Capillary biosensor for glutamate

We have developed and characterized a novel glutamate biosensor which allows biological transduction of glutamate signal during transport of analyte from the sampling site to the detector. This biosensor exploits the high surface-to-volume ratio found in small-diameter fused silica capillaries, Glutamate dehydrogenase (GDH) was attached to the inner surface of a 75 mu m i,d, capillary using biotin-avidin chemistry. In the presence of excess nicotinamide adenine dinucleotide (NAD(+)), GDH converts glutamate to alpha-ketoglutarate while simultaneously reducing NAD(+) to NADH. Detection of NADH was accomplished using laser-induced fluorescence. Perfusion with 30 mu M glutamate in the presence of 3 mM NAD(+) resulted in a strong increase in fluorescence, with a response time of 450 ms, This effect was abolished upon exclusion of NAD(+) from the buffer. The limit of detection is 3 mu M (S/N = 3), with a linear working range from 3 to 300 mu M, Efficiency of the GDH-modified capillary ranged between 20% and 92% and was positively correlated with concentration of glutamate, The effect of linear velocity was also examined and was shown to be indirectly related to efficiency, with maximum response observed at 4.5 cm/ min. In summary, we have demonstrated the successful attachment of glutamate dehydrogenase to the inner wall of a small-diameter fused silica capillary while retaining enzymatic activity. The resulting biosensor exhibits characteristics amenable for in vivo applications. Future efforts will be directed toward the incorporation of this biosensor into current technologies, such as capillary electrophoresis and microdialysis.