Anal. Chem. 1999, 71, 1529-1533
Direct Immobilization of Glutamate Dehydrogenase on Optical Fiber Probes for Ultrasensitive Glutamate Detection Julia Cordek, Xinwen Wang, and Weihong Tan*
Department of Chemistry and UF Brain Institute, University of Florida, Gainesville, Florida 32601
Ultrasensitive glutamate monitoring is important in a variety of areas of biochemical analysis. We have developed a glutamate sensor with micrometer to submicrometer 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 µM 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. Glutamate is the major excitatory neurotransmitter in the central nervous system. Its sensitive determination is of great interest in a variety of areas of biomedical research and biotechnology. There have been many research activities in the development of advanced bioanalytical techniques for the monitoring of a variety of biomolecules,1-13 including glutamate, one of the most (1) Hu, Y.; Mitchell, K. M.; Albahadily, F. N.; Michaelis, E. K.; Wilson, G. S. Brain Res. 1994, 659, 117-125. Hu, Y.; Wilson, G. S. J. Neurochem. 1997, 69, 1484-1490. (2) Pantano P.; Kuhr, W. G. Anal. Chem. 1993, 65, 623-630. (3) Bunin, M. A.; Wightman R. J. Neurosci. 1998, 18, 4854-4860. (4) Garguilo, M. G.; Michael, A. C. J. Neurosci. Methods 1996, 70, 73-82. (5) Ingersoll, C. M.; Bright, F. V. Anal. Chem. 1997, 69, 403A-408A. (6) Wang, A.-J.; Arnold, M. A. Anal. Chem. 1992, 64, 1051-1055. Kar, S.; Arnold, M. A. Anal. Chem. 1992, 64, 2438-2443. (7) Dremel, B. A.; Schmid, R. D.; Wolfbeiss, O. S. Anal. Chim. Acta 1991, 248, 351-359. (8) Pantano, P.; Walt, D. R. Anal. Chem. 1995, 67, 481A-487A. (9) Lada, M. W.; Vickroy, T. W., Kennedy, R. T. Anal. Chem. 1997, 69, 45604565. 10.1021/ac980850l CCC: $18.00 Published on Web 03/16/1999
© 1999 American Chemical Society
studied neurotransmitters. The major techniques for sensitive glutamate detection are microelectrodes,1,2 fiber optic sensors,6,7 and separation techniques coupled with optical or electrochemical detection.9-11 These techniques have been useful for many applications in glutamate monitoring. For example, dual enzymebased microelectrodes have been developed for direct measurement of glutamate release from neurons in the brain.1 The electrodes have a size of a few micrometers, a response time of ∼1 s, and a sensitivity of less than 2 µM. Our goal for this work is focused toward the development of a glutamate biosensor suitable for continuous monitoring of glutamate release from individual cells or subcellular structures, thus resulting in an effective detection technique for the mapping of extracellular glutamate concentration in cell culture during ischemia.14 This has not been possible due to the limitations of available analytical techniques. Current techniques for the measurement of the release of endogenous glutamate into the extracellular space of the central nervous system are relatively slow and have inadequate spatial resolution. A continuous monitoring of the extracellular glutamate release will permit the resolution of the kinetics of neurotransmitter release. However, this can be realized only if the analytical probe in the extracellular space is sufficiently small (submicrometer to ∼5 µm), highly sensitivity (down to µM), and fast (3σ) for detection. The shortest response time of the sensor corresponds to the minimally required optical signal
Figure 5. Glutamate monitoring in a 5-µm polycarbonate membrane hole by a submicrometer GDH probe. Figure 4. Responses of two GDH probes. The responses are shorter than 50 ms. Both NAD+ and glutamate concentrations were 1 mM.
generated by NADH. The standard deviation of the baseline was used to estimate the response time of the sensor. It thus cannot be compared with those reported in the literature.1,6 This definition of response time for the glutamate sensor is appropriate for its intended application. When a neuron releases glutamate, the speed at which the sensor can detect the released glutamate is an important characteristic of the sensor. This property will determine the suitability of the sensor’s application to neurons. A great benefit to our biosensors is the lack of recovery time needed. The GDH-based sensors were ready for experimental measurements directly after rinsing with PBS buffer. This fast response time is due to the direct immobilization of the GDH molecules onto the optical fiber surface and the small size of the sensor. Submicrometer Glutamate Sensors. The direct GDH immobilization method enabled the miniaturization of the glutamate sensor to a submicrometer scale, required for subcellular monitoring. The submicrometer sensor, shown in Figure 5, can easily detect a glutamate concentration of 30 µM in the capillary reactor. To determine the absolute mass detection limit of a submicrometer glutamate sensor, we used 5-µm-diameter polycarbonate membrane holes with a depth of ∼10 µm28 as the reactors for detection. These membrane holes were similar to single biological cells in size, shape, and dispersion on the surface. The glutamate sensor was inserted into one specific hole which was filled with the substrate solution, as shown in Figure 5. The fluorescence intensity from this hole was monitored over time. We were able (28) Tan, W.; Yeung, E. Anal. Chem. 1997, 69, 4242.
to detect glutamate concentration down to 15 µM. This gives our sensor an absolute mass detection limit of ∼3 amol of glutamate (15 µM × 196 × 10-15 L). This experiment imitated living cell monitoring, in which a glutamate sensor is positioned over the cell membrane. We have also confirmed that the sensor is working in preliminary cellular monitoring of glutamate release. CONCLUSION Ultrasensitive glutamate optical sensors, of micrometer to submicrometer sizes, have been developed by directly immobilizing glutamate dehydrogenase onto optical fiber surfaces through covalent binding. Optimal immobilization of GDH occurred between 20 and 25 h in the enzyme solution. The glutamate sensor is small and fast, uses only GDH enzyme molecules, is simple in its preparation, and is easy to use. The GDH molecules on the fiber surface have high enzymatic activity. The sensor’s detection limit is 0.22 µM glutamate, and the absolute mass detection limit is 3 amol for a submicrometer glutamate optical sensor. Our detection system can monitor NADH generated by the sensor within 50 ms. The glutamate sensor is stable and extremely selective. These sensors will be useful in monitoring glutamate release and thus in studying subcellular level neurophysiological responses. ACKNOWLEDGMENT This work is supported by an NSF Career award (CHE9733650) and by the Office of Naval Research Young Investigator Award (N00014-98-1-0621). Received for review July 31, 1998. Accepted January 29, 1999. AC980850L
Analytical Chemistry, Vol. 71, No. 8, April 15, 1999
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