Design of covalently immobilized alpha-glucosidase and glucose oxidase biosensor and kinetic study for maltose hydrolysis

Abstract

A hybrid sol gel modified nanobiosensor has been developed to determine quantity of maltose generated by hydrolysis of starch in the presence of α-amylase. The biosensor was developed by covalent immobilization of α-glucosidase and glucose oxidase enzyme on sol gel modified electrode with ferrocene via carbodiimide reaction. The presence of amine groups provided by APTMOS in sol gel controlled the covalent bonding sites for enzyme. The immobilization conditions such as amount of enzyme, immobilization time and pH in working buffer was studied by using Response Surface Methodology (RSM) through Central Composite Design (CCD) of Design Expert® software. The biosensor showed maximum current response at 20 U/mL of immobilized glucose oxidase and 90 minutes of immobilization time. The optimum pH value of working buffer is pH 7. The biosensor exhibited the optimal response at 45˚C. The calibration curve of GOx and GD biosensor was linear in the range of 0.5 mM to 5 mM of maltose concentration with a sensitivity of 43.15 μAmM-1cm-2 and correlation coefficient of 0.9975. The performance of GOx and GD biosensor was improved by covalent immobilized of GOx and GD on the surface of modified electrode was successfully proven by kinetic study. The Lineweaver-Burk plot gave the value of immobilized GOx and GD enzyme 12.73 mM much lower that GOx and GD molecules in solution which was 24.6 mM. The FTIR measurement was used to establish the GOx and GD biosensor. The FTIR spectra showed the successful immobilized GOx and GD on modified electrode because the wavenumber were essentially the same as native GOx and GD in solution.