New Antifouling Electrochemical Biosensors Based On Functional Peptides

Accurate and sensitive detection of disease markers is important for timely diagnosis and treatment of diseases.Electrochemical biosensors have attracted great attention in quantitative detection of disease markers due to their simple operation and high sensitivity.However,for the practical sensing application in complex systems such as blood,serum,saliva or sweat,electrochemical biosensors are susceptible to biofouling on their sensing interfaces.Therefore,it is quite essential to construct an electrochemical biosensor which can prevent biofouling.As a kind of biomaterial,peptide has the advantages of easy synthesis,structural versatility and outstanding biocompatibility,and it is a kind of promising antifouling material.In this thesis,a series of antifouling peptides was designed,and they were combined with different conducting polymers to construct antifouling biosensors for the detection of disease biomarkers in complex biological fluids.The main content of this thesis includes the following three parts:(1)A biosensor based on peptide nucleic acid and antifouling peptide was proposed for the non-fouling detection of COVID-19 nucleic acid in saliva.In this work,based on the conducting polymer polyaniline(PANI)modified glassy carbon electrode(GCE),linear peptide CPPPPEKEKEKEK was used as an antifouling material and peptide nucleic acid(PNA)was used as a recognition probe to construct a low-fouling electrochemical biosensor for the specific detection of novel coronavirus RNA in saliva.Due to the negatively charged DNA recognition probe usually produces electrostatic adsorption with the positively charged biomolecules,resulting in biofouling of the sensing interface,thus reducing the accuracy and sensitivity of the biosensor.However,the electrically neutral PNA recognition probe has no side-effect on the sensing interface.Most importantly,the target RNA was diluted with 100% saliva and phosphate buffered solution(PBS)respectively,and its sensing performance was highly consistent,indicating that the biosensor constructed by combining antifouling straight-chain peptide and PNA could achieve accurate detection of novel coronavirus RNA in saliva.Therefore,the sensor showed excellent antifouling ability and significant accuracy.It has a wide linear range(1.0 f M-1.0 n M),and the LOD can reach 0.38 f M.In addition,the test process does not require direct contact between health care workers and patients,thus reducing the risk of infection for health care workers,and provides a new idea for the development of a new contactless nucleic acid test.(2)A highly sensitive and low-fouling electrochemical biosensor was developed for immunoglobulin G detection in human serum based on antifouling recognition branched peptide.In this work,in order to improve the recognition efficiency and sensitivity of target RNA,we innovatively designed a branch peptide biosensor with antifouling main chain(EKEKEK)and two recognition branches peptide(FYWHCLDE,FYCHTIDE)for the detection of immunoglobulin G(Ig G)based on conductive polymer(PEDOT)modified GCE.Due to the synergistic effect and the large steric hindrance effect of the two recognition branching peptides,the sensitivity and antifouling ability of the sensor are significantly improved.Under the optimal experimental conditions,the linear response range of the dual-recognition peptide biosensor was 0.1pg/m L to 0.1 μg/m L,and the detection limit was 0.031 pg/m L(about 2 orders of magnitude lower than that of the single-recognition peptide biosensor).In addition,the biosensor is able to detect Ig G in real biological samples of human serum without significant biofouling.This biosensor construction strategy ensures the super sensitivity of target,and effectively avoids biofouling of sensing interface in complex biological media.(3)An antifouling electrochemical biosensor based on cyclic stapled peptide was constructed for the detection of carcinoembryonic antigen in serum.In this work,in order to improve the stability of the antifouling peptide-based biosensor in complex serum and blood,two kinds of unnatural amino acids were introduced to connect the common straight-chain peptide(CPPPPEKEKEK)into a cyclic peptide,which can resist the hydrolysis of protease and thus improve the stability of the antifouling peptide-based biosensor.The mercapto modified stapled peptides were self-assembled onto the surface of the PEDOT polymer modified by gold nanoparticles(Au NPs)via the Au-S bond.Since the cyclic stapled peptide is an all-hydrocarbon cross-linked cyclic peptide withα-helix structure,the stable α-helical structure can resist the hydrolysis of the peptides.Compared with biosensors based the ordinary straight-chain peptides,the cyclic stapled peptide-based electrochemical biosensor showed enhanced stability and antifouling performance.The biosensor was able to detect carcinoembryonic antigen(CEA)sensitively in the concentration range of 1.0 pg/m L to 0.1 μg/m L,with the LOD of 0.49pg/m L(S/N = 3).The strategy of constructing antifouling biosensors with cyclic peptides provides a new way for clinical detection of disease markers in human blood or other complex environments.