Direct immobilization of glutamate dehydrogenase on optical fiber probes for ultrasensitive glutamate detection

Ultrasensitive glutamate monitoring is important in a variety of areas of biochemical analysis. We have developed a glutamate sensor with micrometer to sulbmicrometer diameter. Glutamate dehydrogenase (GDH) has been directly immobilized onto an optical fiber probe surface through covalent binding mechanisms. An optical fiber surface is initially activated by silanization, which adds amine groups (NH2) to the surface. We then affix functional groups CHO to the optical fiber surface by employing a bifunctional cross-linking agent, glutaraldehyde. The amino acids of GDH molecules (or other biomolecules) readily attach to these free CHO groups on the fiber surface. Optimal immobilization of GDH occurred between 20 and 25 h in the enzyme solution. The immobilized GDH enzyme molecules on the fiber surface have shown high enzymatic activity. The sensor is able to detect its substrate, glutamate, by monitoring the fluorescence of NADH, a product of the reaction between NAD(+) and glutamate. The concentration detection limit of the sensor is 0.22 mu M glutamate, and the absolute mass detection limit is 3 amol. Response times of the sensors are fast due to the direct GDH molecule immobilization. The glutamate sensor is selective and stable. A submicrometer glutamate sensor has been tested. Our glutamate probes could be applied to the study of subcellular level neurophysiological responses.