| Recently,the identification of specific biomarkers in complex biological media for determining disease characteristics and circumstances has become a research hot spot in the field of clinical medicine and has also facilitated the advance of high-performance diagnostic technologies.Achieving real-time and timely detection of biomarkers has placed new demands on biochemical analyses,such as nucleic acid analysis,protein analysis and cellular analysis.In the past decades,many responsive,specific,inexpensive,efficient,and fully automated biosensors based on electrochemical methods have been developed for the ultrasensitive detection and real-time monitoring of biomarkers and are expected to be used in clinical analysis.However,a large number of interfering biological substances are present in actual biological samples,such as interfering proteins in serum and hundreds of millions of cells in human blood,etc.Their nonspecific adsorption on the sensing interface will cause signal errors in the sensor,and resulting in a decrease in sensitivity or even failure of the biosensor,which makes it impossible for the normal biosensors to be applied to the clinical detection of actual samples.Designing a sensing interface with antifoulnig function will have direct relevance to the availability for biosensors achieving clinical applications.Peptides are widely applied in the fabrication of biosensors for their natural biocompatibility and functional versatility.In this thesis,taking advantage of the unique properties of peptides,we have developed different peptides with multiple functions including anchoring,doping,antifouling,recognizing,and linking based on a variety of self-designed antifouling peptides.The conductive polymer poly(3,4-ethylenedioxythiophene)(PEDOT)can effectively resolve poor electrical conductivity of the functional peptide modified interface and endow them with excellent electrochemical properties.Further,combined with PEDOT,antifouling electrochemical biosensors based on functional peptides-doped PEDOT have been developed.Antifouling electrochemical aptamer sensor,immunosensor and cellular sensor were constructed respectively,and they all achieved ultra-sensitive detection of specific targets in complex biological environments.The three main parts of this thesis are as follows:(1)An antifouling electrochemical immunosensor capable of detecting breast cancer biomarker CA15-3 was constructed based on the conducting polymer PEDOT planted with designed peptides,which can realize the precise doping of the PEDOT and overcome the shortcoming of poor conductivity of the peptide.The designed peptides containing doping and antifouling sequences were anchored to an electrode surface,followed by the electrochemical polymerization of PEDOT.The negatively charged doping sequence of the peptide was gradually doped into the PEDOT during the polymerization process,and by controlling the polymerization time,it was able to exactly dope the whole doping sequence into the PEDOT film,leaving the antifouling sequence of the peptide stretched out of the PEDOT surface.Therefore,an excellent conducting and antifouling platform was constructed just like planting a peptide tree in the PEDOT soil.With antibodies immobilized on the peptide,an antifouling electrochemical biosensor for the detection of a typical biomarker CA15-3 was developed.Owing to the unique properties of the conducting polymer PEDOT and the antifouling peptide,the electrochemical biosensor displayed high sensitivity and longstanding stability,and it could detect CA15-3 in serum of breast cancer patients without suffering from biofouling.The strategy of planting designed antifouling peptides in conducting polymers offered an effective way to develop electrochemical sensors for practical biomarker assaying in complex biological samples.(2)An antifouling electrochemical aptasensor for biomarker detection in complex serum samples with unique long-term antifouling performance was constructed,based on newly designed multifunctional peptides containing anchoring,doping,linking,and antifouling sequences.The designed peptides were first attached onto an electrode surface with the assistance of the anchoring sequences,and the negatively charged doping sequences as dopants for conducting polymer poly(3,4-ethylenedioxythiophene)(PEDOT)were then precisely doped into the electropolymerized PEDOT to form a conducting and stable substrate,leaving the linking and antifouling sequences exposed on the PEDOT substrate surface.The linking sequence of the peptide between the doping and antifouling parts was designed to be beneficial for enhancing the antifouling performance.After the biorecognizing probe(mi RNA aptamer)immobilization,the obtained biosensor was able to detect targets mi RNA-21 with a low limit of detection of2.3 f M and high specificity in complex biological fluids.More importantly,the electrochemical biosensor exhibited incomparable long-term antifouling performances over previous reports and retained their antifouling capabilities for 20 days,indicating a promising feasibility of this design strategy for the construction of biosensors and bioelectronics to be used or implanted in real biological systems.(3)In order to prevent the biofouling in detecting circulating tumor cells(CTCs),as well as to simplify the construction steps of the biosensor and improve its utility,we have designed a tetrafunctionalized peptide for fabricating a cellular biosensor that can be used for the selective detection of breast cancer cells MCF-7.The tetrafunctionalized peptide combined four functions of anchoring,doping,antifouling and recognizing: this peptide can be covalently bonded to the gold nanoparticles modified on the glassy carbon electrode through Au-S bond by using the N-terminal modified cysteine(C)as the anchoring group;then the doping sequence was used as the dopant for PEDOT electropolymerization to precisely form the PEDOT polymer in the doped peptide part to improve the conductivity of the interface,and maintain the electrical neutrality of the whole interface;then the antifouling functional sequence and recognizing sequence were exposed on the surface of the PEDOT film,which can effectively prevent nonspecific protein adsorption and cell adhesion,meanwhile can generate electrochemical response by specific capture of MCF-7 cells.This work is expected to achieve direct detection of CTCs in the complex human environment and providing a novel idea for liquid biopsy technology to better serve cancer patients. |
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