Biosensors & Bioelectronics 10 (1995) 443-461 !nfluence of graphite powder, additives and enzyme immobilization procedures on a mediatorless HRP- modified carbon paste electrode for amperometric flow-injection detection of H2Oz Ionel Cit~lin Popescu Department of Physical Chemistry, University "Babes-Bolyar', 3400 Cluj-Napoca, Romania. Gunilla Zetterberg & Lo Gorton Department of Analytical Chemistry, University of Lund, P.O. Box 124, S-221 00 Lund, Sweden. (Received 13 June 1994; revised version received 23 November 1994; accepted 28 November 1994) Abstract: The behaviour of horseradish peroxidase modified carbon paste electrodes (HRP-CPEs) for mediatorless amperometric flow-injection (FI) detection of hydrogen peroxide is described. The influence of various commercially available graphite powders and additives was studied, using adsorption and covalent binding (at different pHs) as enzyme immobilization techniques. The HRP-CPE was used as the working electrode in an FI system. An optimal applied potential for electrocatalytic reduction of H202 was found at -50mV vs. Ag/AgC1 (0.1 M KCI). The highest sensitivity (~ 78 ~A cm -2 mM -1) obtained in the FI mode was observed for electrodes made of graphite powder having the highest surface area, with HRP immobilized by adsorption (at pH 8) in the presence of lactitol as enzyme stabilizer. The lowest detection limit (~ 0.1/xM) was noted for HRP-CPE incorporating the graphite powder having the lowest background current. Keywords: modified carbon paste electrodes, additives, graphite powders, peroxidase, hydrogen peroxide, amperometric biosensors INTRODUCTION The detection of hydrogen peroxide plays an important role in the electrochemistry of many enzyme based amperometric biosensors. This is not only because hydrogen peroxide is itself an important analyte in many areas, including industry, the clinical laboratory and the environ- ment, but also because many of the oxidases frequently used to realize amperometric sensors 0956-5663/95/$07.00 © 1995 Elsevier Science Lid 443