Amplification of amperometric biosensor responses by electrochemical substrate recycling Part II. Experimental study of the catechol-polyphenol oxidase system immobilized in a laponite clay matrix

Amperometric catechol biosensors can be constructed by drying onto the surf ace 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 elect rode surface to be coated with composite enzyme-laponite clay films exhibit ing improved adhesion, enhanced mechanical strength and high enzymatic acti vity. Electrodes prepared in this manner can be used to detect catechol in the range 0.5 nM to 10 mu M. 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 funct ion of applied potential, enzyme activity and electrode rotation rate are i n excellent agreement with theory. I;rom a comparison of the experimental r esults with theory, we are able to characterize diffusion and enzyme kineti cs in the enzymatic layer. The results are consistent with a microporous st ructure of the enzymatic layer in which microchannels are distributed. Diff usion of catechol substrate and orthoquinone product occurs within the micr ochannels 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 catech ol substrate while keeping its full enzymatic activity. The other fraction should correspond to inaccessible proteins enclosed within the walls of mic rochannels. (C) 1999 Elsevier Science S.A. All rights reserved.