Two-dimensional enzyme-based biosensors for spatially resolved imaging and monitoring of neurochemicals

This work focuses on the development of a novel two-dimensional (2D) enzyme-based biosensor for the detection of neurochemicals. There is a growing need for the elucidation of neurochemicals such as glutamate and lactate. Enzyme-based biosensors have shown to be an excellent detection technique for these neurochemicals. Current biosensors, which detect neurochemicals by single point measurements, have made much progress towards real time monitoring. However, a single point is not adequate for the complete understanding of many interesting cellular and subcellular events. The development of a spatially resolved 2D biochemical sensor will enable a better understanding of many biological processes by simultaneously imaging a large target area, not just a single point.; Our (2D) biosensor is based on a flat silica plate (FSP) with immobilized enzyme. The immobilization of the enzymes was done through a combination of covalent chemistries established for biosensor development. The enzymes (LDH and GDH for the detection of lactate and glutamate respectively) go through a catalytic reaction with its co-substrate nicotinamide adenine dinucleotide (NAD+) and either lactate or glutamate to yield the fluorescent product NADH. With an intensified charged coupled device (ICCD) detection system, highly sensitive glutamate and lactate monitoring was achieved.; To improve the biosensors detection capabilites, silica nanoparticles were polymerized onto the FSP. Higher silica content for enzyme immobilization increased the sensitivity down to the nanomolar range. Also individual nanoparticles were modified with enzyme and utilized for glutamate and lactate detection. Respectively, these nanoparticles have been covalently assembled onto 2D substrates for bio-analysis. We have used the 2D biosensors for real-time monitoring of lactate released from mouse brain slices. We have studied the impact of KCl on lactate release. The variations of lactate released from the tissue slice have been spatially resolved and monitored.; Moreover, we have used the 2D biosensors for the detection of glutamate release. Nicotine, a well known addictive drug, has been used for the stimulation for glutamate release. We have found that low levels of nicotine can stimulate glutamate release, which gives more information about nicotine addiction.; The detection, monitoring and imaging of neurochemical release has shown that the 2D enzyme-based biosensor is a feasible approach to study cellular communications and possibly other biological processes that require simultaneous temporal and spatial resolutions. The two-dimensional biosensor also has the capability of monitoring many cells.