The Research On Enzyme Biosensor And Enzyme-Linked Fluoroimmunosensing System

Biosensors are of practical significance in clinical diagnosis, food industry, environment monitoring and military affairs, so extensive research has been done by scientists in this field. However, the preparation of the sensitive film, i.e. the immobilization of biomolecules is the key technique. With this in mind, four aspects of the work in the present thesis have been carried out:1. A procedure for fabricating an enzyme electrode has been described based on the effective immobilization of horseradish peroxidase (HRP) to an ultrathin titania layer modified self-assembled gold electrode. The strong affinity of the titania layer for positively charged HRP enzyme has been utilized to realize the immobilization of HRP enzyme. The resulting electrode exhibits excellent electrocatalytical activity to hydrogen peroxide in the presence of hydroquinone as a mediator. The analytical conditions, such as the applied potential of the working electrode, the concentration of hydroquinone were studied in detail by using amperometric method. Under the optimized experimental conditions, the linear response to hydrogen peroxide ranged from 1.0 x 10~-6 mol 1-1 to 7.6 10~-4 mol 1-1 and a detection limit of 7.1 10-7 mol 1-1were obtained.2. Bienzyme sensor has been prepared for glucose assay based on combining Nafion with titania sol-gel prepared by vapor deposition method on glass carbon electrode. Ferrocene acted as an electron mediator and GOD, HRP were immobilized simultaneously in the resulting Nafion/titania sol-gel modified electrode. Because of low working potential, electroactive species such as ascorbic acid and uric acid do not cause any interference. Under the optimized experimental conditions, the linear response to glucose ranged from 5.0 x 10-5 mol 1-1 to 1.5 10-3 mol 1-1 and a detection limit of 1.0 x 10-5 mol 1-1 were obtained.3. Poly(ferulic acid) film modified gold electrode was prepared by electropolymerization. The electrocatalytic reaction involving dopamine at the formed electrode was investigated. The results indicated that the electrode shows excellent electrocatalytic effect on dopamine, and shows a good linear relationship between the peak current and the concentration of dopamine ranged from 1.0 10-6-1.0 10-3 mol 1-1. Moreover, the resulting electrode can suppress the electroactive substance (ascorbic acid) to a certain extent.4. A fluoroimmunosensing system with the use of ferulic acid as the substrate for the determination of transferrin in serum samples has been developed. The results indicated that ferulic acid as the substrate possesses a better stability towards H2O2 as compared to TMB. The analytical conditions, such as pH, the amount of labeled transferrin, incubation time and temperature were studied in detail. Under the optimized experimental conditions, the linear detection ranged from 39.3 to 177.0 ng/ml was obtained. This approach has been applied to clinical samples with satisfactory results.