| Carcinoembryonic antigen (CEA) or a-1-fetoprotein (AFP) is the mainly used tumor markers expressed in many malignancies, such as lung cancer, pancreas carcinoma, breast cancer, hepatocellular carcinoma and others. Thus, the determination of CEA or AFP levels is essential to clinical tumor diagnoses. Many immunoassay methods, based on the measurement of antigen-antibody reactions, have been developed for the detection of CEA or AFP. In recent years, the electrochemical amperometric immunosensors have been developed to detect CEA or AFP due to advantages such as simple pretreatment procedure, fast analytical time, highly sensitive current measurement, lower price and portability. As for the construction of an electrochemical immunosensor, the crucial step is the immobilization of sensing biomolecules onto the electrode surface, which should be simple, fast and effective.In this thesis, we have constructed several immunosensors based on electrodepositing, electrostatic interaction, covalent bonding. Moreover, we use gold nanoparticles not only can firmly adsorb more antibody via the large specific surface area but also provide a mild microenvironment similar to that of proteins in native systems to efficiently retain the activity of proteins.The main works and conclusions are included as follows:1. Preparation of a composite film electrochemically deposited with chitosan and gold nanoparticles for the determination ofα-1-fetoprotein.A novel amperometric immunosensor based on chitosan-gold nanoparticles (Chit-GNPs) composite film and thionine (Thi) was prepared for the determination ofα-1-fetoprotein (AFP). The immunosensor was prepared by electro-depositing a Chit-GNPs composite matrix on the surface of the glass carbon electrode, then Thi was immobilized onto the Chit-GNPs film using glutaraldehyde as a cross-linker. Furthermore, the GNPs were chemisorbed onto Thi film for immobilization ofα-1-fetoprotein antibody (anti-AFP). The procedure of the immunosensor was characterized by means of cyclic voltammograms (CV). The performance and influencing factors (eg. the pH of the working solution, incubation time and temperature) of the resulting immunosensor were studied in details. Under optimal conditions, the immunosensor was highly sensitive to AFP and the linear range covered from 0.40 to 200.0 ng·mL-1 with a detection limit of 0.24 ng·mL-1 at three times background noise. Moreover, the simple and controllable electro-deposition method overcame the irreproducibility for preparing Chit-based immunosensor systems and the proposed immunosensor displayed a satisfactory reproducibility and stability.2. Ultrasensitive amperometric immunosensor for the determination of carcinoembryonic antigen based on porous chitosan and gold nanoparticles functionalized interfaceA novel immunosensor for direct amperometric determination of carcinoembryonic antigen based on porous chitosan (pChit) and gold nanoparticles (GNPs) biocompatibility composite film has been fabricated. Firstly, a pChit film was formed on glassy carbon electrode by an electrodeposition method. Then, thionine as a redox probe was immobilized on the pChit film modified electrode using glutaraldehyde as a cross-linker. Finally, GNPs was adsorbed on the electrode surface to assemble carcinoembryonic antibody (anti-CEA). The surface morphology of the pChit films was studied by means of scanning electron microscope. In addition, the procedure of the immunosensor was further characterized by cyclic voltammograms and electrochemical impedance spectroscopy. The electrochemical behaviors and factors (eg. the pH of the working solution, incubation temperature and time) influencing the performance of the resulting immunosensors were studied in detail. Results showed that the pChit films can enhance the surface coverage of antibody and improved the sensitivity of the immunosensor. Under optimal conditions, the immunosensor was highly sensitive to CEA with a detection limit of 0.08 ng·mL-1 at three times background noise and the linear ranges of 0.2～10.0 and 10.0～160 ng～mL-1. Moreover, the immunosensor exhibited high selectivity, good reproducibility and stability.3. Horseradish peroxidase-functionalized SiO2@Au core/shell structured nanoparticle label for amplified immunoanalysis An electrochemical immunosensors using gold nanoparticles platforms with multi-label secondary antibody-SiO2@Au bioconjugates was prepared for the determination ofα-1-fetoprotein (AFP). Greatly amplified sensitivity was attained by using bioconjugates featuring horseradish peroxidase (HRP) labels and secondary antibodies (Ab2) linked to SiO2@Au at high HRP/Ab2 ratio. The procedure of the immunosensor was characterized by means of cyclic voltammograms (CV). The influencing factors (eg. the pH of the working solution, incubation time) of the resulting immunosensor were studied in details. This approach provided a detection limit of 0.019 ng·mL-1 and the linear range covered from 0.05 to 200 ng·mL-1. Moreover, the immunosensor exhibited high selectivity, good stability. |
PDF Full Text Request