MINIZYMES - A NEW STRATEGY FOR THE DEVELOPMENT OF REAGENTLESS AMPEROMETRIC BIOSENSORS BASED ON DIRECT ELECTRON-TRANSFER PROCESSES

Direct electron transfer between an immobilized biological compound an d an electrode is one of the most interesting transduction processes f or the development of fast responding amperometric biosensors. Differe nt biocatalysts like horseradish peroxidase, cytochrome c, myoglobin, microperoxidase MP-II and haemin, all of them catalyzing the reduction of H2O2, have been investigated aiming on their ability for direct el ectron-transfer reactions when covalently tethered to self-assembled m onolayers (SAMs) on gold. As direct electron-transfer processes are pr edominantly limited by the distance between the active site of the bio compound and the electrode surface, the highest electrocatalytic effic iency with the monolayer-immobilized biocatalysts was observed for the smallest peroxidase-active compounds (e.g., microperoxidase MP-II, ha emin). Although these compounds show a significant lower catalytic act ivity for the reduction of H2O2 in homogeneous solution, the catalytic activity of horseradish peroxidase is by a factor of 3300 higher as c ompared with that of haemin. Haemin exhibits a more than tenfold highe r electrocatalytic activity when immobilized at a monolayer. This trem endous difference between the catalytic activity in homogeneous soluti on and the electrocatalytic activity of the monolayer-immobilized bioc atalyst could be attributed to a higher surface concentration for the smaller compounds, the improved access for the substrate to their acti ve sites and, most significantly, the increased electron-transfer rate due to the decrease of the distance between redox site of the biocata lyst and electrode surface. Hence, for the development of enzyme elect rodes based on direct electron-transfer processes between monolayer-im mobilized biocatalysts and the electrode the size of the biocatalyst i tself should be decreased. Such catalytically-active compounds with de creased protein shell have been called 'minimized enzymes' or 'minizym es'. (C) 1997 Elsevier Science S.A.