29 June 2005 L-glutamate detection using a poly-L-lysine coated ENFET
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Abstract
Synaptic transmission in neuronal networks occur on a very short time scale and is highly specific. Fast, sensitive and in situ detection of single neuron L-glutamate release is essential for the investigation of these events under physiological or pathophysiological conditions. Up till now, amperometry with enzyme-modified electrodes has extensively been used to monitor extracellular glutamate release. However, due to in situ signal amplification, ENzyme-modified Field-Effect Transistors (ENFETs) have the advantage of preserving sensitivity and a fast response time when scaled down to micrometer dimensions. We have realized a L-GLutamate OxiDase (GLOD) functionalized FET to be used for glutamate detection in neuronal cultures. Effective and reproducible immobilization of GLOD on the FET active area is achieved by using Poly-L-Lysine (PLL) as a loading matrix. PLL plays a dual role in the assay: on the one hand this molecule serves as a platform for obtaining high enzyme loading and on the other hand it benefits the survival of the neuronal network on the active area of the FET. Both PLL and enzyme immobilization were characterised by quartz crystal microbalance measurements. A much higher enzyme loading has been achieved by this approach compared to immobilization methods without PLL. The enzyme coating has proven to be extremely durable as it keeps its activity for at least 3 weeks as monitored by a colorimetric assay. FET characterisation curves and glutamate response curves of the ENFET are presented.
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D. Braeken, D. Braeken, C. Zhou, C. Zhou, R. Huys, R. Huys, C. Bartic, C. Bartic, K. De Keersmaecker, K. De Keersmaecker, K. Winters, K. Winters, G. Callewaert, G. Callewaert, G. Borghs, G. Borghs, } "L-glutamate detection using a poly-L-lysine coated ENFET", Proc. SPIE 5839, Bioengineered and Bioinspired Systems II, (29 June 2005); doi: 10.1117/12.608316; https://doi.org/10.1117/12.608316
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