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 exploi ts the high surface-to-volume ratio found in small-diameter fused sili ca capillaries, Glutamate dehydrogenase (GDH) was attached to the inne r surface of a 75 mu m i,d, capillary using biotin-avidin chemistry. I n the presence of excess nicotinamide adenine dinucleotide (NAD(+)), G DH converts glutamate to alpha-ketoglutarate while simultaneously redu cing NAD(+) to NADH. Detection of NADH was accomplished using laser-in duced fluorescence. Perfusion with 30 mu M glutamate in the presence o f 3 mM NAD(+) resulted in a strong increase in fluorescence, with a re sponse 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-modif ied capillary ranged between 20% and 92% and was positively correlated with concentration of glutamate, The effect of linear velocity was al so examined and was shown to be indirectly related to efficiency, with maximum response observed at 4.5 cm/ min. In summary, we have demonst rated the successful attachment of glutamate dehydrogenase to the inne r wall of a small-diameter fused silica capillary while retaining enzy matic activity. The resulting biosensor exhibits characteristics amena ble for in vivo applications. Future efforts will be directed toward t he incorporation of this biosensor into current technologies, such as capillary electrophoresis and microdialysis.