Amplification of amperometric biosensor responses by electrochemical substrate recycling

Amperometric catechol biosensors can be constructed by drying onto the surface of a glassy carbon rotating-disk electrode an aqueous sol of synthetic laponite clay containing controlled amounts of polyphenol oxidase (PPO) and polycationic oligosilasesquioxane additive. The procedure allows the electrode surface to be coated with composite enzyme–laponite clay films exhibiting improved adhesion, enhanced mechanical strength and high enzymatic activity. Electrodes prepared in this manner can be used to detect catechol in the range 0.5 nM to 10 μM. The low detection limit of 0.5 nM results from an efficient signal amplification as a consequence of the electrochemical recycling of catechol substrate. An intrinsic amplification factor of 3.35 has been measured. The observed responses from such an electrode as a function of applied potential, enzyme activity and electrode rotation rate are in excellent agreement with theory. From a comparison of the experimental results with theory, we are able to characterize diffusion and enzyme kinetics in the enzymatic layer. The results are consistent with a microporous structure of the enzymatic layer in which microchannels are distributed. Diffusion of catechol substrate and orthoquinone product occurs within the microchannels filled by electrolyte and can be described using a pinhole model. The study shows that only a fraction of PPO, the one which is entrapped in open micropores with interconnected microchannels, is accessible to catechol substrate while keeping its full enzymatic activity. The other fraction should correspond to inaccessible proteins enclosed within the walls of microchannels.