Enzyme Immobilization on Glass Surface for the Development of Phosphate Detection Biosensors

Alkaline Phosphatase was immobilized on aminated glass fiber disks by covalent bond in the vacuum process. In this procedure, amide bonds were formed between carboxyl groups on the enzyme and amino groups on the glass surface. 10% Glycidoxypropyle trimethoxysilane was the best coupling reagent which could help form bonds between carboxyl and amino group on the glass fiber disk. A 10% concentration of coupling reagent, pH 9.0 and 2 gram of silica were found to be the best conditions for coupling the enzyme over the glass surface showed the highest enzyme activity. The covalent attached immobilized enzyme not only retained its activity but also could be reused at least 4 times after washing without loss of enzyme activity. Immobilized enzyme showed nearly 16% loss of enzyme activity after the first trial. An average of 1.65 mg of reusable alkaline phosphatase was immobilized per gram of glass fiber. Phosphate elements were measured from water, raw milk and raw shrimp sample by the used of this alkaline phosphatase immobilized disk as a biosensor. Immobilized enzyme can converts substrate to product and then will converts it to a measurable signal. This study demonstrates the possibility of using such a glass disk for the development of biosensors application.