Novel Nano-materials As Applied To Sensing-interface Fabrication For Electrochemical Immunosensor

Electrochemical biosensor, which is a novel and developing technique that linking biochemical electrochemical and electronic techniques, has become the main analytical methods in clinical and biochemical analyses and in other areas such as environmental control, food quality control, etc. In the design and fabrication of immunosensor, the development of a simple and effective strategy for the construction of sensitive membrane on the electrode is a crucial step. Nano-crystalline particles, exhibiting a large surface area with diameter less than 100 nm, high surface-activity, good biocompatibility, strong adsorption and great efficiency of catalysis, might be favorable for constructing biosensor. Moreover, the amplified electronic transduction of antigen/antibody process is of importance in the development of a highly sensitive immunosensor. For these reasons, we combined the merits of the unique electrochemical properties of carbon nanotubes, good biocompatibiting of gold nanoparticles and the amplification of enzymes to the antigen-antibody reaction to develop four different immunosensor. The main works are shown as follows:1. A potentiometric immunosensor for the determination ofα-fetoprotein based on Protein A/Gold Nanoparticies/MWNT modified composite membraneA new potentiometric immunosensor for the determination ofα-fetoprotein (AFP) has been developed by means of immobilizingα-fetoprotein antibody onto the protein A/gold nanoparticles/multiwall carbon nanotube (MWNT) - Chitosan (CS) modified composite film. The fabrication procedure of the immunosensor is characterized by electrochemical impedance spectroscopy and cyclic Vohammetry. The introductions of gold nanoparticles not only increase the adsorption amount of protein A, but also retain their biological activites. Protein A, which is used for the proper orientation of antibody, constructs a well-defined antibody surface and enhances the sensitivity of an immunosensor. The detection is based on the change of electrodepotential before and after the antigen—antibody reaction. Under optimal conditions, the proposed immunosensor exhibited a wide linear range from 7.0～190.0 ng/mL relative to AFP concentration. 2. A novel amperometric immunosensor for the determination ofα-fetoprotein based on Gold Nanoparticles and Azure I/Multi-Wall Carbon Nanotubes Composite Membrane.A convenient and effective strategy based on the unique characteristics of Azure I / multi-wall carbon nanotubes (Azure I/MWNT) composite membrane and amplification response of enzyme to the antigen-antibody reaction is used to develop a highly sensitive amperometric immunosensor for detection ofα-fetoprotein (AFP). Azure I/MWNT composite overcome the defect of insulate film and provide a remarkable synergistic augmentation to facilitate electron-transfer process. Second, gold nanoparticles with larger specific interface area, desirable biocomapatibility and high surface free energy can adsorb more antibodies without loss of their biological activities; Due to the electrocatalytic ability of HRP and MWNT towards H₂O₂, they significantly amplify the response of the antigen-antibody reaction. Several techniques, including scanning electron microscopy (SEM), transmission electron microscopy (TEM), atomic force microscopy (AFM), X-ray photoelectron spectroscopy (XPS) and cyclic voltammetry (CV) have been employed to characterize the assembly process. The immunosensor displays high sensitivity, fast analytical time and a broader linear response to AFP in two ranges from 0.1 to 8.0 and 8.0 to 250.0 ng/mL with relatively low detection limit of 0.04 ng/mL.3. Novel amperometric Human Chorionic Gonadotrophin immunosensor based on Multi-Wall Carbon Nanotube MatrixA novel amperometric immunosensor for human chorionic gonadotropin (HCG) assay has been fabricated through incorporating toluidine blue (TB) and hemoglobin (Hb) on the multiwall carbon nanotube (MWNT) - Chitosan (CS) modified glassy carbon electrode, followed by electrostatic adsorption of a conducting gold nanoparticles film as sensing interface. The construction of double hemoglobin membranes amplifiy the electrochemical signal and improve the sensitivity of immunosensor. The characteristics of the modified electrode at different stages of modification were studied by transmission electron microscopy (TEM) and cyclic voltammetry (CV). The decreased percentage of the current is proportional to HCG concentration ranging from 0.8 to 500 mIU/mL with a detection limit of 0.3 mIU/mL (S/N = 3). This approach has some advantage such as simply, a short response time and a satisfied detection range.4. Base on gold nanoparticles and DNA polyion complex membrane modified amperometric immunosensor for the determination of carcinoembryonic antigenA new and effective strategy for constructing a mediator-type amperometric immunosensor based on the merits of the DNA-PDDA polyion complex membrane, toluidine blue O and gold nanoparticles was described. The introduction of DNA-PDDA polyion complex film not only enhances the electrode surface area for construction of efficient biosensors, but also can act as a charge carrier to facilitate the electron transfer. The fabrication process of the immunosensor is characterized by atomic force microscopy (AFM), cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS). The studied immunosensor displays a high-sensitivity and good correlation for detection of carcinoembryonic antigen (CEA) in the range of 0.5 to 120.0 ng/mL with a detection limit of 0.3 ng/mL.