Anal. Chem. 1998, 70, 89-93
Small-Volume On-Line Sensor for Continuous Measurement of γ-Aminobutyric Acid Osamu Niwa,*,† Ryoji Kurita,‡ Tsutomu Horiuchi,† and Keiichi Torimitsu†
NTT Basic Research Laboratories and NTT Advanced Technology, 3-1 Morinosato, Wakamiya, Atsugi, Kanagawa, 243-01, Japan
We report the first on-line electrochemical sensor for the continuous measurement of γ-aminobutyric acid (GABA), which is a well-known inhibitory neurotransmitter in the nervous system. The sensor is composed of a glutamate oxidase (GluOx) and catalase immobilized small-volume enzymatic reactor and a glassy carbon (GC) electrode modified with a top layer film consisting of gabase and GluOx coimmobilized bovine serum albumin and an Ospoly(vinylpyrridine) bottom layer film containing horseradish peroxidase. The response of the sensor depends on the r-ketoglutarate concentration and is almost saturated when its concentration is 100 times higher than GABA. The sensor exhibits a sensitivity of 1.56 nA/µM for GABA under optimized conditions and shows almost no response when 10 µM glutamate is continuously injected. A detection limit of 0.1 µM is obtained with a linear range of 0.1-10 µM. GABA can be measured in the absence of r-ketoglutarate when there is L-glutamate in the sample solution, which is a typical condition for the extracellular measurement of cultured nerve cells. The continuous monitoring of neurotransmitters is a very important technique for studying the physiology of nerve cells. This method is superior to various off-line methods such as liquid chromatography or capillary electrophoresis because the concentration change can be monitored continuously and the measurement can be undertaken in a much shorter time. The electrochemically active transmitters such as catecholamines and indoleamines released from nerve cells can be directly monitored using carbon fiber electrodes.1-3 γ-Aminobutyric acid (GABA) is well-known as a neurotransmitter which regulates inhibitory neurotransmission in mammalian central nervous systems. Extracellular concentrations of GABA have been determined using liquid chromatography combined with pre-/postcolumn derivatization.4 However, the direct monitoring of GABA is very difficult because it is insensitive to electrochemical and UV-visible spectroscopic methods. It is also difficult to detect GABA in combination with an enzymatic reaction †
NTT Basic Research Laboratories. NTT Advanced Technology. (1) Kawagoe, K. T.; Garris, P. A.; Wiedemann, D. J.; Wightman, R. M. Neuroscience 1992, 51, 55-64. (2) Ghasemzadeh, M. B.; Capella, P.; Mitchell, K.; Adams, R. N. J. Neurochem. 1993, 60, 442-448. (3) Bruns, D.; Jahn, R. Nature 1995, 377, 62. (4) Caudill, W. L.; Houck, G.; Wightman, R. M. J. Chromatogr. 1982, 227, 331. ‡
S0003-2700(97)00740-3 CCC: $14.00 Published on Web 01/01/1998
© 1997 American Chemical Society
because neither oxidase nor hydrogenase can be found, unlike the case with other neurotransmitters such as L-glutamate and acetylcholine.5-7 Gabase, which contains mainly two enzymes, γ-aminobutylate ketoglutarate aminotransferase (GABA-T) and succinic semialdehyde dehydrogenase (SSDH), is known to catalyze the two following GABA reactions. GABA-T
GABA + R-ketoglutarate 9 8 succinic SSDH semialdehyde (SSA) + glutamic acid (1) SSA + NADP + H2O f succinate + NADPH
(2)
GABA has been determined by the UV spectrophotometric or colorimetric method using gabase8 based on the optical density change caused by the NADP to NADPH reaction. However, this is not a continuous method because the products generated by the enzymatic reaction must be separated. In addition, it is necessary to add reagents such as NADP to the sample solution to detect GABA. Recently, we developed an on-line L-glutamate sensor, whose detection limit is ∼7-10 nM, by using a small-volume enzymatic reactor consisting of an L-glutamate oxidase (GluOx) and glassy carbon (GC) electrode9 modified with Os-poly(vinylpyrridine)based polymer containing horseradish peroxidase (Os-gelHRP)10,11 or using a BSA-GluOx/Os-gel-HRP bilayer film-modified GC electrode.12,13 Since the reaction of GABA with gabase (GABA-T and SSDH) produces the same amount of glutamic acid as shown in eq 1, the GABA concentration should be continuously detected by measuring the L-glutamate concentration with a gabase and GluOx coimmobilized electrode. In this paper, we describe an on-line electrochemical sensor for the continuous measurement of GABA. The sensitivity, (5) Tamiya, E.; Sugiura, Y.; Amou, Y.; Karube, I.; Ajima, A.; Kado, R. T.; Ito, M. Sens. Mater. 1995, 7, 249-259. (6) Hu, Y.; Mitchell, K. M.; Albahadily F. N.; Michaelis, E. K.; Wilson, G. S. Brain Res. 1994, 659, 117-125. (7) Albery, W. J.; Boutelle, M. G.; Galley P. T. J. Chem. Soc., Chem. Commun. 1992, 900-901. (8) Sethi, M. L. J. Pharm. Biomed. Anal. 1993, 11, 613. (9) Niwa, O.; Torimitsu, K.; Morita, M.; Osborne, P. G.; Yamamoto, K. Anal. Chem. 1996, 68, 1865-1870. (10) Vreeke, M.; Maiden, R.; Heller, A. Anal. Chem. 1992, 64, 3084-3090. (11) Yang, L.; Janle, E.; Huang, T.; Gitzen, J.; Kissinger, P. T.; Vreeke, M.; Heller, A. Anal. Chem. 1995, 67, 1326-1331. (12) Niwa, O.; Horiuchi T.; Torimitsu K. Biosens. Bioelectron. 1997, 12, 311319. (13) Torimitsu K.; Niwa, O. Neuroreport 1997, 6, 1353-57.
Analytical Chemistry, Vol. 70, No. 1, January 1, 1998 89
Figure 1. Schematic representation of GABA detection in an on-line flow sensor.
detection limit, and selectivity against other biological molecules are also discussed using standard solutions. PRINCIPLE: DETERMINATION OF GABA Instead of the optical absorption method shown in eq 1, we propose an amperometric method for measuring GABA which uses gabase and a GluOx-modified GC electrode. The amperometric method is superior to the photoabsorption method because of its low detection limit. The reaction scheme of this method is as follows. GABA-T
GABA + R-ketoglutarate 9 8 SSDH succinic semialdehyde (SSA) + glutamic acid (1) GluOx
glutamic acid + H2O + O2 98 R-ketoglutarate + NH3+ + H2O2 (3)
The hydrogen peroxide generated by the above enzymatic reaction was then reduced electrochemically through an Os-gel-HRP film.10 In this system, L-glutamate contained in the biological sample can also be reacted on the electrode; therefore, we used a smallvolume prereactor in which GluOx and catalase were immobilized to consume all the L-glutamate molecules in the sample solution. Since the oxidation of L-glutamate produces R-ketoglutarate, GABA could be measured without adding any reagents containing R-ketoglutarate in the sample when the L-glutamate concentration was relatively high. This is very important when the sensor is applied to the measurement of biological samples because the addition of other reagents sometimes changes the physiological response of neurons. EXPERIMENTAL SECTION Chemicals. Gabase from Pseudomonas fluorescens. (0.37 unit/ mg of solid) was obtained from Sigma (St. Louis, MO). GluOx from Streptomyces Sp. X119-6 (6.8 mg/mg of solid) was obtained from Yamasa Shoyu (Choshi, Japan), and catalase was obtained from Katayama (1000 units/mg, NK-118). 2-Oxoglutaric disodium salt (R-ketoglutarate) was obtained form Merck. Osmium-poly(vinylpyrridine) wired horseradish peroxidase (Os-gel-HRP) was obtained from Bioanalytical Systems Inc. (BAS) (West Lafayette, IN). It was reported that an Os-gel-HRP-modified electrode reduces hydrogen peroxide at 0 mV vs Ag/AgCl.10,11 90
Analytical Chemistry, Vol. 70, No. 1, January 1, 1998
Bovine serum albumin (BSA) and aspartic acid was purchased from Sigma. Aminopropyl-CPG beads (200/400 mesh) were obtained from Electro Nucleonics Inc. (Fairfield, NJ). Sodium dihydrogen phosphate monohydrate was obtained from Merck (Darmstadt, Germany). Phosphate-buffered saline (PBS) (Gibco), glutaraldehyde (Wako, Tokyo, Japan), L-ascorbic acid (Kanto Chemical, Japan), Nafion (5 wt % solution, Aldrich), GABA, L-glutamic acid, dopamine, and serotonin (Funakoshi, Tokyo, Japan) were used as purchased. Electrode and Prereactor. A GC electrode (6 mm diameter, BAS) was used to fabricate the GABA sensor. The GC electrode surface was first modified with Os-gel-HRP by the casting method. The solution applied to the surface was 7.1 µL/cm2. The enzyme layer was immobilized by covering it with a PBS solution (5 µL) containing 2% BSA, 0.76 unit of GluOx, 0.26-0.33 unit of gabase, and 0.2% glutaraldehyde on the Os-gel-HRP-modified electrode. For voltammetric measurement, we used a 3 mm diameter GC electrode after modifying it with Os-gel-HRP and BSA-GluOxgabase bilayer films. A Teflon tube with an inner diameter of 0.75 mm was divided into two rooms by inserting a plastic filter in its center and used as a prereactor to remove glutamate. The length of the tube was 5 cm (inner volume 22 µL). GluOx and catalase were separately immobilized on beads with glutaraldehyde. The GluOx immobilized beads were packed in the upstream portion of the tube, and the catalase immobilized beads were packed in the downstream portion. Voltammetric Measurement. The 3 mm GC electrode (BAS) modified with bilayer films of BSA-gabase-GluOx and Osgel-HRP was immersed in a PBS solution containing 40.0 mM R-ketoglutarate or 36.1 mM R-ketoglutarate and 9.1 mM GABA. The voltammogram of the modified electrode was measured with a BAS 100B electrochemical analyzer (BAS). The scan rate of the potential was 50 mV/s. Platinum wire and an Ag/AgCl electrode were used as auxiliary and reference electrodes, respectively. Continuous Measurement of GABA. Figure 1 shows a block diagram of the sensor system. A CMA 102 dual-syringe pump (CMA Microdialysis, Stockholm, Sweden) was used to introduce the solutions into the system. Two different solutions, a phosphate buffer containing R-ketoglutarate and a buffer solution containing R-ketoglutarate and GABA were injected separately by using syringes 1 and 2 and mixed upstream of the prereactor as shown in the figure. It was possible to change the GABA
Figure 2. Cyclic voltammograms of a BSA-GluOx-gabase/Os-gelHRP bilayer film modified GC electrode (d ) 3 mm) in (a) PBS, (b) PBS containing 40 mM R-ketoglutarate, and (c) PBS containing 9.1 mM GABA and 36.1 mM R-ketoglutarate. The scan rate is 50 mV/s. An Ag/AgCl electrode is used as a reference.
concentration by controlling the flow rate from the two syringes. In this system, we expected that all the glutamate molecules to be oxidized in the upstream portion of the prereactor and that any hydrogen peroxide molecules generated by the enzymatic reaction of the glutamate would be consumed by the catalase in the downstream portion. In contrast, the GABA molecules would not react in the prereactor and would be detected at the BSAgabase-ChOx- and Os-gel-HRP bilayer-modified GC electrode in the thin-layer radial flow cell, which shows higher sensitivity than the channel flow cell. For calibration, GABA solution ranging in concentration from 0.1 to 10 µM was injected into the sensor at a flow rate of 16 µL/min. We measured the GABA selectivity against glutamate by comparing the signal magnitudes when injecting 10 µM GABA and 10 or 50 µM glutamate into the sensor. We also studied the effects of other neurotransmitters such as dopamine, serotonin, aspartate, and L-ascorbic acid by injecting 1-10 µM of these molecules into the sensor. When studying the effect of L-ascorbic acid, we overcoated the modified electrode surface with Nafion film and changed the detection potential to -50 or -100 mV. RESULTS AND DISCUSSION Voltammetric Response of GABA. Figure 2 shows voltammograms of the modified electrode in (a) PBS solution, (b) PBS solution containing R-ketoglutarate, and (c) PBS solution containing GABA and R-ketoglutarate. The anodic and cathodic peaks assigned to the redox reaction of Os-gel were observed when measuring the voltammogram in PBS. The voltammogram did not change in the PBS containing R-ketoglutarate, indicating that no enzymatic or electrochemical reaction took place. In contrast, the cathodic limiting current increases and the anodic current decreases by mixing with GABA, as shown in Figure 2c. The cathodic current increases by increasing the GABA concentration.
Figure 3. Variation in the 1 µM GABA response as a function of R-ketoglutarate concentration. A syringe pump was used to introduce the sample into the sensor at a flow rate of 16 µL/min. The potential of the GC electrode modified with Os-gel-HRP film and BSA containing gabase and L-glutamate oxidase film was held at 0 mV vs Ag/ AgCl. The R-ketoglutarate concentration was varied from 1 to 100 µM.
This result indicates that the reaction shown in eqs 1 and 3 occurs in the BSA-gabase-GluOx layer only when GABA and R-ketoglutarate coexist and the hydrogen peroxide generated by the enzymatic reaction is reduced electrocatalytically in the Os-gelHRP-modified GC electrode. Sensitivity and Detection Limit of the Sensor in the Flow Cell. Since R-ketoglutarate is needed to transform GABA to L-glutamate, the GABA signal should be dependent on the R-ketoglutarate concentration. Figure 3 shows the variation in the cathodic current of 1 µM GABA on the BSA-gabase-GluOx/ Os-gel-HRP bilayer-modified GC electrode (d ) 6 mm) in the flow cell (Figure 1) when the R-ketoglutarate concentration is increased from 0 to 100 µM. The GABA response is almost 0 nA when the R-ketoglutarate concentration is 0 µM. However, it increases rapidly as the R-ketoglutarate concentration is increased from 0 to 20 µM. The current increases gradually when the concentration is higher than 20 µM and is almost saturated at 100 µM. Therefore, the sensor was calibrated with a solution whose R-ketoglutarate concentration is much higher than its GABA concentration. Figure 4 shows the relationship between GABA concentration and the cathodic limiting current at a flow rate of 16 µL/min. The R-ketoglutarate concentration is 100 µM. The sensitivity of GABA under optimized conditions is 1.56 nA/µM with a linear range from 0.1 to 10 µM. The reproducibility of the response using one sensor is very good (SD
