Investigation of concentration profiles inside operating biocatalytic sensors with scanning electrochemical microscopy (SECM) Bala ´zs Cso ´ka a , Barna Kova ´cs a , Ge ´za Nagy a,b,  a Department of General and Physical Chemistry, University of Pe ´cs, Pe ´cs, Ifju ´sa ´g u. 6., H-7624, Hungary b MTA-PTE Research Group for Chemical Sensors, Pe ´cs, Ifju ´sa ´g u. 6., H-7624, Hungary Received 31 October 2001; accepted 16 May 2002 Abstract Scanning electrochemical microscopy (SECM) with amperometric or potentiometric measuring tips was used to investigate biocatalytic reactions inside the enzyme layer of a biosensor during its operation. The well known glucose oxidase catalyzed oxidation of glucose has been selected for the studies. Local, instantaneous concentration of dissolved oxygen and hydrogen peroxide was studied observing the amperometric current while miniaturized potentiometric tip served for local pH measurements. Liquid enzyme layer immobilized with Cellophane membrane or cross linked polyacrilamide gel membrane containing entrapped enzyme served for biocatalytic media in the SECM imaging. Local maximum of H 2 O 2 and minimum of O 2 profiles were found at approximately 200 mm far from the substrate/enzyme layer boundary. From the experimental findings guidelines to design well functioning biocatalytic sensors could be concluded. The concentration profiles obtained with SECM techniques were compared with the results of simple model calculations carried out with the method of finite changes. Most of earlier made SECM studies dealing with enzyme reactions imaged the electrolyte being in contact with the immobilized enzyme. The data in our investigation, however, were collected inside the working catalytic layer. # 2002 Elsevier Science B.V. All rights reserved. Keywords: Immobilized enzymes; Reaction layer; Biosensor; SECM; Digital simulation 1. Introduction The biosensors are well known analytical tools. They can be used to measure concentration of different species in complex matrices selectively. The selectivity is provided by the biological nature of their working principle. Clark (Clark and Lyons, 1962), Updike (Updike and Hicks, 1967, Guilbault (Guilbault and Montalvo, 1969) and other pioneers of the biosensor research used enzyme catalysis first to develop biosen- sors. Enzymatic transducers are still the most often used ones. As it is well known their measuring function is based on a chemical reaction that takes place in a catalytic reaction layer. The interaction of this reaction and the mass transport processes produce changes inside the reaction layer. In order to describe the function of the different enzyme sensors or to design efficient ones, the distribution of the different species inside the differently made, sized and formed reaction layers during their operation is necessary to be known. Considering the complex interaction of different processes */the distribution of different species between the sample and the reaction layer, the mass transport inside the sample phase and in the reaction layer, and the enzyme catalysis */very complicated spatial and time dependence of concentration of different chemical species can be expected. Since the early times of biosensor research several attempts have been made to describe the concentration profiles inside biocatalytic layers or to explain the function of different kinds of enzyme sensors. However, to give an explicit equation with measurable variables and parameters is a difficult task. Often equations derived are applicable only in special limiting cases, or their use often ends with giving explanation on experi-  Corresponding author. Tel.:  /36-72-327-622/4681; fax:  /36-72- 327-622/4680 E-mail address: g-nagy@ttk.pte.hu (G. Nagy). Biosensors and Bioelectronics 18 (2003) 141 /149 www.elsevier.com/locate/bios 0956-5663/02/$ - see front matter # 2002 Elsevier Science B.V. All rights reserved. PII:S0956-5663(02)00167-7