Signal amplification in enzyme-based amperometric biosensors.

A unique mode of current amplification was investigated in reagentless biosensors based on the clinically significant enzymes including alcohol dehydrogenase, glucose 6-phosphate dehydrogenase, glycerol 3-phosphate dehydrogenase, and glucose oxidase. The biosensors were designed by sandwiching the enzyme-polymer film between an electrode and Nafion film. In particular, each enzyme and its cofactor were covalently attached to the chains of polysaccharide chitosan and mixed with carbon nanotubes on the electrode surface. The coating of such biosensors with Nafion resulted in the current increase by up to 1000%, depending on the enzyme. The results were analyzed considering the interplay between the enzyme activity-pH profiles and the Nafion-induced pH increase in the underlying chitosan film. The data were collected by using the rapid (<5 min) amperometric enzyme assays and pH-sensitive iridium oxide films. The increase in the biosensor current was attributed to the pH-driven increase in the enzyme activity inside the two-film interface. Such signal amplification should also be feasible in other biosensors based on the polyelectrolytes and sandwiched enzymes providing that a proper match is made between the enzyme activity-pH profiles and the pH of buffer solutions.