Concentration of Extracellular l-Glutamate Released from Cultured Nerve Cells Measured with a Small-Volume Online Sensor

Abstract

An online sensor with a low detection limit for L-glutamate was developed in order to monitor the change in the extracellular L-glutamate concentration as a result of stimulated release from cultured nerve cells. The sensor consisted of a microdialysis (MD) probe fixed at the manipulator, a small-volume L-glutamate oxidase enzymatic reactor (0.75 mm i.d. and 2.5 cm long), and an electrochemical detector in a thin-layer radial flow cell with an active volume of 70-340 nL. Glassy carbon bulk or carbon film ring-disk electrodes were used as detectors by modifying them with Os poly(vinylpyridine) mediator containing horseradish peroxidase. The overall efficiency of L-glutamate detection with the sensor is 94% under optimum conditions, due to an efficient enzymatic reaction in the reactor and a high conversion efficiency in the radial flow cell. As a result, we achieved a sensitivity of 24.3 nA/muM and a detection limit of 7.2 nM (S/N = 3). The effect of interferents such as L-ascorbic acid can be minimized effectively by applying a low potential to the electrode for hydrogen peroxide detection (O mV) and via the ring-disk electrode geometry by using the disk for preoxidation. In the in vitro experiment, an MD probe for sampling was connected to a manipulator that controls distance between the probe and the stimulated cells. The cells were stimulated by KCl in a glass capillary or electrically with microarray film electrodes fabricated on a substrate. By using the sensor, we can monitor L-glutamate concentration changes at the submicromolar level caused by KCl stimulation of a single nerve cell and micromolar L-glutamate concentration increases caused by electrical stimulation of a brain slice. An increase in L-glutamate concentration can also be measured by positioning the probe near the cell that is connected synaptically to the stimulated cell.

Micro-flow in vivo Analysis of l-Glutamate with an On-line Enzyme Amplifier Based on Substrate Recycling

“…This method is based on the use of a microdialysis probe in combination with an immobilized enzyme reactor. A similar attempt was achieved for the on-time monitoring of glucose 9,10 and L-glutamate [11][12][13] by using a miniaturized enzyme electrode. However, the concentration of L-glutamate in the extracellular spaces of rat brain was normally at the few µmol dm -3 level, 14,15 and the permeability of L-glutamate into the microdialysis probe was about 10% at a flow rate of 2 µl min -1 when Ringer's solution was pumped as a perfusion solution into the probe.…”

Amperometric l-glutamate biosensor based on bacterial cell-surface displayed glutamate dehydrogenase

“…As a functional amino acid, it is a crucial excitatory neurotransmitter in the central nervous system. For most eukaryon and mammalian, L-glutamate can serve as energy and nitrogen source [6][7][8]. In addition, L-glutamate has been widely used as the flavor enhancer in foodstuffs such as monosodium glutamate (MSG), chicken essence, soy sauce and some snack seasonings.…”

Microfabricated On-Line Sensor for Continuous Monitoring of l-Glutamate

“…However, their slow response and large sampling probe size limits their spatial and temporal resolutions in spite of their high sensitivity and long term stability. 21,22 We reported an on-line L-glutamate sensor for measuring dissociated nerve cell cultures. 23 The sensor consists of a glass capillary for sampling combined with an electrochemical thin layer flow cell.…”

“…These consist of a syringe pump, a microdialysis (MD) sampling probe, an enzymatic reactor and an electrode in a flow cell. [17][18][19][20][21][22] On-line sensors are particularly useful for measuring in vivo L-glutamate, since they are very stable. This is because the flow measurement is very reproducibile and the MD membrane eliminates high molecular weight interferents such as proteins.…”

“…[10][11][12] Therefore, glutamate oxidase has been more often used to fabricate electrochemical sensors for detecting the neurotransmitter, L-glutamate. [13][14][15][16][17][18][19][20][21][22][23][24] Carbon fiber or metal wire microelectrodes modified with glutamate oxidase have been employed for measuring L-glutamate in the brain and in cultured rat brain tissue. [13][14][15][16] It has been reported that L-glutamate increases as the result of KCl and electrical stimulations.…”