| Chapter 1:A biosensor, a analytical device composed of a recognition element of biological origin and a physico-chemical transducer, becomes one of the important research fields of analytical chemistry. The biological element is capable of sensing the presence, activity or concentration of a chemical analyte in solution. The conception, basal constitution, working principles, types, characteristic and application of biosensors were summarized. In addition, research status of enzyme biosensor and detecting methods of L-ascorbic acid were reviewed.Chapter 2:A L-ascorbic acid biosensor using a chitosan/ascorbate oxide/ eggshell membrane as a biological molecular recognition component and a dissolved oxygen electrode as a transducer has been fabricated. The ascorbate oxidase was entrapped between chitosan and eggshell membrane. The effect of phosphate buffer concentration, pH and temperature on the response of the L-ascorbic acid biosensor has been studied in detail. The resulting sensor exhibited a fast response of 100 s, high sensitivity and good storage stability. The linear response is 0.07-0.77 mmol/L of L-ascorbic acid concentration with a detection limit of 16μmol/LChapter 3:A L-ascorbic acid biosensor using an enzyme-immobilized eggshell membrane and oxygen electrode for L-ascorbic acid determination has been fabricated. Ascorbate oxidase was covalently immobilized on an eggshell membrane by chitosan with glutaraldehyde as a cross-linking agent. In the optimization studies of the biosensor, the most suitable enzyme and glutaraldehyde concentration were found to be 6 units and 15%(w/v), respectively. The phosphate buffer of pH 5.0 was chosen as the optimum working condition. The biosensor response has a linear range of L-ascorbic acid concentration between 0.02 mmol/L and 0.82 mmol/L with a detection limit of 12μmol/L (S/N=3). Compare with the membrane without glutaraldehyde, enzyme of the proposed biosensor was immobilized firmly。In the characterization studies of the glucose biosensor, some parameters such as interference effects and storage stability were investigated in detail.Chapter 4:A L-ascorbic acid biosensing assay was developed using the enzyme-immobilized pig bladder membrane and dissolved oxygen electrode. The ascorbate oxidase immobilized with chitosan was bound to bladder membrane by glutaraldehyde as a crosslinking agent. The phosphate buffer (100 mM, pH=5.0), glutaraldehyde concentration (15%), and 0.035 mg enzyme loading were established as the optimum test conditions. This biosensor exhibited a response time of 80 s, a generous linear range of 0.010 mM to 0.88 mM with a detection limit of 10μM, repeatbility (3.1%, n=20), recoveries (98.7-102%), and good stability with a shelf-life of more than 3 months. The reproducibility of fabrication of the biosensors was investigated by using three different membranes (R.S.D.=3.0%). The comparison of the analytical performance between the published and the proposed biosensor methods was made. This biosensor has been applied to determine the L-ascorbic acid content in real samples, and the results were agreement with those obtained by a commercial colorimetric ascorbic acid assay kit.Chapter 5:Porphyrins are recognized as perfect host for molecular recognition owing to their diversity, rigid molecular structures, the position and direction of the substitutes on the porphyrin ring being controllable, Metalloporphyrin is a prototype of metal hexacyanoferrates with remarkable electro-, catalytic-, photo-and biochemical properties. In this paper, a type of iron (Ⅲ)-porphyrin (FeTPP) nanoparticles was prepared and applied for the fabrication of L-ascorbic acid biosensor. The effects of experimental conditions on biosensors'response have been investigated. |
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