Biosensors and Bioelectronics 20 (2005) 1559–1565 Simultaneous detection of the release of glutamate and nitric oxide from adherently growing cells using an array of glutamate and nitric oxide selective electrodes Jaime Castillo a , Sonnur Isik b , Andrea Bl ¨ ochl c , Nazar´ e Pereira-Rodrigues d , Fethi Bedioui d , Elizabeth Cs ¨ oregi a , Wolfgang Schuhmann b , Joshua Oni b,∗ a Department of Biotechnology, Lund University, S-22100 Lund, Sweden b Anal. Chem., Elektroanalytik and Sensorik, Ruhr-Universit¨ at Bochum, Universit¨ atsstr. 150, D-44780 Bochum, Germany c Lehrstuhl f¨ ur Molekulare Neurobiochemie, Ruhr-Universit¨ at Bochum, Universit¨ atsstr. 150, D-44780 Bochum, Germany d Ecole Nationale Sup´ erieure de Chimie de Paris, Laboratoire de Pharmacologie Chimique et G´ en´ etique, Fre Cnrs Enscp 2463 + U Inserm 266, 11 rue Pierre et Marie Curie, 75231 Paris cedex 05, France Received 21 May 2004; received in revised form 24 July 2004; accepted 4 August 2004 Available online 27 September 2004 Abstract The simultaneous detection of nitric oxide and glutamate using an array of individually addressable electrodes, in which the individual electrodes in the array were suitably modified with a highly sensitive nitric oxide sensing chemistry or a glutamate oxidase/redox hydrogel- based glutamate biosensor is presented. In a sequence of modification steps one of the electrodes was covered first with a positively charged Ni porphyrin entrapped into a negatively charged electrodeposition paint followed by the manual modification of the second working electrode by a bienzyme sensor architecture based on crosslinked redox hydrogels with entrapped peroxidase and glutamate oxidase. Adherently growing C6-glioma cells were grown on membrane inserts and placed in close distance to the modified sensor surfaces. The current responses recorded at each electrode after stimulation of glutamate and NO release by means of K + and bradykinin clearly demonstrate the ability of the individual electrode in the array to detect the analyte towards which its sensitivity and selectivity was targeted without interference from the neighbouring electrode or other analytes present in the test mixture © 2004 Elsevier B.V. All rights reserved. Keywords: Electrode array; Biosensor; Glutamate oxidase; Nitric oxide; Modified electrodes; Transmitter release 1. Introduction The simultaneous detection of chemical substances re- leased by adherently growing cells is not only of utmost im- portance for the elucidation of signal transduction pathways in vivo but also for drug testing and the possible replace- ment of animal tests by cell-based assays. Of equal signifi- cance is the simultaneous detection of the release of multiple transmitter substances by living cells upon stimulation with the appropriate agents. Neurotransmitters are chemical sub- stances that aid in transmitting impulses between nerve cells ∗ Corresponding author. Tel.: +49 234 322 5464; fax: +49 234 321 4683. E-mail address: joshua.oni@ruhr-uni-bochum.de (J. Oni). or between a nerve cell and a muscle (Dox et al., 1979). Thus the highly sophisticated mechanism of communica- tion between populations of neurons is largely kept oper- ational by neurotransmitters. A complex interplay between the variations in the concentration levels of several trans- mitter substances is one of the many factors that can lead to the manifestation of neurological and psychiatric disor- ders (Bradford, 1986; Marsden and Fahn, 1982; Bird and Iversen, 1974). The ability to carry out simultaneous detec- tion of neurotransmitters will go a long way in shedding more light onto the complex mechanisms involved in the devel- opment of neuropsychiatric disorders and considerably im- prove the procedure of diagnosis and management of these diseases. 0956-5663/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2004.08.021