Photoelectrochemical immunosensor based on dendritic peptide tetramer as well as preparation method and application of photoelectrochemical immunosensor

Abstract

The invention belongs to the technical field of biosensors, and discloses a photoelectrochemical immunosensor based on a dendritic peptide tetramer as well as a preparation method and application of the photoelectrochemical immunosensor. The dendritic peptide tetramer is adopted to construct the photoelectrochemical immunosensor for the first time, and the method comprises the following steps: preparing an HsGDY / Cu2O photocathode, and sequentially anchoring an antibody probe and the dendritic peptide tetramer on the surface of the photocathode. Through specific binding of a target object and the antibody probe, a significant steric hindrance effect is generated on an electrode interface, interface charge transfer is hindered, photocurrent signal change is caused, and detection of the target object staphylococcus aureus enterotoxin A is realized. The photoelectrochemical immunosensor provided by the invention is simple, convenient and efficient in preparation process, has excellent food matrix biological pollution resistance, can effectively inhibit non-specific adsorption so as to significantly improve the sensitivity, specificity and accuracy of detection, is suitable for the field of food safety detection, and is suitable for market popularization and application.

Description

Technical Field

[0001] This invention belongs to the field of biosensor technology, and relates to a photoelectrochemical immunosensor based on dendritic peptide tetramers, its preparation method and application. Background Technology

[0002] Foodborne illnesses remain a major public health problem globally, with pathogens and their toxins posing a serious threat to human health and causing significant economic losses to the food industry. Among these toxins, Staphylococcus aureus enterotoxin A (SEA) is one of the most potent and widely distributed enterotoxins produced by Staphylococcus aureus. Even trace amounts of SEA at the nanogram level can cause acute gastrointestinal symptoms such as nausea, vomiting, abdominal cramps, and diarrhea. Therefore, sensitive and reliable detection of SEA is crucial for ensuring food safety and maintaining public health. Currently, analytical techniques used for SEA determination mainly include enzyme-linked immunosorbent assay (ELISA), mass spectrometry, fluorescence analysis, and lateral flow immunoassay. However, these methods typically suffer from cumbersome sample pretreatment processes, long detection cycles, low sensitivity, and susceptibility to biocontamination in food matrices, limiting their widespread application in practice. Therefore, there is an urgent need to develop a simple, efficient sensor with excellent anti-contamination capabilities for the accurate detection of SEA in real samples.

[0003] Among existing detection strategies, photoelectrochemical (PEC) immunosensing technology has become a highly promising alternative due to its advantages such as high sensitivity, low background noise, fast response speed, and ease of integration with portable analytical devices. In PEC detection, photoactive semiconductor electrodes convert light signals into electrical signals, which can be modulated through specific antigen-antibody interactions, thereby achieving sensitive detection of SEA and effectively alleviating many limitations of traditional detection methods. Despite these advantages, the practical application of PEC immunosensing in food safety monitoring is still constrained by two key issues: (i) insufficient stability of semiconductor photoelectrodes under operating conditions; and (ii) non-specific adsorption of sample matrix components on the electrode surface, leading to significant signal drift and false positive results. Solving these problems urgently requires the rational integration of highly stable photoactive materials with advanced anti-fouling strategies.

[0004] Photoelectrodes are generally classified into photoanodes and photocathodes. Compared to photoanodes, photocathode-based immunosensors rely on electron reduction reactions occurring at the electrode / electrolyte interface, which can effectively resist interference from reducing substances in biological samples, thus showing significant practical potential. Photocathodes are typically constructed based on p-type semiconductors, where holes act as the primary charge carriers in charge transport. Cu₂O is a promising p-type semiconductor material with strong light absorption, a narrow bandgap, and a high theoretical photocurrent density. However, Cu₂O still faces challenges in practical applications, such as rapid electron-hole recombination and severe photocorrosion, which limit its photoelectrochemical performance. In recent years, hydrogen-substituted graphyne (HsGDY) has attracted widespread attention as a novel carbon-based semiconductor material, possessing a highly conjugated π-electron network, tunable electronic structure, and excellent chemical stability under aqueous and photoluminescence conditions. The ultrathin porous structure of HsGDY not only facilitates the formation of uniform films on copper substrates but also serves as a protective layer to effectively enhance the structural stability of the photocathode, promote carrier separation, and accelerate electron transport. These properties make HsGDY an ideal functional material for improving the performance and durability of photocathode-type PEC systems.

[0005] Due to the high complexity of food matrices, which typically contain multiple components such as proteins, carbohydrates, lipids, and reducing agents, accurate quantification of SEA in real food samples remains a significant challenge, as these components often interfere with the analytical signal. To address the long-standing problem of nonspecific adsorption, the introduction of antifouling materials has become a widely adopted and effective strategy. Among numerous antifouling materials, zwitterionic peptides are considered to possess excellent antifouling potential due to their overall electroneutrality and strong hydrophilicity. Studies have shown that zwitterionic peptides can form a certain degree of hydration interface on the electrode surface, thereby inhibiting the nonspecific adsorption of proteins and other hydrophilic biomolecules. However, in practical applications, existing antifouling materials, especially antifouling molecular systems represented by linear zwitterionic peptides, still have several shortcomings: linear zwitterionic peptides or traditional antifouling molecules have limited interfacial stability in complex matrices, are prone to desorption or interfacial rearrangement, and are difficult to effectively inhibit non-specific adsorption; some antifouling materials require high coverage modification to achieve antifouling effects, but their antifouling performance is difficult to maintain for a long time and may weaken the photoelectric properties of the electrode surface, thus affecting sensitivity; in addition, the preparation process of some antifouling materials is complex and costly, which is not conducive to large-scale preparation and practical application. Summary of the Invention

[0006] In view of this, one of the objectives of this invention is to address the problems existing in the prior art by providing a photoelectrochemical immunosensor based on dendritic peptide tetramers with high sensitivity and excellent resistance to biocontamination, as well as a method for its preparation. This photoelectrochemical immunosensor has excellent resistance to biocontamination of food matrices and can effectively inhibit non-specific adsorption, thereby significantly improving the sensitivity, specificity, and accuracy of detection. It is suitable for the field of food safety testing and can be used to detect Staphylococcus aureus enterotoxin A (SEA) in food.

[0007] It should be noted that the dendritic peptide tetramer has a multi-level branched topology and four equivalent terminal arms, which is beneficial for enhancing intramolecular hydrogen bonding interactions and promoting the formation of a stable and dense hydration layer at the electrode interface, thereby improving the interfacial structural stability and enhancing anti-fouling performance. This invention is the first to use dendritic peptide tetramers to construct a photoelectrochemical immunosensor, comprising: preparing an HsGDY / Cu2O photocathode and sequentially anchoring an antibody probe and a dendritic peptide tetramer onto the surface of the photocathode. Through the specific binding of the target analyte and the antibody probe, a significant steric hindrance effect is generated at the electrode interface, hindering interfacial charge transfer and causing a change in the photocurrent signal, thus achieving the detection of the target analyte, Staphylococcus aureus enterotoxin A. The photoelectrochemical immunosensor based on dendritic peptide tetramers described in this invention has a simple and efficient fabrication process, excellent resistance to food matrix biocontamination, and can effectively suppress non-specific adsorption, thereby significantly improving the sensitivity, specificity, and accuracy of detection. It is suitable for the field of food safety testing and is suitable for commercial promotion and application.

[0008] To achieve the above objectives, the technical solution of the present invention is as follows:

[0009] A photoelectrochemical immunosensor based on dendritic peptide tetramers is disclosed. The immunosensor is fabricated by sequentially anchoring an antibody probe that specifically recognizes Staphylococcus aureus enterotoxin A (SEA) and a dendritic peptide tetramer (DPT) with anti-biocontamination properties in food matrices onto a photocathode. The photocurrent signal is detected by utilizing the significant steric hindrance effect of Staphylococcus aureus enterotoxin A on the charge transfer of the sensor.

[0010] It should be noted that the present invention proposes to use dendritic peptide tetramers (DPT), whose highly ordered branched structure can enhance intramolecular hydrogen bonding and promote the formation of a more stable hydration layer, thereby significantly improving interfacial stability and antifouling performance, effectively inhibiting nonspecific adsorption, and taking into account the simplicity of synthesis and controllable cost, thus meeting the demand for high-performance antifouling materials in the field of analytical testing.

[0011] Furthermore, the dendritic peptide tetramer is characterized by having a multi-level branched topology and four equivalent terminal arms, with the amino acid sequence (N3-)KPPPPEK(EK(EKEKEK)2)2; wherein the rigid anchoring sequence (N3-)KPPPPEK is the backbone; four identical EKEKEK are the main branches, and the main chain is connected by two secondary branch EK units.

[0012] Furthermore, relevant data have demonstrated that the EKEKEK peptide sequence is composed of alternating positively charged lysine (K) and negatively charged glutamic acid (E), resembling a zwitterionic polymer. This sequence exhibits high hydrophilicity and overall electroneutrality, enabling the formation of a stable hydration layer on the sensor surface and effectively resisting the non-specific adsorption of biomolecules such as proteins through electrostatic and hydrophobic interactions. Compared to traditional linear peptides, dendritic peptide tetramers (DPTs) possess a highly ordered branched structure, enhancing intramolecular hydrogen bonding and promoting the formation of a more stable hydration layer, thus exhibiting superior stability and antifouling properties, demonstrating excellent resistance to biofouling.

[0013] Another objective of this invention is to provide a method for preparing the above-mentioned photoelectrochemical immunosensor based on dendritic peptide tetramers. The preparation method disclosed in this invention is simple, rapid, efficient, and easy to operate, and is suitable for promotion and application.

[0014] To achieve the above objectives, the present invention adopts the following technical solution: A method for preparing a photoelectrochemical immunosensor based on dendritic peptide tetramers, comprising the following steps: (1) Preparation of HsGDY / Cu2O photocathode: Using Cu2O nanorod array electrodes that absorb visible light as a substrate, hydrogen-substituted graphyne HsGDY, a carbon-based semiconductor material with excellent conductivity, is modified on the surface of the substrate as a sensitizer to prepare HsGDY / Cu2O photocathode. (2) Staphylococcus aureus enterotoxin A (SEA) antibody probe and dendritic peptide tetramer (DPT) are successively anchored to the HsGDY / Cu2O photocathode prepared in step (1) to obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

[0015] Preferably, in step (1), Cu2O nanorod array electrodes are prepared by anodizing and high-temperature annealing: the copper substrate is anodized with 3.0 M NaOH solution in a constant current manner, and then annealed at 550 °C in a tube furnace under nitrogen protection to obtain Cu2O nanorod array electrodes; HsGDY was grown on the surface of the Cu2O nanorod array electrode via a Glaser coupling reaction: 2.5 mg of triacetylenebenzene and 5 μL of piperidine were dissolved in 5 mL of pyridine and placed in a brown conical flask; the Cu2O nanorod array electrode was immersed in the pyridine reaction solution, and oxygen was introduced before sealing; the system was heated to 60 °C in an oil bath and kept at a constant temperature to allow the Glaser coupling reaction to proceed fully on the electrode surface; after the reaction was completed, the electrode was washed sequentially with pyridine, dichloromethane and methanol, and dried under a nitrogen gas flow to obtain the desired HsGDY / Cu2O photocathode.

[0016] It should be noted that the electrode material used in the HsGDY / Cu2O photocathode is environmentally friendly, biocompatible, and has a simple preparation process. It also exhibits a very obvious photocurrent signal response and good photochemical stability.

[0017] More preferably, the Cu2O nanorod array electrode is prepared using a method of 8~12 mA / cm². 2 The constant current density is maintained, the anodizing time is 3-10 minutes, and the annealing time is 2-6 hours; and when preparing the HsGDY / Cu2O photocathode, oxygen is introduced for 2-10 minutes before sealing, and the Glaser coupling reaction time is 8-15 hours.

[0018] Preferably, in step (2), the SEA antibody is first modified by azidation: the SEA antibody and N3-PEG4-NHS ester are mixed at a mass ratio of 1:1 and incubated in a shaker at 4 °C for 2-4 hours; after the reaction, excess reagents are removed by ultrafiltration and dissolved in phosphate buffer solution to obtain the azidated SEA antibody; then, an immunosensor is assembled using an HsGDY / Cu2O photocathode: the azidated SEA antibody is dropped onto the HsGDY / Cu2O photocathode prepared in step (1), incubated at 4 °C, and then washed with phosphate buffer solution to obtain a photocathode modified with SEA antibody probe; next, DPT is dropped onto the surface of the photocathode and incubated at 4 °C to form an anti-biocontamination interface through efficient click chemistry reaction; unreacted or physically adsorbed DPT is removed by washing with phosphate buffer solution to finally obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

[0019] It should be noted that the above-mentioned photoelectrochemical immunosensor based on dendritic peptide tetramers not only has mild construction conditions, simple and rapid process steps, and low biological sample consumption, but also has simple operation for detecting the target analyte SEA, requires no purification, and is highly sensitive, resistant to biocontamination, accurate, and rapid.

[0020] Further preferred, the concentration of SEA azide for low-temperature incubation is 50~300 μg / mL, and the low-temperature incubation time is 1~4 hours; the concentration of DPT for low-temperature incubation is 0.1~0.5 mg / mL, and the low-temperature incubation time is 1~4 hours.

[0021] As an example, the preferred preparation method of the present invention is as follows: (1) Using Cu2O nanorod array electrodes that absorb visible light as a substrate, hydrogen-substituted graphyne (HsGDY), a carbon-based semiconductor material with excellent conductivity, is modified on the surface of the substrate as a sensitizer to prepare an HsGDY / Cu2O photocathode. 1) Preparation of Cu2O nanorod array electrodes by anodic oxidation and high-temperature annealing: The copper substrate was annealed in 3.0 M NaOH solution at 10 mA / cm 2 Anodizing was performed at a constant current density for 5 minutes, followed by annealing the anodized copper substrate at 550 °C for 4 hours in a tube furnace under nitrogen protection to obtain Cu2O nanorod array electrodes.

[0022] 2) HsGDY was grown on the surface of the Cu2O nanorod array electrode via Glaser coupling reaction: 2.5 mg of triacetylenebenzene and 5 μL of piperidine were dissolved in 5 mL of pyridine and placed in a brown conical flask; the Cu2O nanorod array electrode was immersed in the pyridine reaction solution, and oxygen was introduced for 3 minutes before sealing; the system was heated to 60 °C in an oil bath and kept at that temperature for 12 hours to allow the Glaser coupling reaction to proceed fully on the electrode surface; after the reaction was completed, the electrode was washed sequentially with pyridine, dichloromethane and methanol, and dried under a nitrogen gas flow to obtain the HsGDY / Cu2O photocathode.

[0023] (2) The Staphylococcus aureus enterotoxin A antibody probe and dendritic peptide tetramer are successively anchored to the HsGDY / Cu2O photocathode prepared in step (1) to obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

[0024] 1) First, the SEA antibody was modified by azidation: The SEA antibody and N3-PEG4-NHS ester were mixed at a mass ratio of 1:1 and incubated on a shaker at 4 °C for 2 hours. After the reaction, excess reagents were removed by ultrafiltration, and the mixture was dissolved in phosphate buffer solution to obtain the azidated SEA antibody. N3-PEG4-NHS ester is a bifunctional linker containing an azido group (N3-) and an N-hydroxysuccinimide (NHS) ester group, wherein the NHS ester can react with the primary amino group of the antibody to form a stable amide bond.

[0025] 2) Assembling an immunosensor using an HsGDY / Cu2O photocathode: The 150 μg / mL azide-modified SEA antibody was added to the HsGDY / Cu2O photocathode prepared in step (1), incubated at 4 °C for 2 hours, and then washed with phosphate buffer solution to obtain a photocathode modified with SEA antibody probe; then, DPT at a concentration of 0.2 mg / mL was added to the surface of the photocathode and incubated at 4 °C for 2 hours to form an anti-biocontamination interface through efficient click chemistry reaction; unreacted or physically adsorbed DPT was removed by washing with phosphate buffer solution to finally obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

[0026] Another objective of this invention is to provide the application of the above-mentioned photoelectrochemical immunosensor based on dendritic peptide tetramers in foodborne pathogen detection products.

[0027] In some application scenarios, the photoelectrochemical immunosensor based on dendritic peptide tetramers is also used to detect Staphylococcus aureus enterotoxin A (SEA) in milk powder samples.

[0028] Furthermore, the testing environment for the photoelectrochemical immunosensor based on dendritic peptide tetramers is a buffer solution with a pH of 6.5 to 7.5, and dissolved oxygen is used as the electron acceptor.

[0029] As can be seen from the above technical solution, compared with the prior art, the present invention provides a photoelectrochemical immunosensor based on dendritic peptide tetramers, its preparation method and application, which has the following superior effects: 1) The immunosensor prepared in this invention is the first to use dendritic peptide tetramers resistant to biocontamination to construct a photoelectrochemical immunosensor. Its highly ordered branched structure can enhance intramolecular hydrogen bond interactions and promote the formation of a more stable and dense hydration layer. Compared with traditional linear peptides, it further improves the sensor's resistance to biocontamination, sensitivity and accuracy.

[0030] 2) The immunosensor prepared by the present invention has the significant advantages of simple device, convenient operation, fast signal response, low background interference and self-powered system. At the same time, it utilizes the excellent anti-biocontamination sensing interface of dendritic peptide tetramer and the obvious steric hindrance effect of target analyte SEA on the obstacle of sensor charge transfer to realize photocurrent signal detection.

[0031] 3) The photoelectrochemical immunosensor based on dendritic peptide tetramers provided by this invention has a simple and efficient preparation process, mild conditions, easy operation, and low cost. It can detect the target analyte SEA without the need for actual sample purification, which is convenient and fast. Moreover, this immunosensor has the potential to accurately and sensitively detect SEA in actual food matrices, making it suitable for market promotion and application. It has profound significance for the detection of foodborne pathogens. Attached Figure Description

[0032] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments 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 The image shows the photocurrent signal response of the HsGDY / Cu2O photocathode for different Glasser coupling reaction times in Example 1.

[0034] Figure 2 The image shows the photocurrent signal response of the modified photocathode corresponding to different incubation concentrations of SEA antibody probe in Example 2.

[0035] Figure 3 The image shows the photocurrent signal response of the modified sensing electrode corresponding to different incubation concentrations of the anti-pollution element DPT in Example 3.

[0036] Figure 4 The images show scanning electron microscope (SEM) and transmission electron microscope (TEM) images of the HsGDY / Cu2O photocathode fabrication process in Example 4.

[0037] Figure 5 The images show the X-ray photoelectron spectrum and Raman spectrum of the HsGDY / Cu2O photocathode preparation process in Example 4.

[0038] Figure 6 The images show the photocurrent signal response and photocurrent stability during the fabrication process of the HsGDY / Cu2O photocathode in Example 5.

[0039] Figure 7 The images show the photocurrent signal and electrochemical impedance spectroscopy of the photoelectrochemical immunosensor fabrication process described in Example 6.

[0040] Figure 8 The above are the signal response curves and standard curves of the photoelectrochemical immunosensor for the detection of the target analyte described in Example 7.

[0041] Figure 9 This is an optical microscopic image of the fluorescent protein adsorption of the photoelectrochemical immunosensor described in Example 8.

[0042] Figure 10 This is a graph showing the test data of the anti-contamination performance of the photoelectrochemical immunosensor described in Example 8 on a food matrix.

[0043] Figure 11 This is a graph showing the test data of the photoelectrochemical immunosensor's specific recognition performance of the target analyte described in Example 9. Detailed Implementation

[0044] 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.

[0045] First, it should be noted that the current signal response in the following examples was tested on a photoelectrochemical system, as detailed below: It emits white light with a wavelength of 200 nm to 2500 nm and a light intensity of 150 W / m. 2 A xenon lamp was used as the excitation source, and it was turned on and off every 10 seconds. The photocurrent was recorded by an electrochemical workstation. When testing the current signal response, a three-electrode system was used: a photoelectrode with a modified area of ​​0.5 cm × 0.5 cm as the working electrode, a platinum wire electrode as the counter electrode, and an Ag / AgCl electrode as the reference electrode; the system was self-powered without an external voltage, and the test solution was phosphate buffer (pH 7.4, 10 mM).

[0046] Example 1 Since the photocurrent signal response of the HsGDY / Cu2O photocathode has a significant impact on the detection sensitivity of the final dendritic peptide tetramer-based photoelectrochemical immunosensor, the main fabrication process parameters of the HsGDY / Cu2O photocathode were optimized: Since the amount of HsGDY modification on the photoelectrode can be adjusted by the Glaser coupling reaction time, the Glaser coupling reaction time is optimized as follows: 2.5 mg of triacetylenebenzene and 5 μL of piperidine were dissolved in 5 mL of pyridine and placed in a brown conical flask. The Cu2O nanorod array electrode was immersed in the pyridine reaction solution, and oxygen was introduced for 3 minutes before sealing. The system was heated to 60 °C in an oil bath and kept at the temperature for 9, 10, 11, 12, 13 and 14 hours respectively to allow the Glaser coupling reaction to proceed fully on the electrode surface. After the reaction was completed, the electrode was washed with pyridine, dichloromethane and methanol in sequence, and dried under a nitrogen gas flow to obtain the HsGDY / Cu2O photocathode.

[0047] The results can be obtained by performing photocurrent characterization tests, as shown in the attached figure. Figure 1 As shown, a Glaser coupling reaction time of 12 hours resulted in the optimal photocurrent signal response of the HsGDY / Cu2O photocathode. This is because an appropriate thickness of HsGDY can improve the surface conductivity of the electrode and promote charge separation at the electrode interface; while an excessively thick HsGDY layer would prolong the carrier transport path, partially block light absorption, hinder interface charge transfer, and thus weaken the photocurrent output. Therefore, a Glaser coupling reaction time of 12 hours was chosen as the optimal fabrication process parameter.

[0048] Example 2 Since the amount of SEA antibody probe modified on the photocathode has a significant impact on the quantitative detection range of the final photoelectrochemical immunosensor based on dendritic peptide tetramers, the preparation process parameters for the amount of SEA antibody probe modified on the photocathode were optimized: Since the amount of modification of the SEA antibody probe on the photocathode can be reflected by its incubation concentration on the photocathode, the incubation concentration of the SEA antibody probe was optimized.

[0049] 20 μL of azide-modified SEA antibody at concentrations of 50, 100, 150, 200, and 250 μg / mL was added to the surface of the prepared HsGDY / Cu2O photocathode, incubated at 4 °C for 2 hours, and then washed with phosphate buffer (pH 7.4, 10 mM) to obtain a photocathode modified with SEA antibody probe.

[0050] The results can be obtained by performing photocurrent characterization tests, as shown in the attached figure. Figure 2 As shown, the incubation concentration of the azide-SEA antibody needs to be greater than or equal to 150 μg / mL to ensure that the SEA antibody probe is fully anchored on the photocathode and to obtain the optimal quantitative detection range. Therefore, an SEA antibody probe concentration of greater than or equal to 150 μg / mL is selected as the optimal incubation concentration.

[0051] Example 3 Since the amount of DPT modification on the sensing electrode has a significant impact on the anti-biofouling performance of the final photoelectrochemical immunosensor based on dendritic peptide tetramers, the preparation process parameters for the amount of DPT modification on the sensing electrode were optimized: Since the amount of DPT modification on the sensing electrode can be reflected by its incubation concentration on the sensing electrode, the incubation concentration of DPT was optimized.

[0052] 20 μL of DPT at concentrations of 0.05, 0.1, 0.15, 0.2, 0.25, and 0.3 mg / mL was added to the surface of the sensing electrode modified with the SEA antibody probe and incubated at 4 °C for 2 hours to form an anti-biocontamination interface through efficient click chemistry. Unreacted or physically adsorbed DPT was removed by washing with phosphate buffer (pH 7.4, 10 mM) to finally obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

[0053] The results can be obtained by performing photocurrent characterization tests, as shown in the attached figure. Figure 3 As shown, the incubation concentration of the antifouling element DPT needs to be greater than or equal to 0.2 mg / mL to ensure that DPT is fully anchored on the sensing electrode and obtains the best antifouling performance. Therefore, a DPT concentration of greater than or equal to 0.2 mg / mL is selected as the optimal incubation concentration.

[0054] Example 4 Fabrication of HsGDY / Cu2O photocathode Cu₂O nanorod array electrodes were prepared by anodic oxidation and high-temperature annealing. A copper substrate with an exposure area of ​​0.5 cm × 0.5 cm was then subjected to annealing in 3.0 M NaOH solution at a rate of 10 mA / cm². 2 Anodizing was performed at a constant current density for 5 minutes, followed by annealing of the anodized copper substrate at 550 °C for 4 hours under nitrogen protection in a tube furnace to obtain a Cu2O nanorod array electrode. HsGDY was grown on the surface of the Cu2O electrode via a Glaser coupling reaction. 2.5 mg of triacetylenebenzene and 5 μL of piperidine were dissolved in 5 mL of pyridine and placed in a brown conical flask. The Cu2O electrode was immersed in the pyridine reaction solution, and oxygen was introduced for 3 minutes before sealing. The system was heated to 60 °C in an oil bath and held at that temperature for 12 hours to allow the Glaser coupling reaction to proceed fully on the electrode surface. After the reaction, the electrode was sequentially cleaned with pyridine, dichloromethane, and methanol, and then dried under a nitrogen atmosphere to obtain the desired HsGDY / Cu2O photocathode.

[0055] Scanning electron microscope images are attached. Figure 4As shown in Figure a, the annealed Cu₂O sample exhibits a clear and densely packed nanorod morphology, with a length of approximately 4–6 μm and a diameter of approximately 200 nm. Each nanorod is composed of tightly packed nanoparticles with a size of approximately 40–50 nm. Scanning electron microscope images are attached. Figure 4 As shown in figure b, the overall morphology of the Cu2O nanorods remains largely unchanged after HsGDY modification, but their surface becomes significantly rougher. This indicates that a thin HsGDY coating layer was successfully formed on the nanorod surface, proving the successful fabrication of the HsGDY / Cu2O photocathode. A transmission electron microscope image is attached. Figure 4 As shown in Figure c, the figure further confirms that the Cu2O nanorods are coated with an HsGDY coating with a thickness of approximately 90 nm. The elemental mapping results in the inset show that the carbon element distribution range is significantly expanded, which corroborates the coating structure of the Cu2O nanorods by HsGDY and further proves the successful preparation of the HsGDY / Cu2O photocathode.

[0056] The X-ray photoelectron spectrum of the HsGDY / Cu2O photocathode is shown in the attached figure. Figure 5 As shown in figure a, the results indicate the presence of Cu, O, and C elements in the sample, with Cu 2p 3 / 2 and Cu 2p 1 / 2 The peaks are located at approximately 933 eV and 953 eV, respectively, indicating that copper mainly exists in the +1 valence state. Compared with pure Cu₂O, the carbon signal intensity in the HsGDY / Cu₂O sample is significantly enhanced, indicating a significant increase in its surface carbon content, which corroborates the successful fabrication of the HsGDY / Cu₂O photocathode. The Raman spectrum of the HsGDY / Cu₂O photocathode is attached. Figure 5 As shown in b, at 2212 cm 1 A strong peak appears at 1580 cm⁻¹, which can be attributed to the stretching vibration of the C≡C alkyne bond; at 1580 cm⁻¹... 1 The peak at [location] corresponds to the G band of the aromatic ring. Furthermore, at 1991 cm⁻¹... 1 A weaker characteristic peak was also observed, which can be attributed to the stretching vibration of Cu–C≡C. This further confirms the successful fabrication of the HsGDY / Cu2O photocathode.

[0057] Example 5 Characterization of photoelectrochemical performance of HsGDY / Cu2O photocathode The changes in photocurrent signal response during the construction of the HsGDY / Cu2O photocathode are shown in the attached figure. Figure 6As shown in curve a, HsGDY alone produces a weak photocurrent response (curve a), which is due to the low charge separation efficiency of HsGDY itself. After in-situ growth of Cu2O nanorod arrays on a copper foil substrate, the Cu2O electrode exhibits a significant cathode photocurrent response (curve b), which is consistent with the characteristics of Cu2O as an intrinsic p-type semiconductor. When HsGDY is coated onto the Cu2O surface to form an HsGDY / Cu2O photocathode, the photocurrent response is further significantly enhanced (curve c), with the HsGDY / Cu2O photocathode showing an increase of approximately 64% compared to the Cu2O electrode. This performance improvement can be attributed to the well-matched heterostructure between Cu2O and HsGDY. This structure not only broadens the light absorption range of the photocathode but also effectively promotes the rapid separation and transport of photogenerated carriers through a stepped band structure, extending the lifetime of photogenerated carriers. The photochemical stability characterization of the HsGDY / Cu2O photocathode is attached. Figure 6 As shown in b, it can be observed that after dozens of consecutive on / off light cycles, the photocurrent intensity did not show a significant decrease, indicating that HsGDY can effectively suppress the photocorrosion behavior of Cu2O in aqueous solution, thus endowing the photocathode with excellent working stability.

[0058] Example 6 Fabrication of photoelectrochemical immunosensors based on dendritic peptide tetramers First, the SEA antibody was modified by azidation. 0.5 mL of 300 μg / mL SEA antibody was mixed with 1 mL of 150 μg / mL N3-PEG4-NHS ester and incubated at 4 °C for 2 hours on a shaker. After the reaction, excess reagents were removed by ultrafiltration, and the volume was adjusted to 1 mL with phosphate buffer to obtain the azidated SEA antibody. Subsequently, an immunosensor was assembled using an HsGDY / Cu2O photocathode. 20 μL of the 150 μg / mL azidated SEA antibody was added dropwise to the prepared HsGDY / Cu2O photocathode and incubated at 4 °C for 2 hours. The photocathode was then washed with phosphate buffer to obtain a photocathode modified with the SEA antibody probe. Next, 20 μL of 0.2 mg / mL DPT was added dropwise to the electrode surface and incubated at 4 °C for 2 hours, forming an anti-biocontamination interface on the sensing electrode through an efficient click chemistry reaction. Unreacted or physically adsorbed DPT is removed by washing with phosphate buffer solution, finally yielding the photoelectrochemical immunosensor based on dendritic peptide tetramer.

[0059] The successful fabrication of the photoelectrochemical immunosensor based on dendritic peptide tetramers was verified using photocurrent signal response testing, as shown in the attached figure. Figure 7As shown in curve a, HsGDY possesses a highly delocalized π-electron system, which promotes efficient electron transport from Cu2O to the outside world, thus significantly enhancing the cathode photocurrent and demonstrating the excellent PEC performance of the HsGDY / Cu2O photocathode (curve a). With the sequential anchoring of the antibody probe and the anti-fouling element DPT, the steric hindrance and insulation effects introduced by these biomolecules hinder interfacial electron transport to some extent, leading to a gradual decrease in photocurrent (curves b and c). When the antibody probe on the electrode surface specifically recognizes the target analyte SEA, a large immune complex is formed, further enhancing the steric hindrance of the sensing interface and inhibiting electron transport, thereby causing a significant decrease in photocurrent (curve d). Therefore, the photocurrent signal response test proves the successful fabrication of the photoelectrochemical immunosensor based on dendritic peptide tetramers.

[0060] The successful fabrication of the photoelectrochemical immunosensor based on dendritic peptide tetramers was further verified by electrochemical impedance spectroscopy, as shown in the attached figure. Figure 7 As shown in curve b, due to the excellent electronic conductivity of HsGDY, the HsGDY / Cu2O photocathode exhibits a low charge transfer resistance (curve a). After the antibody probe and the anti-fouling element DPT are sequentially anchored, the charge transfer resistance gradually increases (curve bc), reflecting the hindering effect of the insulating properties of the biomolecular layer on interfacial electron transport. Further incubation with the target compound SEA results further significantly increases the charge transfer resistance (curve d), indicating that specific antigen-antibody recognition has occurred and an immune complex with significant steric hindrance has formed. Therefore, the electrochemical impedance spectroscopy results are highly consistent with the photocurrent signal response results, further confirming the successful fabrication of the photoelectrochemical immunosensor based on dendritic peptide tetramers.

[0061] Example 7 The prepared photoelectrochemical immunosensor detects the target analyte SEA. The photoelectrochemical immunosensor based on dendritic peptide tetramers prepared in Example 6 was incubated with 20 μL of target substance SEA at different concentrations for 2 hours at room temperature, allowing the target substance SEA to undergo a specific recognition reaction with the antibody probe immobilized on the surface of the sensing electrode. After washing with phosphate buffer, the photoelectrochemical immunosensor based on dendritic peptide tetramers incubated with target substance SEA was finally tested for photocurrent signal in phosphate buffer (pH 7.4, 0.1 M). The photocurrent signal was detected by utilizing the significant steric hindrance effect of target substance SEA on the obstacle of charge transfer in the sensor.

[0062] The detection results show that as the concentration of the target analyte SEA increases, the corresponding photocurrent detection signal gradually weakens, as shown in the attached figure. Figure 8As shown in Figure a; and within the SEA concentration range of 50 pg / mL to 5000 ng / mL, the photocurrent detection signal exhibits a good linear relationship with the logarithm of the SEA concentration, as shown in the attached figure. Figure 8 As shown in b; the linear fitting equation is I= The results showed that the photoelectrochemical immunosensor based on dendritic peptide tetramers prepared according to this invention has high sensitivity for the detection of the target analyte SEA, with a linear correlation coefficient of 0.9983 and an experimental limit of detection of 18.63 pg / mL.

[0063] Example 8 Anti-biocontamination performance test of the prepared photoelectrochemical immunosensor for detecting SEA First, the anti-biocontamination performance of the prepared photoelectrochemical immunosensor for detecting SEA was evaluated through a fluorescent protein adsorption experiment. The prepared HsGDY / Cu2O photocathode, the antibody probe-anchored photocathode, and the sensing electrode further anchored by the anti-contamination element DPT were incubated with 1.0 mg / mL fluorescein isothiocyanate-labeled bovine serum albumin (BSA-FITC) for 2 hours under light-protected conditions. After washing with phosphate buffer, optical imaging analysis was performed. A confocal laser scanning microscope image of the HsGDY / Cu2O photocathode is attached. Figure 9 As shown in Figure a, the bright green fluorescence on the electrode surface indicates significant nonspecific protein adsorption; a confocal laser scanning microscope image of the photocathode anchored to the antibody probe is attached. Figure 9 As shown in b, the fluorescence intensity on the electrode surface is significantly reduced, indicating that non-specific protein adsorption is suppressed to some extent; a confocal laser scanning microscope image of the sensing electrode further anchored by the anti-fouling element DPT is attached. Figure 9 As shown in c, the electrode surface shows almost no green fluorescence, indicating that its non-specific adsorption to proteins is extremely low, highlighting its excellent anti-fouling performance.

[0064] The photoelectrochemical immunosensor for detecting SEA was further evaluated for its anti-biocontamination performance in a real food matrix using photocurrent signal response testing. The DPT-modified sensing electrode prepared in Example 6 was incubated with 20 μL of milk powder samples at different dilutions for 2 hours at room temperature. After washing the electrode with phosphate buffer, photocurrent signal response testing was performed. For comparison, sensing electrodes without DPT modification and those modified with the linear peptide CLP (peptide sequence (N3-)KPPPPEKEKEKEKEK) were also incubated with milk powder samples at different dilutions under the same conditions and then washed. The changes in photocurrent signals are shown in the attached figure. Figure 10As shown, the DPT-modified sensing electrode exhibited minimal signal change (less than 5%) even after incubating with 15% (w / v) milk powder samples, demonstrating its significant resistance to biocontamination. In contrast, the linear peptide CLP-modified sensor showed a significantly increased signal change, approaching its performance limit even after incubation with 5% (w / v) milk powder samples; while the unmodified peptide sensor showed the greatest signal change, indicating that the sensor surface was more susceptible to non-specific adsorption. These comparative results fully demonstrate the excellent resistance to biocontamination of the DPT-modified immunosensor in real food matrices.

[0065] Example 9 Specific recognition performance test of the prepared photoelectrochemical immunosensor for detecting SEA To demonstrate the excellent specificity of the aforementioned photoelectrochemical immunosensor for detecting SEA, representative interfering molecules—Staphylococcal enterotoxin B (SEB), Staphylococcal enterotoxin C1 (SEC1), T-2 mycotoxin (T-2), aflatoxin B1 (AFB1), serum albumin (BSA), immunoglobulin G (IgG), and casein—were selected as typical interferants for specificity verification experiments. The specific procedures are as follows: Take 1 ng / mL of the target analyte SEA and 10 ng / mL of interfering molecules SEB, SEC1, T-2, AFB1, BSA, IgG, and Casein, either separately or in combination, and add them to phosphate buffer (pH 7.4, 10 mM). Detect SEA using the photoelectrochemical immunosensor prepared according to this invention, following the method described above. The changes in photocurrent signals are shown in the attached figure. Figure 11 As shown in the figure. The results indicate that, relative to the detection of the target analyte SEA, the changes in photocurrent generated by interfering molecules SEB, SEC1, T-2, AFB1, BSA, IgG, Casein, and their mixtures are negligible. This demonstrates that the photoelectrochemical immunosensor for detecting SEA prepared in this invention has excellent specific recognition ability for the target analyte SEA and is unaffected by interfering proteins.

[0066] Example 10 Application of the prepared photoelectrochemical immunosensor for detecting SEA in milk powder samples The practical application capability of the photoelectrochemical immunosensor for detecting SEA prepared in this invention was evaluated through a spiked recovery experiment in milk powder samples. The specific operation is as follows: First, a 10% (w / v) skim milk powder solution was prepared and divided into five groups. Four groups were supplemented with SEA standard samples at concentrations of 500 ng / mL, 100 ng / mL, 20 ng / mL, and 1 ng / mL, respectively, while the other group was not supplemented with SEA. Using the photoelectrochemical immunosensor based on dendritic peptide tetramers prepared in Example 6, 20 μL of the above-mentioned milk powder spiked samples at different concentrations were incubated at room temperature for 2 hours. After cleaning the electrode, the photocurrent signal was measured in phosphate buffer (pH 7.4, 0.1 M) and compared with the photoelectrochemical detection performance of Example 7.

[0067] The results of the spiked recovery experiment in milk powder samples are shown in Table 1. The recovery rate of the spiked samples ranged from 94.3% to 105.6%, and the relative standard deviation of the test results was less than 4.6%. This proves that the prepared photoelectrochemical immunosensor based on dendritic peptide tetramer has excellent application potential for accurate detection of target analytes SEA in actual complex food matrices, and can achieve rapid, sensitive, accurate and efficient detection of target analytes.

[0068] Table 1. Detection data of target substances in milk powder samples by the photoelectrochemical immunosensor described in Example 10.

[0069] 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. A photoelectrochemical immunosensor based on dendritic peptide tetramers, characterized in that, The photoelectrochemical immunosensor is fabricated by sequentially anchoring an antibody probe that specifically recognizes Staphylococcus aureus enterotoxin A (SEA) and a dendritic peptide tetramer (DPT) with anti-biocontamination properties on a food matrix onto a photocathode. The photocurrent signal is detected by utilizing the significant steric hindrance effect of Staphylococcus aureus enterotoxin A to impede charge transfer in the sensor.

2. The photoelectrochemical immunosensor based on dendritic peptide tetramers according to claim 1, characterized in that, The dendritic peptide tetramer has a multi-branched topology and four equivalent terminal arms, with the amino acid sequence (N3-)KPPPPEK(EK(EKEKEK)2)2; wherein the rigid anchoring sequence (N3-)KPPPPEK is the backbone; four identical EKEKEK are the main branches, and the main chain is connected by two secondary branch EK units.

3. A method for preparing a photoelectrochemical immunosensor based on dendritic peptide tetramers as described in claim 1, characterized in that, The specific steps include: (1) Preparation of HsGDY / Cu2O photocathode: Using Cu2O nanorod array electrodes with visible light absorption as substrate, hydrogen-substituted graphyne (HsGDY), a carbon-based semiconductor material with excellent conductivity, is modified on the surface of the substrate as a sensitizer to prepare HsGDY / Cu2O photocathode; (2) Staphylococcus aureus enterotoxin A (SEA) antibody probe and dendritic peptide tetramer (DPT) are successively anchored to the HsGDY / Cu2O photocathode prepared in step (1) to obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

4. The method for preparing a photoelectrochemical immunosensor based on dendritic peptide tetramers according to claim 3, characterized in that, In step (1), Cu2O nanorod array electrodes are prepared by anodizing and high-temperature annealing: the copper substrate is anodized in 3.0M NaOH solution in a constant current manner, and then the anodized copper substrate is annealed in a tube furnace under nitrogen protection to obtain Cu2O nanorod array electrodes. HsGDY was grown on the surface of the Cu2O nanorod array electrode via a Glaser coupling reaction: 2.5 mg of triacetylenebenzene and 5 μL of piperidine were dissolved in 5 mL of pyridine and placed in a brown conical flask; the Cu2O nanorod array electrode was immersed in the pyridine reaction solution, and oxygen was introduced before sealing; the system was heated to 60 °C in an oil bath and kept at a constant temperature to allow the Glaser coupling reaction to proceed fully on the electrode surface; after the reaction was completed, the electrode was washed sequentially with pyridine, dichloromethane and methanol, and dried under a nitrogen gas flow to obtain the desired HsGDY / Cu2O photocathode.

5. The method for preparing a photoelectrochemical immunosensor based on dendritic peptide tetramers according to claim 4, characterized in that, The Cu₂O nanorod array electrodes were prepared using 8–12 mA / cm². 2 The constant current density, anodizing time of 3~10 minutes, annealing time of 2~6 hours, and annealing temperature of 550 °C; Furthermore, when preparing the HsGDY / Cu2O photocathode, oxygen is introduced for 2-10 minutes before sealing, and the Glaser coupling reaction takes 8-15 hours.

6. The method for preparing a photoelectrochemical immunosensor based on dendritic peptide tetramers according to claim 3, characterized in that, In step (2), the SEA antibody is first modified by azidation: the SEA antibody and N3-PEG4-NHS ester are mixed at a mass ratio of 1:1 and incubated in a shaker at 4 °C for 2 to 4 hours; after the reaction is completed, excess reagents are removed by ultrafiltration and dissolved in phosphate buffer solution to obtain the azidated SEA antibody. Then, an photoelectrochemical immunosensor was assembled using an HsGDY / Cu2O photocathode: the azide-treated SEA antibody was dropped onto the HsGDY / Cu2O photocathode prepared in step (1), incubated at 4 °C, and then washed with phosphate buffer solution to obtain a photocathode modified with SEA antibody probe; next, dendritic peptide tetramer (DPT) was dropped onto the surface of the photocathode and incubated at 4 °C to form an anti-biocontamination sensing interface through efficient click chemistry reaction; unreacted or physically adsorbed dendritic peptide tetramer (DPT) was removed by washing with phosphate buffer solution to finally obtain the photoelectrochemical immunosensor based on dendritic peptide tetramer.

7. The method for preparing a photoelectrochemical immunosensor based on dendritic peptide tetramers according to claim 6, characterized in that, The concentration of SEA azidiones for low-temperature incubation is 50–300 μg / mL, and the incubation time is 1–4 hours; the concentration of dendritic peptide tetramers (DPT) for low-temperature incubation is 0.1–0.5 mg / mL, and the incubation time is 1–4 hours.

8. The application of a photoelectrochemical immunosensor based on dendritic peptide tetramer as described in claim 1, or a photoelectrochemical immunosensor based on dendritic peptide tetramer prepared by any one of claims 3 to 7, in the detection of foodborne pathogens.

9. The application according to claim 8, characterized in that, The application of the photoelectrochemical immunosensor based on dendritic peptide tetramers in detecting Staphylococcus aureus enterotoxin A (SEA) in milk powder samples.

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