On-Line Electrochemical Sensor for Selective Continuous

Anal. Chem. 1998, 70, 1126-1132

On-Line Electrochemical Sensor for Selective Continuous Measurement of Acetylcholine in Cultured Brain Tissue Osamu Niwa,*,† Tsutomu Horiuchi,† Ryoji Kurita,‡ and Keiichi Torimitsu†

Downloaded by UNIV OF CAMBRIDGE on September 2, 2015 | http://pubs.acs.org Publication Date (Web): February 10, 1998 | doi: 10.1021/ac970257o

NTT Basic Research Laboratories and NTT Advanced Technology, 3-1 Morinosato, Wakamiya, Atsugi, Kanagawa 243-0198, Japan

An on-line acetylcholine (ACh) sensor was developed in order to determine extracellular ACh concentrations without interference from choline (Ch). The sensor is composed of a small-volume enzymatic prereactor (22µL inner volume) in which choline oxidase and catalase are immobilized in series. Carbon electrodes were modified with an acetylcholine esterase (AChE), choline oxidase (ChOx), and osmium poly(vinylpyridine)-based redox polymer containing horseradish peroxidase (Os-gelHRP). The sensor sensitivity was 43.7 nA/µM ((0.15, n ) 3) for ACh under optimized conditions. Almost no response was seen when 100 µM Ch was continuously injected. The detection limit for ACh with the sensor was comparable to that obtained using liquid chromatography with electrochemical detection combined with an enzymatic reactor. The electrical stimulation of cultured rat hippocampal tissue resulted in an extracellular ACh increase of 20 nM ((11 nM, n ) 3). This increase was observed continuously with our online sensor combined with a microcapillary sampling probe located very close to the tissue. The continuous measurement of ACh and Ch using a split disk carbon film dual electrode in which one electrode surface was modified with ChOx/Os-gelHRP and the other with AChE-ChOx/Os-gel-HRP bilayer film was also demonstrated to improve the response time by eliminating the prereactor. The continuous monitoring of neurotransmitters is very important for studying the physiology of nerve cells, particularly for monitoring time-dependent changes in transmitter concentration near a nerve cell. Much effort has been focused on the detection of catecholamines and serotonin using carbon fiber electrodes, and this has been successfully applied to monitoring catecholamine release from a single cell.1-3 On the other hand, various enzyme-modified electrodes have been developed for monitoring electroinactive neurotransmitters such as glutamate, †

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) Chen, T.-K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (3) Bruns, D.; Jahn, R. Nature 1995, 377, 62-65. ‡

1126 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

acetylcholine (ACh), and choline (Ch).4-6,8 ACh is widely distributed in the body, and in vertebrates only trace amounts are needed to transfer important biological information through motor neurons in the spinal cord and at nerve skeletal junctions. ACh has also attracted considerable attention as an important transmitter involved in learning and memory in relation to the mammalian central nervous system.7 Therefore, the continuous monitoring of ACh in brain tissue is required in order to understand the function of neurons. Enzyme sensors have been developed which consist of an electrode co-immobilized with acetylcholine esterase (AChE) and choline oxidase (ChOx).8-12 However, these sensors cannot be used for measuring ACh in a biological sample due to the existence of various interferences. With regard to electroactive interferences such as catecholamines and L-ascorbic acid, various methods have been developed to increase selectivity. These include the use of an electron mediator to lower the detection potential and a modified anionic polymer film to reject anionic interferences such as L-ascorbic acid and DOPAC. Compared with the various techniques for rejecting electroactive interferences, the selective determination of ACh in the presence of Ch is very difficult. This is because the Ch concentration is much higher than that of ACh, particularly for in vivo measurement, and both ACh and Ch react enzymatically at an AChE and ChOx-modified electrode or an immobilized reactor.13 Therefore, the in vivo (and in vitro) ACh concentrations have been measured using liquid chromatography/electrochemistry (LCEC) with a postcolumn enzyme reactor.14-18 However, these measure(4) Garguilo, M. G.; Michael A. C. J. Am. Chem. Soc. 1993, 115, 12218-12219. (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) Hasselmo, M. E.; Bower, J. M. Trends Neurosci. 1993, 16, 218-222. (8) Navera, E. N.; Suzuki, M.; Tamiya, E.; Takeuchi, T.; Karube, I. Electroanalysis 1993, 5, 17-22. (9) Marty, J. L.; Sode, K.; Karube, I. Anal. Chim. Acta 1989, 228, 49-53. (10) Morelis, R. M.; Coulet, P. R. Anal. Chim. Acta 1990, 231, 27-32. (11) Hale, P. D.; Wightman, R. M. Mol. Cryst. Liq. Cryst. 1988, 160, 269-279. (12) Ruiz, B. L.; Dempsey, E.; Hua, C.; Smyth, M. R.; Wang, J. Anal. Chim. Acta 1993, 273, 425-430. (13) Guzman, R. G.; Kendrick, K. M.; Emson, P. C. Brain Res. 1993, 622, 147154. (14) Potter, P. E.; Meek, J. L.; Neff, N. H. J. Neurochem. 1983, 41, 188-194. (15) Fossati, T.; Colombo, M.; Castiglioni, C.; Abbiati, G. J. Chromatogr. B 1994, 656, 59-64. S0003-2700(97)00257-6 CCC: $15.00

© 1998 American Chemical Society Published on Web 02/10/1998


ments need a long separation column and take a relatively long time to separate ACh and Ch. The on-line sensor has been used for both in vivo and in vitro measurements of neurotransmitters.19-21 Although the sensor response is slower than that of a modified microelectrode, it has the advantages of high sensitivity, a low detection limit, and a long lifetime. The low detection limit is particularly important for transmitters because of their low extracellular concentration. The selectivity can also be improved by inserting a prereactor or pre-electrolysis electrode to eliminate the interferences between the sampling probe and the detector. In this paper, we report a small-volume online sensor which can continuously monitor extracellular ACh selectively in a brain tissue culture. A high selectivity between ACh and Ch has been reported by using a small-volume prereactor in which choline oxidase (ChOx) and catalase were immobilized and an electrode modified with osmium poly(vinylpyridine) mediator containing horseradish peroxidase (HRP).22 A detection limit of low nanomolar concentration was achieved, which is comparable to that of LCEC. EXPERIMENTAL SECTION Chemicals. Acetylcholine esterase (AChE) from an electric eel (970 units/mg solid), choline oxidase (ChOx) from an alcaligenes species (14 units/mg solid), bovine serum albumin, acetylcholine (ACh), choline (Ch), and papain were purchased from Sigma (St. Louis, MO). Osmium poly(vinylpyridine) solution containing horseradish peroxidase (Os-gel-HRP) and Kathon CG reagent (1%) were obtained from Bioanalytical Systems Inc.17,22 (BAS) (West Lafayette, IN). Catalase was obtained from Katayama (1000 units/mg, NK-118). Aminopropyl-CPG beads (200/400 mesh) were obtained from Electro Nucleonics Inc. (Fairfield, NJ). Sodium dihydrogenphosphate monohydrate was obtained from Merck (Darmstadt, Germany). Phosphate-buffered saline (PBS) (Gibco, Gaithersburg, MD), plastic culture dishes coated with laminin and poly-D-lysine (Nunc), and Dulbecco’s Modified Eagle’s medium (Gibco) were used as purchased. Brain-derived neurotrophic factor (BDNF) was obtained from Iwaki (Tokyo, Japan). Nafion (5 wt %) was obtained from Aldrich (Milwaukee, WI). Other chemicals, such as nerve growth factor (NGF) and glutaraldehyde, were obtained from Wako (Tokyo, Japan). Electrode and Prereactor. A glassy carbon (GC) electrode (6-mm diameter, BAS) and a carbon film-based split disk electrode were used to fabricate ACh sensors.23 The carbon film-based split disk electrodes were fabricated on a glass substrate by using chemical vapor deposition, photolithography, and a dry-etching (16) Ajima, A.; Nakagawa, T.; Kato, T. J. Chromatogr. 1989, 494, 297-302. (17) Huang, T.; Yang, L.; Gitzen, J.; Kissinger, P. T.; Vreeke, M.; Heller, A. J. Chromatogr. B 1995, 670, 323-327. (18) Niwa, O.; Horiuchi, T.; Morita, M.; Huang, T.; Kissinger, P. T. Anal. Chim. Acta 1996, 318, 167-173. (19) Albery, W. J.; Boutelle, M. G.; Galley P. T. J. Chem. Soc., Chem. Commun. 1992, 900-901. (20) Zilkha, E.; Koshy, A.; Obrenovitch, T. P.; Bennetto, H. P.; Symon, L. Anal. Lett. 1994, 27, 453-473. (21) Niwa, O.; Torimitsu, K.; Morita, M.; Osborne, P. G.; Yamamoto, K. Anal. Chem. 1996, 68, 1865-1870. (22) Vreeke, M.; Maidan, R.; Heller, A. Anal. Chem. 1992, 64, 3084-3090. (23) Niwa, O.; Morita, M.; Solomon, B. P.; Kissinger, P. T. Electroanalysis 1996, 8, 427-433.

Figure 1. Structures of the modified electrodes used for the measurement of Ach. The GC electrode was first modified with Osgel-HRP and then co-immobilized with AChE and ChOx. With the split disk electrode, one surface was modified with BSA-ChOx and the other with BSA-AChE-ChOx after both surfaces had been modified with Os-gel-HRP.

technique as we previously reported.24 Figure 1 shows the structures of the ACh sensors. The GC electrode surface was first modified with osmium poly(vinylpyridine) redox polymer containing HRP (Os-gel-HRP) by the casting method.17,22 The solution was applied to the surface at 7.1 µL/ cm2. The enzyme layer was immobilized by covering it with an aqueous solution (4 µL) containing 2% bovine serum albumin (BSA, Sigma), AChE, ChOx, and 0.2% glutalaldehyde on the Osredox polymer-HRP-modified electrode. With the split disk electrode, one surface was modified with BSA-ChOx/Os-gel-HRP bilayer film and the other with BSA-AChE-ChOx/Os-gel-HRP film. When evaluating the selectivity of the sensor, the modified electrode was covered with Nafion solution (2.5% in 2-propanol) by the spin-coating method. A 5-cm-long Teflon tube with an inner diameter of 0.75 mm was divided transversely into two volumes by inserting a plastic filter in its center and used as a prereactor to remove Ch. ChOx and catalase were separately immobilized on beads with glutaraldehyde. The ChOx-immobilized beads were packed in the upstream portion of the tube, and the catalase-immobilized beads were packed in the downstream portion. Sensor System for Standard Solution. Figure 2 shows a block diagram of the sensor system we used for standard ACh or ACh/Ch solutions to study the performance of the sensor. 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 and a buffer solution containing ACh or ACh/Ch, can be injected independently by using syringes 1 and 2 and mixed upstream of the prereactor, as shown in the figure. The ACh or ACh/Ch concentration can be changed by controlling the flow rate from the two syringes. In this system, we expected all the Ch molecules to be oxidized in the upstream portion of the prereactor and that any hydrogen peroxide molecules generated by the enzymatic reaction of Ch would be consumed by the catalase in the downstream portion. (24) Niwa, O.; Tabei, H. Anal. Chem. 1994, 66, 285-289.

Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

1127


Figure 2. Schematic representation of the online sensor for the measurement of standard solutions. The prereactor was a polyethylene tube (0.75-mm i.d. and 22-µL inner volume) packed with ChOx and catalase-immobilized aminopropyl-CPG beads. The modified electrode shown in Figure 1 was installed in a thin-layer radial flow cell. The sensor was oeprated at 0 mV (vs Ag/AgCl).

In contrast, we expected that the ACh molecules would not react in the prereactor and would be detected at the BSA/AChE/ ChOx and Os-gel-HRP bilayer modified GC electrode in the thinlayer radial flow cell. To calibrate the ACh, ACh solutions ranging from 2.5 nM to 2 µM were injected into the sensor at flow rates from 6 to 16 µL/min. The potential of the modified electrode was held at 0 V (vs Ag/AgCl) during the measurement, and the current was measured with an LC4C electrochemical detector (BAS) and recorded with a SR6335 analogue recorder (Graphtec, Tokyo, Japan). The ACh selectivity against Ch was measured by comparing the magnitude of the signal when injecting 1 µM ACh and 1-100 µM Ch through the sensor. Preparation of Brain Slice Culture. We dissected hippocampus from a 2-day-old postnatal rat (Wistar). It was sliced to a thickness of 300 µm and cultured on laminin- and poly-D-lysinecoated plastic culture dishes. The slice were maintained for a week in Dulbecco’s Modified Eagle’s medium containing 5% heatinactivated fetal bovine serum, 5% heat-inactivated horse serum, 20 ng/mL 7S NGF, and 20 ng/mL BDNF at 37 °C in a watersaturated 10% CO2 atmosphere. Before measurement, the cultured slice was rinsed twice with medium containing 148 mM NaCl, 2.8 mM KCl, 2 mM MgCl2, 2 mM CaCl2, and 10 mM glucose in HEPS, pH 7.4. The measurements were undertaken in the same solution. Measurement of the Concentration of Extracellular ACh from Rat Hippocampal Slice Culture. Figure 3 shows a schematic representation of our method for the continuous measurement of the concentration of extracellular ACh released from a hippocampal slice. We used a glass microcapillary to sample the solution near the cell by controlling the probe position with a manipulator (Narishige, Tokyo, Japan). The inner diameter of the glass capillary tip was about 20 µm. A Teflon tube (120µm i.d., CMA) was inserted into the glass capillary, sealed with epoxy resin to minimize the dead volume, and used as a sampling probe. The other side of the tube was connected through ChOx and catalase-immobilized prereactor to the inlet port of a thinlayer radial flow cell. The outlet port of the flow cell was connected to a syringe which pulled by a syringe pump (CMA). The ACh released from the slice was measured by stimulating the slice with a tungsten dual microelectrode located near the microcapillary sampling probe. The slice was stimulated for 5 s with 30-ms, 500-µA pulses between the two microelectrodes, and the extracellular solution was continuously injected into the sensor 1128 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

Figure 3. Schematic representation of our online sensor for the continuous monitoring of ACh in a cultured brain slice. The glass capillary sampling probe approaches the slice, and the tungsten dual microelectrode attached to the slice is used for stimulation. The extracellular solution is pulled at a flow rate of 6 µL/min by the syringe pump.

at 4 or 6 µL/min. This suction mode sampling did not damage the tissue during the measurement. RESULTS AND DISCUSSION Selectivity of the Sensor. The ability to select ACh is very important for continuous ACh measurement since the Ch concentration is much higher than that of ACh in a biological sample. Since several groups reported that Os-gel-HRP-based biosensors can be operated at 0 mV (vs Ag/AgCl),4,17,21,22 electroactive neurotransmitters such as dopamine and norepinephrine or DOPAC, which is a dopamine metabolite, should not influence the sensor response. In contrast, it is known that the oxidation of L-ascorbic acid at the carbon electrode starts at a potential lower than 0 V, and the L-ascorbic acid in vivo concentration is high. However, Gargulio and Michael reported that an overcoating of Nafion film on their Os-gel-HRP-based Ch sensor greatly reduces the effect of L-ascorbate.4 We have also reported that the effect of L-ascorbic acid is reduced by pre-electrolysis with a second electrode located upstream of the sensor.22

In contrast, in this study the selective detection of ACh in the presence of Ch was very difficult to achieve without using a column separation. Figure 4 shows a typical sensor response when 1 µM ACh and 10 and 100 µM Ch were continuously injected into the sensor at a flow rate of 16 µL/min. We observed a large reductive current with a magnitude of more than 20 nA when 1 µM ACh standard solution was continuously injected. In contrast, we observed almost no cathodic current increase when 10 and 100 µM Ch standard solutions were injected. Only a small baseline change was observed when the buffer solution was switched to the 100 µM Ch standard. This indicates that all the Ch molecules flowing into the sensor were consumed in the prereactor by the following enzymatic reactions: ChOx

Ch + 2O2 + H2O 98 betaine + 2H2O2 catalase


2H2O2 98 O2 + 2H2O

(1) (2)

In contrast, Ach was detected as in following reactions at the modified electrode, since it passes through the prereactor without enzymatic reaction: AChE

ACh + H2O 98 Ch + CH3COOH ChOx

Ch + 2O2 + H2O 98 betaine + 2H2O2 HRP

(3) (4)

2Os2+ + H2O2 + 2H+ 98 2Os3+ + 2H2O

(5)

Os3+ + e- f Os2+

(6)

Figure 4. Response of the sensor to a buffer containing 1 µM ACh or 10 or 100 µM Ch. The electrochemical detector was a 6-mm GC electrode modified with an Os-gel-HRP layer and a BSA layer containing AChE and ChOx. The flow rate was 16 µL/min.

The sensitivity of ACh is revealed to be almost 10 000 times higher than that of Ch by comparing the 1 µM ACh signal with the small peak obtained when measuring 100 µM Ch. Huang et al. reported that the basal concentration of ACh in the brain dialysate was detemined to be 31 ( 5 fmol/5 µL.17 From the result, the basal ACh concentration in the brain was calculated to 62 ( 10 nM when we assumed that the recovery of their MD probe was 10%. In contrast, the Ch level is several hundred times higer than the ACh level, indicating that Ch concentration in the brain is several micromolar.25 Therefore, this high selectivity is sufficient even for the in vivo measurement of ACh. The cathodic current did not increase when we injected 10 µM choline over a much longer time. When the Ch concentration was 1 mM, the cathodic current did not increase when Ch was injected for a short time, but the current started to increase when the Ch was injected for a long time. This indicates that the rate of Ch supply exceeds the consumption when the concentration is higher than 100 µM. The prereactor will consume a much higher concentration of Ch if the flow rate is lowered to reduce the Ch supply, and this will increase the time in the enzymatic prereactor. Sensitivity and Detection Limit. High sensitivity is required for measuring neurotransmitters because of their low extracellular concentration. It was reported that the basal level of Ach in a rat brain is several tens nanomolar.17 Liquid chromatography with electrochemical detection (LCEC), combined with an enzymatic

reactor, offers a low detection limit. However, even with this LC measurement, an AChE inhibitor is needed to measure the basal ACh level since the inhibitor increases the extracellular ACh concentration. Recently, basal ACh was determined in a rat brain without using the inhibitor by using an Os-gel-HRP-modified GC electrode combined with an LCEC-AChE/ChOx enzymatic reactor.17,26 These papers reported a detection limit of 10 fmol (5-µL sample). The previously reported sensors exhibit higher detection limits than the present ACh measurements using LCEC. To improve the sensitivity and detection limit of the sensor, we used a thin-layer radial flow cell which shows a high analyte conversion efficiency.21 The GC electrode was modified with bilayer film consisting of Os-gel-HRP and BSA-AChE-ChOx layers. The top layer works as a reactor, since the sample solution containing ACh flows through the thin-layer cell at a relatively slow rate. Almost all generated hydrogen peroxide was reduced by the HRP in the bottom layer. Figure 5 shows the flow rate dependence of the limiting current caused by the enzymatic reaction of ACh and the conversion efficiency of the analyte at the bilayer-modified electrode. The conversion efficiency was calculated by dividing the magnitude of the observed current by the current when all the injected ACh molecules had been converted to betaine by the enzymatic reaction and the generated hydrogen peroxide had also been reduced at the modified electrode. With 100% conversion efficiency, the current is theoretically proportional to the flow rate.

(25) Damsma, G.; Westerink, B. H. C.; de Boer, P.; de Vries, J. B.; Horn, A. S. Life Sci. 1988, 43, 1161-1168.

(26) Kato, T.; Liu, J. K.; Yamamoto, K.; Osborne, P. G.; Niwa, O. J. Chromatogr. B 1996, 682, 162-166.


1129


Figure 6. Response of the ACh online sensor when injecting extracellular fluid with and without the ChOx/catalase prereactor. The extracellular fluid was first sampled and injected by using syringe 2 as shown in Figure 2. Figure 5. Flow rate dependence of the limiting current for 1.0 µM ACh and the conversion efficiency in the flow cell at an BSA-AChEChOx/Os-gel-HRP-modified GC electrode.

However, the cathodic current for ACh at the BSA-AChE-ChOx/ Os-gel-HRP bilayer-modified electrode decreased gradually with decreasing flow rate in the higher flow rate region and decreased rapidly at the lower flow rate. The theoretical current of 1 µM Ach at a flow rate of 20 µL/min is 129 nA. Since the current at the flow rate of 20 µL/min is 60.1 nA, the conversion efficiency is 0.47. This can be increased by decreasing the flow rate, as also shown in Figure 5. More than 90% conversion was achieved at a flow rate lower than 4 µL/min. These results indicate that the reduction in the limiting current caused by decreasing the flow rate is partially compensated for by increasing the conversion efficiency at the modified electrode in the thin-layer radial flow cell. This is because ACh molecules have more time to flow through the enzyme layer when the flow rate is slow. The relatively high sensitivity, even at the lower flow rate, is advantageous for measuring ACh in a small area because the lower sampling rate makes it easier to obtain samples from a small area without severely disturbing the solution around the cell. The limiting currents at flow rates of 16 and 6 µL/min were proportional to the ACh concentration from 10 nM to 10 µM. The sensitivity was 43.7 ((0.15, n ) 3) and 33.4 nA/µM (( 2.7, n ) 3) at flow rates of 16 and 6 µL/min, respectively. A detection limit of 4.8 nM (S/N ) 3, n ) 2) was obtained. In addtion, a signal could be observed with 2.5 nM ACh solution with some of the sensors when the background noise level was low. This lowest detection limit is comparable to that of LCEC using an immobilized enzyme reactor whose detection limit is typically 10 fmol in a 5-µL sample (2 nM),17 although the concentration in the LCEC cell is actually lower due to the dilution caused by the chromatographic process. The low detection limit of the sensor was enhanced by the high conversion efficiency of the analyte at the modified electrode in the thin-layer radial flow cell and the low background current at 0 mV. Extracellular ACh Concentration Measurement with the Sensor. The measurement of neurotransmitters in a cultured nerve cell is different from that in an in vivo system. Although the concentration of interferences in the cultured sample can be reduced by controlling the medium, the extracellular neurotrans1130 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

mitter concentration is not as high as that in an in vivo measurement. This is because the volume of the medium (2 mL) was larger than the size of the tissue (a few millimeters diameter and 300 µm thick), and the chemicals released from the tissue were diluted by diffusion into the medium. The extracellular ACh concentration is usually measured using the microdialysis (MD) sampling technique and LCEC. We have applied the MD sampling technique to measure the extracellular glutamate in a cultured rat cortex.21 However, we cannot apply this technique directly to the measurement of ACh in a cultured cell since the ACh concentration is lower than that of glutamate and the recovery of the MD probe (typically 10%) dilutes the ACh concentration below the detection limit of the sensor. Instead of the MD sampling technique, we employed direct sampling with a glass microcapillary, as shown in Figure 3. Although the analyte is also diluted by using direct sampling, we have already confirmed that the dilution of the analyte is suppressed compared with MD sampling.27 Before undertaking the capillary suction sampling measurement, the extracellular solution was sampled and injected into the sensor by using syringe 2 shown in Figure 2. The responses of the sensor with and without using the ChOx/catalase precolumn are shown in Figure 6. The cathodic current increased and reached a steady state as a result of injecting extracellular fluid without the prereactor. In contrast, almost no cathodic current was observed with the prereactor, indicating that most of the increase was caused by the extracellular Ch and almost all Ch molecules were consumed in the prereactor. Figure 7 shows the variation in extracellular ACh concentration in a rat hippocampal slice caused by electrical stimulation. The extracellular solution was introduced as shown in Figure 3. The background current before stimulation was higher than that measured in the buffer without the slice sample, suggesting that there was a small amount of ACh in the extracellular fluid before stimulation. The ACh and Ch levels before stimulation could also be confirmed by conventional LCEC with an enzymatic reactor. The cathodic current could be increased reproducibly by electrically stimulating the slice, again with a tungsten dual (27) Niwa, O.; Horiuchi, T.; Torimitsu, K. Biosens. Bioelectron. 1997, 12, 311319.


Figure 7. Variation in extracellular ACh concentration released from a rat hippocampal slice culture (300 µm) by electrical stimulation. The slice was stimulated for 5 s at 30 ms, 500 µA between two tungsten electrodes attached to the slice. The flow rate was 6 µL/min.

microelectrode when the sensor was operated in the suction mode at a flow rate of 6 µL/min. The current increases could be the direct result of the extracellular ACh increase from the rat hippocampal slice. The ACh concentration after stimulation increased 20 nM ((11 nM, n ) 3). The fluctuation of the response after stimulation was not due to the reproducibility of the sensor since the SD of the sensor with the standard sample was much lower. Therefore, this may be due to the conditions of sampling or electrical stimulation. To confirm that the peak after stimulation indicates ACh release, the effect of interferences such as other neurotransmitters, L-ascorbic acid, and oxygen should be considered when measuring brain samples. The injection of 1-10 µM of L-aspartate, glutamate, GABA, and glutamine, which are electroinactive amino compounds, caused almost no signal. When electroactive neurotrasmitters such as dopamine and serotonin were injected, a baseline change was sometimes observed in the cathodic direction. However, the baseline did not increase upon increasing the concentration of these compounds, and the oxidation potentials of catecholamines and serotonin were higher than 0 V, which is the operating potential of the sensor. Therefore, the smaller baseline changes may be due to the difference of the ionic strength of the buffer and dopamine and serotonin standard solution. Since ChOx was used, the sensor response should be influenced by the oxygen concentration. We measured 1 µM standard ACh solution with or without oxygenating the solution. There was a difference of 5.5% for both solutions, and this increased with increasing concentration. In Figure 7, the effect of oxygen could be low since the ACh peak is at the 10 nM level. The effect of L-ascorbic acid is more important, since the oxidation of the acid starts near 0 V. However, the concentration of the acid in our case should be much lower than that in a brain since the sensor showed a cathodic background current of a few nanoamperes during the measurement. With our sensor, the anodic signal with a magnitude of 15 nA was observed when we injected a buffer containing 10 µM L-ascorbic acid. This low extracellular acid concentration occurred because we changed the medium before the measurement. However, O’Neill et al. reported that the extracellular L-ascorbic acid level increases upon increasing the extracellular concentration of excitatory neurotrans-

Figure 8. Response of 0.5 µM ACh and Ch at the modified split disk electrode at the flow rate of 16 µL/min. Both electrodes were held at 0 mV. The electrode was sandwiched between a plastic block and a counter block, and the inlet port was adjusted to the center of the two electrodes. (a) BSA-AChE-ChOx/Os-gel-HRP-modified electrode in the split disk. (b) BSA-ChOx/Os-gel-HRP-modified electrode in the split disk.

mitters such as L-glutamate.28 Since electrical stimulation to the hippocampal slice of our experiment may increase not only extracellular ACh but also glutamate concentrations, we consider the effect on the sensor response if L-ascorbic acid concentration increases after the electrical stimulation. The ACh peaks shown in the Figure 7 are reduced by the effect of L-ascorbic acid concentration increase, which reverses the signal direction from ACh. We also measured standard L-ascorbic acid solution with the ACh sensor after overcoating it with Nafion since many applications require the sensor to be used in the presence of large amounts of L-ascorbic acid. The sensitivity of the acid at the Nafion-overcoated ACh sensor was only 0.1-0.2 nA/µM when we continuously injected 10 µM L-ascorbic acid. This indicates that the sensor can be used for the measurement of biological samples containing L-ascorbic acid. In the ACh measurement in the slice, the sensor was operated at a relatively high flow rate, since a lower flow rate would reduce the electrode response. A lower flow rate is, however, more useful (28) O’Neill, R. D.; Fillenz, M.; Sundstrom, L.; Rawlins, J. N. P. Neurosci. Lett. 1984, 52, 227-233.


1131


since it allows the concentration of ACh in a small area to be evaluated. A lower flow rate with a low detection limit will be achieved by reducing the thickness of the flow cell and the electrode size. However, the time delay caused by the dead volume between a sampling capillary and the electrode in the flow cell was increased by reducing the flow rate. The time delay is about 6 min, even at a flow rate of 6 µL/min. The dead volume existed in the prereactor (22 µL) and in connections between the reactor and tube. However, the sensor has advantages over conventional ACh measurement with LC, since LCEC usually requires a much longer analysis time and is not a continuous measurement. For example, the analysis time for the experiment shown in Figure 7 was less than 31 min. We also performed a similar experiment by stimulating the slice and determining ACh in the extracellular fluid with commercially available LCEC by sampling the solution every 3 min (total six samples sampled every 3 min). When LCEC is used, the total analysis requires more than 2 h (about 20 min for one analysis with LC), excluding the time for sampling from the cultured slice. In addition, the sensor could be employed for the continuous in vivo measurement of ACh since the sensor can continuously consume a much higher concentration of Ch (100 µM) than that in the brain (a few tens micromolar). Determination of ACh and Ch at the Modified Split Disk Electrode. It is very important to improve the response time of the sensor for neurotransmitter measurement in a cultured sample. The dual electrode, in which one electrode is modified with ChOx and the other with AChE and ChOx, can be used to monitor the ACh continuously by subtracting the response at the AChE/ChOx-modified electrode from that at the ChOx-modified electrode. It may be very difficult to use this idea to obtain good accuracy for in vivo applications since the difference between the ACh and Ch concentrations in the brain is very large (ACh, several tens nanomolar; Ch, a few tens micromolar). With a cultured sample, the extracellular concentration of Ch is much lower than that in the brain. Therefore, it will be possible to measure both ACh and Ch with the modified dual electrode. This is very

1132 Analytical Chemistry, Vol. 70, No. 6, March 15, 1998

important in terms of improving the response time, since the ChOx and catalase-immobilized prereactor, which has a relatively large volume, can be eliminated. Figure 8 shows the response of 0.5 µM ACh and Ch at a split disk electrode in which one semicircular electrode was modified with a BSA-ChOx/Os-gel-HRP bilayer-modified electrode and the other with a BSA-AChE-ChOx/Os-gel-HRP bilayer-modified electrode. When the Ch was injected into the sensor, both modified electrodes showed very similar responses. In contrast, only the electrode modified with BSA-AChE-ChOx/Os-gel-HRP showed a response when 0.5 µM ACh was injected. When a mixed solution of ACh and Ch was injected, it was possible to measure the ACh concentration by subtracting the response at the AChE/ChOxmodified electrode from that at the ChOx-modified electrode. This method may not be sufficient for an in vivo sample, since the concentration difference between ACh and Ch is very large. However, this could be used for measuring the extracellular ACh concentration in a cultured sample, since the Ch can be lowered by adjusting the culture medium. The sensor response is less than 1 min (about 30 s), which is much lower than that with a prereactor. The structure of the split disk electrode has certain advantages compared with a commercially available dual disk electrode with regard to improving sensitivity since its conversion efficiency is higher than that at a dual disk electrode. ACKNOWLEDGMENT The authors thank Drs. N. Matsumoto, M. Morita, and A. Kawana for encouraging this project and Drs. H. Kamioka and P. J. Charlety for preparing the culture. The authors also thank Dr. T. Kato (Yokohama City University), Dr. P. G. Osborne, and Mr. K. Yamamoto (BAS) for fruitful discussions, Miss J. Araki (BAS) for immobilizing the enzyme on the beads, and Miss H. Ishibashi for preparing the figures. Received for review March 6, 1997. Accepted January 5, 1998. AC970257O

On-Line Electrochemical Sensor for Selective Continuous

Recommend Documents