Developing novel biosensors for detection of L-aspartate and N-acetyl aspartate

Microelectrode biosensors have proven to be an invaluable tool in direct measurement of extracellular concentration of analytes. Real-time measurement of released neuroactive compounds with simultaneous recording of electrophysiological activity has been made possible because of enzyme based sensors specific for their analytes. By using the sol-gel coating method of sensor fabrication, enzyme-based sensors for L-aspartate (L-Asp) and N-acetyl aspartate (NAA) were fabricated on a Platinum (Pt) microelectrode. The L-Asp sensor was made using the enzyme L-aspartate oxidase (LAO) and was fully characterised. The sensor achieved a steady state response to L-Asp within 15 s, had a lower detection limit of 3 μM with a sensitivity of 0.0016 μA/μM/cm2 (R=0.99). The L-Asp sensor also detected real-time release of L-Asp in area CA1 of hippocampal brain slices. L-Asp release was enhanced by independent application of excitatory amino acid transporter blocker DL-threo-β-Benzyloxyasparticacid (TBOA) and L-albizziine (L-Alb) an inhibitor for L-Asp metabolising enzyme. L-Alb also induced seizure-like activity with accompanying L-Asp release. These results bolster the idea of L-Asp as a possible co-transmitter or neuromodulator. NAA is a derivative of L-Asp and is the second most abundant compound in the brain after L-glutamate. NAA is also a marker for stroke and traumatic brain injury. NAA sensor was fabricated using the enzymes aspartoacylase (ASPA) and LAO. A novel method for detection of ASPA activity and proof of concept data for the NAA sensor is presented. The principle and methodology for NAA sensor fabrication can be extended to develop integrated electrochemical sensor array such as SMARTChip. Both the L-Asp and NAA sensor can provide valuable insight into the role of these compounds in functional aspects of brain physiology