposite material on a bare glassy carbon electrode.

4. The method for preparing the antifouling interface according to any one of claims 2-3, characterized in that, include: 1) Electrode treatment: The glassy carbon electrode was polished with 0.3μm and 0.05μm alumina powder in sequence, and then ultrasonically cleaned with deionized water, anhydrous ethanol and deionized water in sequence. 2) Electrode modification: The pretreated glassy carbon electrode, saturated calomel electrode, and platinum wire electrode were first placed in an aqueous solution of a conductive polymer doped with negatively charged groups, such as sodium citrate or sodium alginate, for electrochemical polymerization using the it method. The potential was set to 1.0 V and the running time was 10-30 s to obtain the modified electrode. The obtained conductive nanomaterial modified electrode, saturated calomel electrode, and platinum wire electrode were then placed in a chloroauric acid solution and modified with gold nanoparticles by cyclic voltammetry (segments: 2-8) to complete the final modification of the electrode. 3) Preparation of antifouling interface: The mixed peptide solution was dropped onto the electrode interface modified with gold nanoparticles and incubated at room temperature for 8-12 hours to obtain the antifouling interface.

5. An electrochemical biosensor, characterized in that, The electrochemical biosensor contains the antifouling hybrid peptide of claim 1 or the antifouling interface of any one of claims 2-3.

6. The electrochemical biosensor according to claim 5, characterized in that, The electrochemical biosensor is obtained by co-incubating a nucleic acid aptamer and the antifouling mixed peptide of claim 1 on a glassy carbon electrode in one step; the glassy carbon electrode is obtained by electrochemically polymerizing a conductive nanocomposite material on a bare glassy carbon electrode.

7. A method for preparing the electrochemical sensor according to any one of claims 5-6, characterized in that, include: 1) Electrode treatment: The glassy carbon electrode was polished with 0.3μm and 0.05μm alumina powder in sequence, and then ultrasonically cleaned with deionized water, anhydrous ethanol and deionized water in sequence. 2) Electrode modification: The pretreated glassy carbon electrode, saturated calomel electrode, and platinum wire electrode were first placed in an aqueous solution containing a conductive polymer doped with negatively charged groups such as sodium citrate or sodium alginate for electrochemical polymerization using the I-method. The potential was set to 1.0 V and the running time was 10-30 s to obtain the modified electrode. The obtained conductive nanomaterials were then used to modify the electrode, saturated calomel electrode, and platinum wire electrode. The gold nanoparticles were placed in a chloroauric acid solution and modified by cyclic voltammetry (segments: 2~8) to complete the final modification of the electrode. 3) Preparation of electrochemical biosensor: A mixed solution containing mixed peptides and aptamers was dropped onto the interface of the gold nanoparticle modified electrode described above, and incubated at room temperature for 8-12 hours to obtain the electrochemical biosensor.

8. The preparation method according to claim 7, characterized in that, The aptamer is designed or screened with different sequences depending on the target analyte.

9. The use of the mixed peptide of claim 1, or the antifouling interface of claim 2 or 3, in the preparation of electrochemical biosensors.

10. A method for improving the antifouling properties and stability of electrochemical biosensors, characterized in that, The method involves modifying a conductive nanocomposite material onto a glassy carbon electrode and immobilizing the mixed peptide described in claim 1 onto the electrode interface.

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