A new biosensor development based on pencil graphite electrode-ascorbate oxidase for determination of l-ascorbic acid(vitamin c)

In this thesis, in order to provide an inventive and novel perspecive, a new assay for detection of L-ascorbic acid was developed using PGE. Ascorbat oxidase was cross bounded using gelatine and gluteraldehyde, fixed to the surface of pencil graphite electrode and designed biosensor was used for the detection of ascorbic acid. L-ascorbic acid measurements were done amperometrically at -0.7 V using the developed biosensor. Measurements were done using amperometric assay, determining the decrease of the current value which is proportional to the concentration of consumed oxygen. Fort he optimization studies of the prepared biosensor first of all optimum enzyme concentration, optimum gelatin amount, optimum time for incubation in gluteraldehyde and other parameters like the optimum repeat cycle fort he gluteraldehyde stratification process were determined. Temperature, pH and buffer concentrations were optimized for the optimization of working conditions. And then, for the characterization studies some parameters investigated as like linear detection range, repeatibility, reproducibility, substrate specifity, storage stability and sample analysis. Fort he PGE/gelatine-ascorbate oxidase/glutaraldehyde modified biosensor ascorbate oxidase concentration, gelatine amount, incubation time in glutaradehyde and glutaraldehyde stratification repat cycle were found to be 1.5 U/mL, 20 mg, 3minutes and 3 times tabakalandırma respectively after optimization studies. Potassium phosphate buffer ( 50 mM, pH:7.0) and 30ºC temperature determined to provide optimum working conditions. After the characterization studies, 25 µM - 500 µM detection range was found to be linear for PGE/gelatine-ascorbate oxidase/glutaraldehyde modified biosensor. According to the results standart deviation and variation coefficient % values were found to be ±1.46 and 0.44 % respectively. It was confirmed that 75 % of activity is conserved after 4 weeks of storage.