Full Paper Microfabricated Amperometric Cells for Multicomponent Analysis Ying Liu, a, e Ro ´ bert E. Gyurcsa ´nyi, b, c Gyula Ja ´gerszki, b John D. DeNuzzio, d Erno ˝ Lindner a * a Joint Graduate Program in Biomedical Engineering, The University of Memphis and The University of Tennessee Health Science Center, Memphis, TN 38152, USA *e-mail: elindner@memphis.edu b Department of Inorganic and Analytical Chemistry, Budapest University of Technology and Economics, H-1111 Budapest, Hungary c Research Group for Technical Analytical Chemistry of the Hungarian Academy of Sciences, Budapest University of Technology and Economics, H-1111, Budapest, Hungary d Becton and Dickinson Diagnostics – Diagnostic Systems Sparks, MD 21152, USA e Current address: Department of Physical Medicine and Rehabilitation, University of Pittsburg, PA 15213, USA Received: January 13, 2009 Accepted: March 13, 2009 Abstract Towards the development of multianalyte electrochemical immunoassays three individually addressable micro- electrode array (MEA) type working electrodes and a reference electrode were integrated into a 4 mL volume, planar electrochemical cell. To model the simultaneous determination of multiple antigens in the cell with enzyme linked immunosorbent assays (ELISAs) glucose oxidase (GOx), alkaline phosphatase (ALP), and b-galactosidase (b-GAL) were immobilized site specifically onto the individual MEA surfaces and the biocatalitic activity of these surface confined enzymes were evaluated by measuring the products of the enzyme catalyzed reactions directly on the gold MEA surfaces by chronoamperometry or by imaging the enzyme patterned microelectrode array surfaces by Scanning Electrochemical Microscopy (SECM). ALP and b-GAL were selected as model enzymes because they are the most commonly used enzymes labels in ELISAs. In these measurements glucose, ascorbic acid phosphate (AAP), and p-aminophenyl-b-d-galactopyranoside (PAPG) served as enzyme substrates, respectively. The electrochemical surface area of the gold MEAs did not change during the multistep immobilization process. All enzyme modified MEAs presented selective and proportional responses to their substrates and the response characteristics of the enzyme modified sensors were identical in separate and simultaneous calibration protocols, i.e., there was no cross- contamination between the closely placed MEAs. The SECM images of the enzyme patterned MEA surfaces suggest that nonspecific adsorption is negligible on the insulating polyimide surface of the MEA separating the individual microelectrode sites. Keywords: Microelectrode array, Multicomponent analysis, Enzymes, SECM imaging, Immobilized enzymes, Biocatalysis, Chronoamperometry DOI: 10.1002/elan.200904614 Presented at the International Conference on Electrochemical Sensors Ma ´trafüred 2008 Dedicated to the memory of Professor Erno ˝ Pungor and His Legacy to Analytical Chemistry 1. Introduction Microfabrication technologies can provide highly reprodu- cible structures. They have unique advantages in manufac- turing planar electrochemical cells equipped with micro- electrodes and microelectrode arrays (MEAs) [1 – 6]. Planar electrochemical cells with individually addressable micro- electrodes or MEAs provide possibilities for real-time imaging of surface processes with high spatial and temporal resolution [7]. Microelectrode arrays are also adequate for multianalyte electrochemical analysis in minute solution volumes [8 – 11]. When microelectrodes are bundled into MEAs with certain geometrical constraints [12], the advan- tageous properties of individual microelectrodes can be retained but the current measurement becomes less de- manding because the measured current is the sum of the currents flowing through the individual microelectrodes in the array [13 – 17]. In biomedical applications, the microelectrode surfaces are commonly modified with multiple layers. These layers boost selectivity, control sensitivity, and provide a biocom- patible interface between the sensor and its environment. In enzyme linked immunosorbent assays (ELISAs) a layer of immobilized antibodies (primary antibodies) capture the sample antigens. The fraction of primary antibodies that bind antigens is proportional to the antigen concentration in the unknown sample. In sandwich ELISAs, the primary antibodies bound antigens are assessed with the help of an enzyme labeled secondary antibody that binds to another epitope of the antigen. In the presence of its substrate the 1944 Electroanalysis 2009, 21, No. 17-18, 1944 – 1954  2009 WILEY-VCH Verlag GmbH&Co. KGaA, Weinheim