Studies On Novel Electrochemical Immunosensors For Carcinoembryonic Antigen And Cancer Antigen 15-3

Carcinoembryonic antigen(CEA)or Cancer antigen 15-3(CA 15-3)is mainly used as a tumor maker for diagnosing a disease,evaluating curative effect or judging recrudescence.The CEA or CA 15-3 level in serum is related to lung cancer,breast cancer,ovarian carcinoma, Cystadenocarcinoma,liver carcinoma,hepatocellular and others.Thus,the fast-and accurate determination of CEA or CA 15-3 is very helpful to clinical tumor diagnoses.A number of immunoassay methods for the determination of CEA or CA 15-3 level in serum have been reported such as enzyme-linked immunosorbent assay(ELISA),radioimmunoasSay(RIA)and chemiluminescence immunoassay(CLIA),yet these methods suffer from disadvantages such as using complex time-consuming procedures and/or requiting potentially dangerous or expensive materials.Due to the advantages of simple preparation,low cost,short response time and yet accurate and sensitive platform,electrochemical immunosensors have been widely applied in the areas of human and animal health,food and fermentation industries,environmental monitoring, agriculture and defense.In recent years,some researchers in national and worldwide have achevived much process by using different methods to immobilize antibodies to the electrodes surface to detect tumor makers,but the stability,sensitivity and selectivity of these biosensors can still not meet the practical need.Therefore,exploring new,simple,and sensitive diagnostic methods with real-time output and a low cost is of considerable interest.The method and material used to immobilize biomolecules is one of the crucial factors for improving the stability,sensitivity and selectivity of biosensor.In this thesis,we have constructed several immunosensors based on electropolymerization,electrostatic interaction,covalent bonding and nano-particle(nano-Au),nano composite material(DNA/nano-Ag composite).At the same time,we use dsDNA,a biological macromolecule with a three dimensional structure not only can enlarge the surface area of the electrode for more loading the antibodies but also can provide good biocompatibility with the environment for avoiding the proteins denaturalization,to improve the sensitivity and life span of the immunosensors.The main works and conclusions are included as follows:1.A new dual-amplification strategy of electrochemical signaling from antigen-antibody interactions was proposed via back-filling gold nanoparticles on(3-mercaptopropyl) trimethoxysilane sol-gel(MPTS)functionalized interface.The MPTS was employed not only as a building block for the electrode surface modification but also as a matrix for ligand functionalization with first amplification.The second signal amplification strategy introduced in this study was based on the back-filling immobilization of nanogold particles to the immunosensor Surface.Several coupling techniques,such as with nanogold but not MPTS or with MPTS but not nanogold,were investigated for the determination of carcinoembryonic antigen(CEA)as a model, and a very good result was obtained with nanogold and MPTS coupling immunosensor.With the non-competitive format,the formation of the antigen-antibody complex by a Simple one-step immunoreaction between the immobilized anti-CEA and CEA in sample solution introduced membrane potential change before and after the antigen-antibody interaction.Under optimal conditions,the proposed immunosensor exhibited a good electrochemical behavior to CEA in a dynamic concentration range of 4.4 to 85.7 ng·mL^-1with a detection limit of 1.2 ng·mL^-1(at 3δ). Moreover,the precision,reproducibility and stability of the as-prepared immunosensor were acceptable.Importantly,the proposed methodology would be valuable for diagnosis and monitoring of carcinoma and its metastasis.2.A novel amperometric immunosensor for the determination of carcinoembryonic antigen (CEA)based on DNA/nano-Ag composite and poly-thione has been fabricated.The composite, which contains DNA(negatively charged)and nano-Ag,can not only be immobilized tO the glass carbon electrode surface modified with poly-thione(positively charged)through negatively charged DNA,but also adsorb carcinoembryonic antibodies through nano-Ag particles with large specific surface areas and good biocompatibility.The modification procedure was electrochemically monitored by cyclic voltarnmetry.The performance and influencing factors of the resuiting immunosensor were studied in detail.Under the optimal conditions,the developed immunosensor exhibited fast response to CEA and the linear ranges were 1.0—10.0 ng·mL^-1and 10.0—80.0 ng·mL^-1with a detection limit of 0.24 ng·mL^-1.3.A novel amperometric immunosensor for the determination of cancer antigen 15-3(CA 15-3) was prepared by immobilizing anti-CA 15-3 on double-layer gold nanoparticles and double-strand DNA(dsDNA)modified gold electrodes.Prussian blue.(PB),as a good mediator,was firstly electro-deposited on Au electrode surface to form a stable and homogeneous interface,which could be encapsulated via electrochemical reduction of HAuCl₄ solution,then dsDNA was attached to the nanogold particles,and then the second-layer gold nanoparticles were deposited on the dsDNA surface to amplify the surface coverage of biomolecules,finally anti-CA 15-3 antibodies were absorbed onto the surface of nanostructured gold particles.The fabrication process of the immunosensor was characterized by scanning electron microscopy(SEM)and cyclic voltammetry (CV).Under the optimized conditions,the proposed immunosensor exhibited a good electrochemical behavior to CA 15-3 in the ranges of 1.0 to 15.0 ng·mL^-1and 15.0 to 240.0 ng·mL^-1 with a detection limit of 0.6 ng·mL^-1.The immunosensor showed high sensitivity,selectivity and stability.Importantly,this fabrication methodology could be further developed for the immobilization of other proteins and biocompounds.