Lactate Amperometric Biosensor Based on an Electrosynthesized Bilayer Film with Covalently Immobilized Enzyme

Abstract

An alternative approach to the fabrication of an amperometric biosensor, which combines electrochemical polymerization of a bilayer film and covalent binding of the enzyme, is presented. A Pt electrode modified by an anti-interferent poly(pyrrole) layer is covered by a poly(tyramine) film, exposing amino groups that are then used for the covalent attachment of lactate oxidase after activation with glutaraldehyde. In this way, a fabrication procedure is obtained that combines the advantages of electrochemical polymerization (high spatial control, no restrictions in electrode shape and dimension, reproducible film thickness even in the sub-micrometre range) with those of covalent binding of the enzyme (no enzyme loss, higher response stability and increased lifetime of the sensor). The interferent rejection efficiency is dictated by the diffusional barrier provided by the bilayer membrane and by the degree of permselectivity that can be built up in the poly(pyrrole) film. A peculiar property was displayed by the bilayer, i.e., the ability to separate L-lactate and residual interferent responses in the time domain in both flow injection and batch addition experiments. The resulting L-lactate amperometric biosensor was characterized in terms of sensitivity, response time, linear response range, stability and rejection of electroactive interferents.