Biosensors and Bioelectronics 24 (2009) 2384–2389 Contents lists available at ScienceDirect Biosensors and Bioelectronics journal homepage: www.elsevier.com/locate/bios Glutamate sensing with enzyme-modified floating-gate field effect transistors D. Braeken a,∗ , D.R. Rand a , A. Andrei a , R. Huys a , M.E. Spira c , S. Yitzchaik d , J. Shappir e , G. Borghs a , G. Callewaert b , C. Bartic a a IMEC v.z.w., Kapeldreef 75, 3001 Leuven, Belgium b Research Team Neurodegeneration, Campus Kortrijk, KULeuven, E. Sabbelaan 53, 8500 Kortrijk, Belgium c Department of Neurobiology – Hebrew University of Jerusalem, Givat Ram Campus, 91904 Jerusalem, Israel d Department of Inorganic and Analytical Chemistry – Hebrew University of Jerusalem, Givat Ram Campus, 91904 Jerusalem, Israel e School of Engineering – Hebrew University of Jerusalem, Givat Ram Campus, 91904 Jerusalem, Israel article info Article history: Received 12 September 2008 Received in revised form 5 December 2008 Accepted 5 December 2008 Available online 14 December 2008 Keywords: Glutamate Glutamate oxidase ENFET Quartz crystal microbalance abstract Neurotransmitter release is the key factor of chemical messaging in the brain. Fast, sensitive and in situ detection of single cell neurotransmitter release is essential for the investigation of synaptic transmission under physiological or pathophysiological conditions. Although various techniques have been devel- oped for detecting neurotransmitter release both in vitro and in vivo, the sensing of such events still remains challenging. First of all, the amount of neurotransmitter released during synaptic transmission is unknown because of the limited number of molecules released and the fast diffusion and reuptake of these molecules after release. On the other hand, advances in microelectronic biosensor devices have made possible the fast detection of various analytes with high sensitivity and selectivity. Specifically, enzyme-modified field-effect (ENFET) devices are attractive for such applications due to their fast response, small dimensions and the possibility to integrate a large number of sensors on the same chip. In this paper, we present a floating-gate FET device coated with glutamate oxidase (GLOD) layer. The surface chemistry was optimized for maximal enzyme loading and long-term stability, and characterized by quartz crystal microbalance and colorimetric assays. Enzyme loading was largest on poly-l-lysin-based surfaces combined with glutaraldehyde. The surface chemistry showed excellent stability for at least one month in Tris buffers stored at 4 ◦ C. A glutamate detection limit of 10 -7 M has been obtained with the GLOD-coated FET and our sensor proved to be selective to glutamate only. We show that this biosensor is a promising tool for the in vitro detection of glutamate and can be extended to other neurotransmitters. © 2008 Elsevier B.V. All rights reserved. 1. Introduction Changes in synaptic efficacy, including long-term potentiation and long-term depression of excitatory synaptic transmission, are considered to be the neuronal bases for learning and memory and are regulated by glutamate, amongst other neurotransmit- ters (Linden and Connor, 1992; Manahan-Vaughan et al., 2003). Pathological conditions related to signal transmission, such as Alzheimer’s disease, require investigation at the synaptic level in order to understand the defects that occur in neuronal signaling (Walsh et al., 2002; Bell et al., 2003). Therefore, there is a large interest to investigate in situ glutamate levels released by neuronal cells. The in vivo extracellular glutamate concentration in brain tissue measured by microdialysis is estimated to be in the range of 1–2 M ∗ Corresponding author. Tel.: +32 162 88942; fax: +32 162 81097. E-mail address: dries.braeken@imec.be (D. Braeken). (Benveniste et al., 1984; Parrot et al., 2004; Zhang et al., 2005). How- ever, these glutamate levels are believed to be an overestimation since the diameter of the microdialysis electrodes (200–500 m) is 10,000-fold larger than the width of the synaptic cleft (20–50 nm) (Zuber et al., 2005). More sensitive and trustworthy techniques are therefore needed to measure glutamate release under these condi- tions. Biosensor technology has proven to be very promising for the sensitive and selective detection of single analytes. Enzymatic detection of glutamate is based on its conversion to side prod- ucts that can be measured by various electrochemical techniques. Among these, ion-sensitive field effect transistors (ISFETs) have many important advantages over conventional amperometric elec- trodes. Since their introduction by Bergveld (1970), they have been proven to be promising tools in various domains of research, includ- ing DNA genotyping, food screening and multi-analyte detection for biomedical applications. Their small dimensions, fast response and compatibility with conventional integrated circuits make them an excellent choice for biosensor applications (Alonso et al., 2003; 0956-5663/$ – see front matter © 2008 Elsevier B.V. All rights reserved. doi:10.1016/j.bios.2008.12.012