| Electrochemical biosensor is attractive because of their potential utility as simple, specific and direct detection techniques compared with conventional assay techniques. The key of the electrochemical biosensor is that immobilization of the biorecognition layer. This dissertation focused on the designing and fabrication of electrochemical sensors for chiral drug, immunoassay and enzyme application.1. Capacitive sensor based on molecularly imprinted polymers and poly(p-aminobenzene sulfonic acid) film for direct detection of pazufloxacin mesilateMolecularly imprinted polymers (MIPs) was an important analytical tool. It was low cost with good mechanical, thermal and chemical properties. All these merits mentioned above makes these synthetic materials appear ideal chemoreceptors. Recently, some novel sensors based on MIPs such as chemiluminescence sensor, quartz crystal microbalance (QCM) sensor and electrochemical sensor were reported. Among these sensors, capacitive sensor based on MIPs evoked much attention because it possess of the merit of high sensitivity and label free. Nevertheless, the reported fabrication of the capacitive sensor based on MIPs is complex and the cost is expensive. Thus, it is important to develop high sensitive capacitive sensor based on MIPs.A novel capacitive sensor for pazufloxacin mesilate (pazufloxacin) determination was developed by electropolymerizing p-aminobenzene sulfonic (p-ABSA) and molecularly imprinted polymers (MIPs), which was synthesized through thermal radical copolymerization of metharylic acid (MAA) and ethylene glycol dimethacrylate (EGDMA) in the presence of pazufloxacin template molecules, on the gold electrode surface. And 1-dedecanethiol was used to insulate the modified electrode. Alternating current (ac) impedance experiments were carried out with a Model IM6e to obtain the capacitance responses. Under the optimum conditions, the sensor showed linear capacitance response to pazufloxacin in the range of 5 ng·mL-1 to 5μg·mL-1 with a relative standard deviation (RSD) 5.3% (n=7) and a detection limit of 1.8 ng·mL-1. The recoveries for different concentration levels of pazufloxacin samples varied from 94.0% to 102.0%. Electrochemical experiments indicated the capacitive sensor exhibited good sensitivity and selectivity and showed excellent parameters of regeneration and stability.2. Electrochemcial immunosensors and enzyme sensor(1) One-off PVC membrane based potentiometric immunosensor for label-free detection of alpha-fetoproteinA poly(vinylchloride) (PVC) membrane based potentiometric immunosensor for the direct detection of alpha-fetoprotein (AFP) has been developed. First, Au colloid particle was chemisorbed upon amino groups of o-phenylenediamine, which was dissolved in plasticized PVC membrane. Then alpha-fetoprotein antibody (anti-AFP) was immobilized upon the surface of the Au colloid particle to prepare a potentiometric AFP immunosensor. The Au colloid particle modified PVC membrane was characterized by digital photo and transmission electron microscope (TEM). The immunosensor exhibited fast potentiometric response (≤4 min) and showed specific response to AFP in the range of 4.9 to 158.5 ng·mL-1 with a correlation coefficient of 0.9971 and a detection limit of 1.6 ng·mL-1. The factors influencing the performance of the immunosensor were also studied in detail. Moreover, the proposed method is economical and efficient as well as potentially attractive for clinical immunoassays.(2) Development of a simple capacitive immunosensor for direct detection of alpha-fetoprotein at the picogram level A capacitive immunosensor based on the layer of the mixture of nafion and 1-dodecanethiol on gold electrode has been developed. Alpha-fetoprotein (anti-AFP) was immobilized on the mixture layer via the opposite-charged adsorption technique. Then the prepared electrode was modified with 1% gelatin as a protective film to obtain a capacitive immunosensor. The insulating properties of the different layers of the immunosensor were investigated by using cyclic voltammetry in the presence of electro-active species as indictor. The factors influencing the performance of the immunosensor were also studied in detail. Under optimum conditions, the alpha-fetoprotein (AFP) was determined with a detection limit of 0.33 pg·mL-1 and a linear detection range of 1.0 pg·mL<sup>-1～100.0 ng·mL-1. Moreover, the proposed method is economical and efficient, making it potentially attractive for clinical immunoassays.(3) Capacitive Immunosensor based on Poly(p-aminobenzene sulfonic acid) film and Electrostatic Attraction for Detection of Cancer embryo antigenA high sensitivity and easy fabricative capacitive immunosensor was proposed for direct detection of cancer embryo antigen (CEA). Firstly, a gold electrode is modified with electropolymerized film of p-aminobenzene sulfonic acid (p-ABSA) in pH 7.0 acetate buffer solution (ABS). And then anti-CEA molecular was adsorbed on the negatively-charged p-ABSA film through electrostatic attraction. To increase the insulation degree of the layer, the prepared electrode was treated with 1-dodecanethiol ethanol solution. Cyclic voltammetry (CV) was used to study the electrochemical properties of the polymer film. Alternating current (ac) impedance experiments were carried out with a Model IM6e to obtain capacitive response signals. Under optimum conditions, the CEA was determined with a detection limit of 0.16 pg·mL-1 and a linear detection range of 0.5 pg·mL-1 5.0 ng·mL-1. And the correlation coefficient is 0.9917.(4) An amperometric immunosensor based on potential control of enzyme immobilization and enzyme-labeled antibody technique for detection of hepatitis B surface antigenA novel amperometric immunosensor for hepatitis B surface antigen (HBsAg) determination was developed by immobilizing horseradish peroxidase (HRP) labeled hepatitis B surface antibody (HBsAb) on cysteine modified gold electrode. First, bare gold electrode was modified with cysteine. Then The resulted gold electrode was used as working electrode under the controlled potential for 30 min to attach HRP labled HBsAb through covalent bond and electrostatic attraction. Cyclic voltammetry (CV), electrochemical impedance spectrum (EIS) and atomic force microscopy (AFM) were employed to characterize the process of the biosensor construction. Under optimum conditions, hepatitis B surface antigen (HBsAg) was determined with a detection limit of 48 pg/ml and a linear detection range of 128.0 pg·mL-1～1.267 ng·mL-1 and 2.532 ng·mL-1～210.00 ng·mL-1. Chronoamperometry was employed to characterize the properties of the immunosensor. Moreover, the proposed method is economical and efficient, making it potentially attractive for clinical immunoassays.(5) Controlled potential of HRP immobilization and its application as enzyme biosensorA third-generation horseradish peroxidase (HRP) biosensor was developed by immobilizing HRP on gold electrode modified with a well-ordered, self-assembled cysteine film. The gold electrode modified with cysteine was used as working electrode under the controlled potential for 30 min to attach HRP through covalent bond and electrostatic attraction. The process of the biosensor construction was characterized by cyclic voltammetry and Atomic force microscopy (AFM). CV and Chronoamperometry techniques were employed to characterize the properties of the enzyme biosensor. The enzyme electrode achieved 95% of the steady-state current within 25 s and had a 1.2×10-9 mol·L-1 detection limit of hydrogen peroxide. Steady-state cathodic currents respond to hydrogen peroxide concentration within the range from 3.5×10-9 mol·L-1 to 3.7×10-3 mol·L-1 was observed. The RSD were calculated to be 3.7%. Potential interfering materials do not interfere with the response of the sensor.
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