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CN-121978183-A - Photoelectrochemical immunosensor based on dendritic peptide tetramer, and preparation method and application thereof

CN121978183ACN 121978183 ACN121978183 ACN 121978183ACN-121978183-A

Abstract

The invention belongs to the technical field of biosensors, and discloses a photoelectrochemical immunosensor based on dendritic peptide tetramers, and a preparation method and application thereof. The invention adopts the dendritic peptide tetramer to construct the photoelectrochemical immunosensor for the first time, and comprises the steps of preparing HsGDY/Cu 2 O photocathode, and sequentially anchoring an antibody probe and the dendritic peptide tetramer on the surface of the photocathode. Through the specific combination of the target object and the antibody probe, a remarkable steric hindrance effect is generated at the electrode interface, interface charge transfer is hindered, photocurrent signal change is caused, and the detection of the target object staphylococcus aureus enterotoxin A is realized. The photoelectrochemical immunosensor has simple and efficient preparation process, has excellent capability of resisting food matrix biological pollution, can effectively inhibit nonspecific adsorption, thereby remarkably improving the sensitivity, specificity and accuracy of detection, is suitable for the field of food safety detection, and is suitable for popularization and application in the market.

Inventors

  • LI WEI
  • FAN GAOCHAO
  • LUO XILIANG

Assignees

  • 青岛科技大学

Dates

Publication Date
20260505
Application Date
20260130

Claims (9)

  1. 1. A photoelectrochemical immunosensor based on dendritic peptide tetramers, which is characterized in that the photoelectrochemical immunosensor is prepared by sequentially anchoring an antibody probe for specifically recognizing staphylococcus aureus enterotoxin A (SEA) and Dendritic Peptide Tetramer (DPT) with anti-food matrix biological pollution performance to a photocathode, and photocurrent signal detection is realized by utilizing the obvious steric hindrance effect of the staphylococcus aureus enterotoxin A on the blocking effect of sensor charge transfer.
  2. 2. The photoelectrochemical immunosensor based on dendrimers according to claim 1, wherein the dendrimer has a multi-branched topology and four equivalent terminal arms, the amino acid sequence is (N 3 -)KPPPPEK(EK(EKEKEK) 2 ) 2 ; wherein the rigid anchoring sequence (N 3 -) KPPPPEK is the backbone, four identical EKEKEK branches are the main branches, and the backbone is linked by 2 secondary branch EK units.
  3. 3. A method of preparing a dendrimer-based photoelectrochemical immunosensor according to claim 1, comprising the specific steps of: (1) Preparing HsGDY/Cu 2 O photocathode, namely preparing HsGDY/Cu 2 O photocathode by taking a Cu 2 O nanorod array electrode absorbed by visible light as a substrate, and modifying carbon-based semiconductor material hydrogen with excellent conductivity on the surface of the substrate to replace graphite alkyne (HsGDY) as a sensitizer; (2) And (3) sequentially anchoring a staphylococcus aureus enterotoxin A (SEA) antibody probe and a Dendritic Peptide Tetramer (DPT) to the HsGDY/Cu 2 O photocathode prepared in the step (1), so as to obtain the photoelectrochemical immunosensor based on the dendritic peptide tetramer.
  4. 4. The method for preparing the photoelectrochemical immunosensor based on the dendrimer peptide according to claim 3, wherein in the step (1), the Cu 2 O nanorod array electrode is prepared by anodic oxidation and high-temperature annealing, wherein a copper substrate is subjected to anodic oxidation in a 3.0M NaOH solution in a constant current mode, and then the anodized copper substrate is subjected to annealing under the protection of nitrogen in a tube furnace, so that the Cu 2 O nanorod array electrode is obtained; Growing HsGDY on the surface of the Cu 2 O nano-rod array electrode through a glass coupling reaction, dissolving 2.5 mg triacetyl benzene and 5 mu L of piperidine in 5 mL pyridine, placing the pyridine in a brown conical flask, immersing the Cu 2 O nano-rod array electrode in the pyridine reaction solution, introducing oxygen before sealing, heating the system to 60 ℃ in an oil bath and keeping the temperature constant, fully carrying out the glass coupling reaction on the surface of the electrode, cleaning the electrode with pyridine, dichloromethane and methanol in sequence after the reaction is finished, and drying the electrode by blowing under a nitrogen flow to obtain the required HsGDY/Cu 2 O photocathode.
  5. 5. The method for preparing the photoelectrochemical immunosensor based on the dendrimer peptide tetramer according to claim 4, wherein the constant current density of 8-12 mA/cm 2 is adopted for preparing the Cu 2 O nanorod array electrode, the anodic oxidation time is 3-10 minutes, the annealing time is 2-6 hours, and the annealing temperature is 550 ℃; And when the HsGDY/Cu 2 O photocathode is prepared, oxygen is introduced for 2-10 minutes before sealing, and the time for performing a Glaser coupling reaction is 8-15 hours.
  6. 6. The preparation method of the photoelectrochemical immunosensor based on the dendrimer peptide tetramer, which is characterized in that in the step (2), firstly, the SEA antibody is subjected to azide modification, wherein the SEA antibody and N 3 -PEG4-NHS ester are mixed according to the mass ratio of 1:1, and are incubated for 2-4 hours by a4 ℃ shaking table; then, utilizing HsGDY/Cu 2 O photocathode to assemble the photoelectrochemical immunosensor, dropwise adding the azide SEA antibody to the HsGDY/Cu 2 O photocathode prepared in the step (1), incubating at 4 ℃, then washing with phosphate buffer solution to obtain a photocathode modified by SEA antibody probes, then, dropwise adding Dendritic Peptide Tetramer (DPT) to the surface of the photocathode, incubating at 4 ℃, forming an anti-biological pollution sensing interface through efficient click chemistry reaction, and washing and removing unreacted or physically adsorbed Dendritic Peptide Tetramer (DPT) through phosphate buffer solution to finally obtain the photoelectrochemical immunosensor based on the dendritic peptide tetramer.
  7. 7. The preparation method of the photoelectrochemical immunosensor based on the dendrimer peptide according to claim 6, wherein the concentration of the low-temperature incubation of the azide SEA is 50-300 mug/mL, the low-temperature incubation time is 1-4 hours, the concentration of the low-temperature incubation of the Dendrimer Peptide Tetramer (DPT) is 0.1-0.5 mg/mL, and the low-temperature incubation time is 1-4 hours.
  8. 8. Use of a dendrimer-based photoelectrochemical immunosensor as claimed in claim 1 or prepared by a method as claimed in any one of claims 3 to 7 in the detection of food borne pathogenic bacteria.
  9. 9. The use according to claim 8, characterized in that the photoelectrochemical immunosensor based on dendrimers detects staphylococcus aureus enterotoxin a (SEA) in a milk powder sample.

Description

Photoelectrochemical immunosensor based on dendritic peptide tetramer, and preparation method and application thereof Technical Field The invention belongs to the technical field of biosensors, and relates to a photoelectrochemical immunosensor based on dendritic peptide tetramers, and a preparation method and application thereof. Background Food-borne diseases remain a major public health problem worldwide, where pathogenic bacteria and toxins produced thereby pose a serious threat to human health and bring about a significant economic loss to the food industry. Among these toxins, staphylococcus aureus enterotoxin a (SEA) is one of the most toxic and most widely distributed enterotoxins produced by staphylococcus aureus. Even nanogram-grade trace SEA can cause acute gastrointestinal symptoms such as nausea, vomiting, abdominal cramps and diarrhea. Therefore, sensitive and reliable detection of SEA is of great importance for guaranteeing food safety and maintaining public health. The analytical techniques currently used for SEA assays mainly include enzyme-linked immunosorbent, mass spectrometry, fluorescence analysis and lateral flow immunoassay. However, these methods generally have problems of complicated sample pretreatment process, long detection period, low sensitivity, and susceptibility to biological contamination of food substrates, and limit popularization in practical application. Therefore, there is an urgent need to develop a sensor that is simple to operate, efficient, and has excellent anti-contamination capability for achieving accurate detection of SEA in a real sample. Among existing detection strategies, photoelectrochemical (PEC) immunosensory technology has become a potential alternative due to its advantages of high sensitivity, low background noise, fast response speed, and easy integration with portable analytical devices. In the PEC detection process, the photoactive semiconductor electrode converts an optical signal into an electrical signal, and the signal can be modulated through specific antigen-antibody interaction, so that the sensitive detection of SEA is realized, and the limitations of the traditional detection method are effectively relieved. Despite the above advantages, the practical application of PEC immunosensor in food safety monitoring is limited by two key problems, namely (i) insufficient stability of semiconductor photoelectrodes under working conditions and (ii) non-specific adsorption of sample matrix components on electrode surfaces, resulting in significant signal drift and false positive results. There is a need to reasonably integrate high stability photoactive materials with advanced anti-fouling strategies to solve the above problems. Photoelectrodes can be generally classified into photoanode and photocathode. Compared with a photo-anode, the photo-cathode-based immunosensor relies on electron reduction reactions occurring at the electrode/electrolyte interface, and is capable of effectively resisting interference of reducing substances in biological samples in mechanism, so that the photo-cathode-based immunosensor has remarkable practical potential. The construction of photocathodes is generally based on p-type semiconductors, in which holes as the main carriers participate in the charge transport process. Cu 2 O is a p-type semiconductor material with good application prospect, and has strong light absorption capability, narrow band gap and higher theoretical photocurrent density. However, cu 2 O still faces problems of fast electron-hole recombination rate, serious photo-corrosion, etc. in practical application, thereby limiting its photoelectrochemical properties. In recent years, hydrogen-substituted graphite alkynes (HsGDY) have received widespread attention as a novel carbon-based semiconductor material having a highly conjugated pi-electron network, a controllable electron structure, and excellent chemical stability under aqueous phase and light conditions. HsGDY is not only favorable for forming a uniform film on the surface of a copper substrate, but also can be used as a protective layer to effectively improve the structural stability of a photocathode, promote carrier separation and accelerate the electron transmission process. The above characteristics make HsGDY an ideal functional material for improving the performance and durability of the photocathode PEC system. Because of the high complexity of the food matrix itself, which typically contains a variety of components, such as proteins, carbohydrates, lipids, and reducing substances, accurate quantification of SEA in real food samples remains a significant challenge, and these components tend to interfere with analytical signals. In order to solve the long-standing problem of non-specific adsorption, the introduction of an anti-fouling material has become a widely adopted and effective strategy. Among the numerous anti-fouling materials, zwitterionic peptides are believed to have excel