The Construction And Biosensing Application Of Antifouling Sensing Interfaces Functionalized With Peptides

Electrochemical biosensors have broad application prospects in food safety,environmental detection,early disease diagnosis and many other fields,owing to their advantages of simple operation,low cost and high sensitivity.However,in many cases,electrochemical biosensors need to be performed in complex biological environments(serum,whole blood,urine,etc.)to detect biomarkers,and the concentrations of those biomarkers(specific proteins)tend to be very low compared to a large number of coexisting background materials(such as cells,proteins,nucleic acids,etc.).This will require the electrochemical biosensors to be of high sensitivity,and severe biofouling may even cause malfunction of the biosensors.Therefore,it is necessary to develop biosensors with excellent antifouling performance.In this thesis,a series of functional peptides combined with conductive composite materials were designed to construct antifouling sensing interfaces.It can realize specific recognition of biomarkers(IgG,HER2,etc.)in complex biological fluids,and expand the application of biosensors in clinical sample analysis.The main contents of this thesis are as follows:(1)Alpha-aminoisobutyric acid incorporated peptides for the construction of electrochemical biosensors with high stability and low fouling in serum for the detection of IgG.An effective strategy to construct low fouling electrochemical biosensors for assaying serum biomarkers was proposed based on specially designedα-aminoisobutyric acid(Aib)incorporated peptides.The Aib-peptides were designed to be of antifouling properties,and at the same to incorporate Aib residues in their interior to enhance the hydrolytic stability.In order to construct the electrochemical biosensor,two kinds of Aib-peptides labelled with biotin were modified on the electrode surface:One with cysteine terminal for easy attachment to the electrode modified with gold nanoparticles,the other with unique terminal peptide sequence for specific binding of immunoglobulin G(IgG),and they were connected through the streptavidin-biotin affinity system.Owing to the interposition of Aib residues,the peptides as well as the constructed biosensors showed excellent antifouling performances and enhanced stability against enzymatic degradation in serum.Furthermore,the IgG biosensor constructed with the Aib-peptides displayed a very low detection limit(29.5 pg mL-1)and a broad linear range(100 pg mL-1-10 μg mL-1),and it was able to assay IgG in clinical human sera with decent accuracy and reliability.This strategy provides a new path for the construction of stable antifouling biosensors based on functional peptides for practical biomarker assaying in real clinical samples.(2)An antifouling electrochemical biosensor based on two kinds of antifouling materials of designed recognizing peptide and PEG for the detection of human epidermal growth factor receptor-2.A simple tactic for electrochemical determination of a typical biomarker for breast cancer,human epidermal growth factor receptor-2(HER2),was presented via the construction of a low fouling sensing interface functionalized with PEG and peptide.The HER2 biosensor was developed based on an electrode modified by the conducting polymer poly(3,4-ethylenedioxythiophene)(PEDOT)and Au nanoparticles(AuNPs)as the sensing substrate,and followed by the immobilization of an antifouling PEG and a peptide with both recognizing and antifouling properties.Thanks to the combined antifouling effect of the PEG and peptide,and the specific recognizing ability of the peptide to the target HER2,the developed electrochemical biosensor exhibited strong antifouling performances in complex biofluids,such as human blood and serum,and it was capable of assaying target HER2 within a very wide linear range(1.0 pg mL-1 to 1.0 μg mL-1),with an ultralow limit of detection(0.44 pg mL-1).The combination of two kinds of antifouling biomaterials(PEG and peptide)offered an effective strategy for the development of low fouling sensing platforms suitable for practical assay in complex biotic environments.(3)An antifouling electrochemical biosensor based on peptide functionalized conducting polymer PEDOT doped with PEG for the detection of extracellular signal-regulated kinase-2.Kinases are important cancer biomarkers that regulate cellular physiological processes based on phosphorylation of multiple substrate proteins.However,biosensors based on catalytic activity lack good selectivity.Therefore,an electrochemical method for sensing kinases based on the interaction between docking sites and specific proteins was proposed.The biocompatible and easily modified PEG-doped conductive polymer PEDOT was codeposited on the electrode surface.The conductive composite(PEDOT-PEG)has strip nanostructure,large surface area and excellent stability.This structure is conducive to the fixation of multifunctional peptides for more sensitive detection.The biosensor designed in this study can identify the biomarker extracellular signal regulated kinase-2(ERK2)in the complex biological fluids with high selectivity,wide detection range(10.0 pg mL-1 to 10.0 μg mL-1)and low detection limit(3.548 pg mL-1).ERK2 biosensor based on antifouling materials(PEG)doped with conductive polymer(PEDOT),and combined with multifunctional peptide,which has a broad application prospect in clinical environment.