Microelectrode Array Biochip: Tool for In Vitro Drug Screening Based

Dong Qin , Younan Xia , George M Whitesides. Nature Protocols 2010 5, ... Kelly L. Adams , Maja Puchades , Andrew G. Ewing. Annual Review of Analytica...
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Anal. Chem. 2006, 78, 6347-6355

Microelectrode Array Biochip: Tool for In Vitro Drug Screening Based on the Detection of a Drug Effect on Dopamine Release from PC12 Cells Hui-Fang Cui,† Jian-Shan Ye,† Yu Chen,‡ Ser-Choong Chong,‡ and Fwu-Shan Sheu*,†,§

Department of Biological Sciences, National University of Singapore, 14 Science Drive 4, Singapore 117543, Institute of Microelectronics, 11 Science Park Road, Science Park II, Singapore 117685, and The University Scholars Programme, National University of Singapore, 10 Kent Ridge Crescent, Singapore 119260

Novel, yet simple detection techniques of drug effect, including the effect of a vesicular monoamine transporter inhibitor (reserpine), a dopamine precursor (L-dopa), and a dopamine transporter inhibitor (nomifensine), on dopamine release from dopaminergic PC12 cells were developed based on a microelectrode array (MEA) biochip. Upon multi-injections of KCl solution into the culture of PC12 cells attached on a MEA biochip, the K+-stimulated dopamine release was temporally and amperometrically recorded by biochip microelectrodes. Two parameters in the recorded amperometric spectra were defined in this study: the peak current of the first KCl injection (Max1), and the steady current after the fourth KCl injection (St4). Statistically significant effects of L-dopa and reserpine were demonstrated by comparing both Max1 and St4 of the second detections in drugs with those of the control without drug treatment. The values of both Max1 and St4 in the first detections were normalized as 1. In contrast, the statistically significant effect of nomifensine was detected by comparing the ratios of St4 to Max1 in the first detections in drug with those of the control. The reason for using different analytical methods for measurements between L-dopa/reserpine and nomifensine lies in the different mechanisms of action on PC12 cells among these drugs. The novel analytical methods developed use the same detection setup and parameters, and the data analysis for the effect of drugs becomes simple. The methods hence may provide a high-throughput in vitro drug screening approach for dopamine-related psychiatric disorders. Dopamine (DA) is the prevalent catecholamine (a collective term for the hormones epinephrine, norepinephrine, and DA) neurotransmitter in the brain. DA neurotransmission in the brain constitutes at least three important dopaminergic pathways.1,2 * Corresponding author. E-mail: [email protected]. Tel: (0065) 6874-2857. Fax: (0065) 6779-2486. † Department of Biological Sciences, National University of Singapore. ‡ Institute of Microelectronics. § The University Scholars Programme, National University of Singapore. (1) Gille G.; Riederer P. In Dopamine and glutamate in psychiatric disorders; Schmidt, W. J., Reith, M. E. A., Eds.; Human Press Inc.: Totowa, NJ, 2005; pp 415-445. 10.1021/ac060018d CCC: $33.50 Published on Web 08/18/2006

© 2006 American Chemical Society

The dysfunction of these pathways, i.e., abnormal neurotransmission of DA, can lead to neurological, psychological, and endocrinological diseases like, e.g., Parkinson’s disease, schizophrenia, major depressive disorder, attention deficit hyperactive disorder, and Huntington’s disease. Consequently, in vitro screening of compounds for the effect on extracellular DA level may find medication candidates for DA-related psychiatric disorders and reduce the costs and lengths of animal trials. For the purpose of low costs and short periods, a rapid, sensitive, high-throughput, easy-to-operate method of DA detection is the prerequisite for in vitro drug screening. In addition, as the extracellular DA level is time dependent due to a dynamic process of DA exocytosis, reuptake, diffusion, and autoxidation, an in situ real-time recording of extracellular DA level is needed. DA levels can be detected by ultraviolet (UV) light spectrometry as well as by an electrochemical method. Compared to the UV method, the electrochemical method offers the advantages of higher sensitivity and selectivity,3 higher spatial resolution, especially at micro- and nanoelectrodes, and the ability to operate in turbid and colored solutions. By using amperometry or fastscan voltammetry, carbon fiber microelectrodes have been used in real-time detection of DA exocytosis from PC12 cells in vitro.4-10 However, this technique is unsuitable for high-throughput drug screening. First, to record DA release from a single cell, the carbon fiber microelectrode must be pushed against the cell. This procedure is invasive to cells and needs very careful and skillful operation. In addition, the one by one detection cannot meet the requirement of high throughput in drug screening. Finally, the data treatment and analysis in this technique is troublesome. Over the past 30 years, the advent of integrated circuit fabrication technologies has resulted in the production of multi(2) Picetti R.; Saiardi A.; Abdel S. T.; Bozzi Y.; Baik J. H.; Borreli E. Crit. Rev. Neurobiol. 1997, 11, 121-142. (3) Oni, J.; Westbroek, P.; Nyokong, T. Electroanalysis 2002, 15, 847-854. (4) Kozminski, K. D.; Gutman, D. A.; Davila, V.; Sulzer, D.; Ewing, A. G. Anal. Chem. 1998, 70, 3123-3130. (5) Pothos, E.; Desmond, M.; Sulzer, D. J. Neurochem. 1996, 66, 629-636. (6) Chen, T. K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (7) Zerby, E. S.; Ewing, G. A. J. Neurochem. 1996, 66, 651-657. (8) Pothos, E. N.; Przedborski, S.; Davila, V.; Schmitz, Y.; Sulzer, D. J. Neurosci. 1998, 18, 5575-5585. (9) Colliver, T. L.; Pyott, S. J.; Achalabun, M.; Ewing, A. G. J. Neurosci. 2000, 20, 5276-5282. (10) Sombers, L. A.; Hanchar, H. J.; Colliver, T. L.; Wittenberg, N.; Cans, A.; Arbault, S.; Amatore, C.; Ewing, A. G. J. Neurosci. 2004, 24 (2), 303-309.

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microelectrode devices on planar substrates. With the integration of a fixed microelectrode array (MEA) with silicon or glass substrate and a cell culture chamber, many laboratories have established the capability to perform noninvasive, multisite, extracellular potential recordings from electrogenic cultures, including PC12 cells, embryonic stem cells,11 cardiomyocytes, neurons,12-21 and brain slices.22-25 The recording of extracellular action potentials spontaneously generated from electrogenic cell cultures of a variety of cell types, most often primary neurons and cardiac myocytes, by using MEA biochips has been used for biosensing.26-28 However, the biosensing system based on extracellular potential recordings depends on the effect of compounds on special receptors and ion channels. As drugs affecting dopaminergic pathways take effect by several mechanisms, including inhibition of vesicular monoamine transporter (VMAT), dopamine transporter (DAT), monoamine oxidase, or catechol-O-methyltransferase, supplying DA precursor, and activation of DA receptors, the approach of recording extracellular potentials is obviously unsuitable for drug screening for DA-related psychiatric disorders. Nevertheless, the capability of multisite recording with MEA and the ability of direct cell culture on MEA biochips may be used to develop a real-time, high-throughput, and easy-to-operate detection assay of DA exocytosis for in vitro drug screening. We have reported the fabrication of a silicon wafer and gold microelectrode-based MEA biochip.29 This MEA biochip has been proven convenient for in situ temporal detection of DA exocytosis from L-dopa-incubated MN9D cells, a mouse mesencephalic dopaminergic cell line.29 However, since the level of DA exocytosis from MN9D cells without L-dopa incubation was undetectable by the MEA biochip,29 the study of drug effects on MN9D cells was limited in a previous study. It has been known that catecholamine (11) Bieberich, E.; Guiseppi-Elie, A. Biosens. Bioelectron. 2004, 19, 923-931. (12) Stenger, A. D.; Gross, W. G.; Keefer, W. E.; Shaffer, M. K.; Andreadis, D. J.; Ma, W.; Pancrazio, J. J. Trends Biotechnol. 2001, 19, 304-309. (13) Gross, G. W.; Williams, A. N.; Lucas, J. H. J. Neurosci. Methods 1982, 5, 13-22. (14) Thie´baud, P.; Lauer, L.; Knoll, W.; Offenha¨user, A. Biosens. Bioelectron. 2002, 17, 87-93. (15) Gross, G. W.; Wen, W. Y.; Lin, J. W. J. Neurosci. Methods 1985, 15, 243252. (16) Thomas, C. A.; Springer, P. A.; Leob, G. E.; Berwald-Netter, Y.; Okun, L. M. Exp. Cell Res. 1972, 74, 61-66. (17) Maher, P. M.; Pine, J.; Wright, J.; Tai, Y. C. J. Neurosci. Methods 1999, 87, 45-56. (18) Selinger, V. J.; Pancrazio, J. J.; Gross, W. G. Biosens. Bioelectron. 2004, 19, 675-683. (19) James, C. D.; Spence, A. J. H.; Dowell-Mesfin, N. M.; Hussain, R. J.; Smith, K. L.; Craighead, H. G.; Isaacson, M. S.; Shain, W.; Turner, J. N. IEEE Trans. Biomed. Eng. 2004, 51, 1640-1648. (20) Martinoia, S.; Bonzano, L.; Chiappalone, M.; Tedesco, M. Sens. Actuators, B 2005, 108, 589-596. (21) Martinoia, S.; Bonzano, L.; Chiappalone, A.; Tedesco, A.; Marcoli, A.; Maura, G. Biosens. Bioelectron. 2005, 20, 2071-2078. (22) Thie´baud, P.; Beuret, C.; Koudelka-Hep, M.; Bove, M.; Zimmer, J.; Dupont, Y. Biosens. Bioelectron. 1999, 14, 61-65. (23) van Bergen, A.; Papanikolaou, T.; Schuker, A.; Moller, A.; Schlosshauer, B. Brain Res. Protoc. 2003, 11, 123-133. (24) Heuschkel, M. O.; Fejtl, M.; Raggenbass, M.; Bertrand, D.; Renaud, P. J. Neurosci. Methods 2002, 114, 135-148. (25) Kristensen, B. W.; Noraberg, J.; Thiebaud, P.; Koudelka-Hep, M.; Zimmer, J. Brain Res. 2001, 896, 1-17. (26) Gross, G. W.; Rhoades, B.; Russell, J. Sens. Actuators, B 1992, 6, 1-8. (27) Kovacs, G. T. A. Proc. IEEE 2003, 91, 915-929. (28) Pancrazio, J. J.; Whelan, J. P.; Borkholder, D. A.; Ma, W.; Stenger, D. A. Ann. Biomed. Eng. 1999, 27, 697-711. (29) Cui, H. F.; Ye, J. S.; Chen, Y.; Chong, S. C.; Liu, X.; Lim, T. M.; Sheu, F. S. Sens. Actuators, B 2006, 115, 634-641.

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vesicles in PC12 cells, a dopaminergic cell line established from a transplantable rat adrenal pheochromocytoma, contain much more catecholamine molecules than those in neurons.5,6 Hence in this study, the K+-stimulated DA exocytosis from PC12 cells was able to be detected with the MEA biochip. In addition, novel, yet simple detection techniques of drug effect on the DA exocytosis from PC12 cells were developed based on the MEA biochip. The drugs studied included a VMAT inhibitor (reserpine), a DA precursor (L-dopa), and a DAT inhibitor (nomifensine). EXPERIMENTAL SECTION Materials and Chemicals. 3,4-Dihydroxyphenylacetic acid (DOPAC) was obtained from Fluka Chemia (Buchs, Switzerland). 3,4-Dihydroxyphenethylamine (dopamine), L-3,4-dihydroxyphenylalanine (L-dopa), 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid (HEPES), serotonin, epinephrine, norepinephrine (NE), homovanillic acid (HVA), type 1 collagen, Ham’s F12 medium, reserpine, and nomifensine were from Sigma (St. Louis, MO). PC12 cells were purchased from American type culture collection (ATCC). Fetal bovine serum (FBS), penicillin-streptomycin (p/s; 10 000 unit mL-1 penicillin sodium and 10 000 µg mL-1 streptomycin sulfate in 0.83% saline), and trypsin (0.05% trypsin, 0.53 mM EDTA‚4Na) were obtained from Gibco (Grand Island, NY). All other chemicals were of analytical grade or higher and were used without further purification. Deionized water obtained from a Millipore water system was used throughout the experiment. Solution Preparation. A 1 mM reserpine stock solution and a 40 mM nomifensine stock solution were prepared by dissolving reserpine and nomifensine, respectively, into DMSO and stored at -20 °C. A 2 mg mL-1 collagen stock solution was prepared by dissolving collagen into 0.2% acetic acid and filtering the solution through a syringe filter of 5 µm in pore size and finally a syringe filter of 0.45 µm in pore size. The collagen stock solution was stored at 4 °C until needed. The preparation of a balanced salt solution (BSS), a salt solution containing either 40 or 120 mM KCl, a phosphate buffer solution (PBS), and a catecholamine stabilizing mixture were reported in our previous paper,29 and are detailed in the Supporting Information. High-Performance Liquid Chromatography (HPLC) Analysis of the K+-Stimulated Exocytosis from PC12 Cells. The procedures of sample collection and HPLC detection of K+stimulated exocytosis from dopaminergic cells were reported previously29 and are described in the Supporting Information. External standard samples containing DA, L-dopa, NE, serotonin, epinephrine, HVA, and DOPAC were detected under the same condition as the K+-stimulated exocytosis sample. The resulting chromatograms showed that the K+-stimulated exocytosis from PC12 cells contained only DA (data not shown). This result is consistent with that reported by Pothos et al.5 Fabrication of MEA Biochip. MEA biochips were fabricated from two 6-in. silicon wafers ∼670 µm thick by using standard semiconductor processes. The steps involved in the fabrication were reported previously29 and are described in the Supporting Information. Briefly, a passivated silicon base plate was patterned with a Ti/Au double layer using standard photoresist and wet etching, forming an array of planar Au disk microelectrodes on the 1-mm center of the silicon base plate. Subsequently, a passivated silicon cover plate was patterned and then etched

Figure 1. (A) Microscopic picture of the array of a MEA biochip (microelectrode diameter, 30 µm). (B) Diagram shown as the cross section of a MEA biochip, excluding the parts of connector pads, wire bonding, and printing circuit board.

through the plate areas at the chip chamber and connector pads. Followed by aligning and binding the silicon cover plate with the base plate, the bonded wafer was diced and wire bound with a printing circuit board to form a biochip. Parts A and B in Figure 1 show the microscopic picture of an integrated MEA biochip (microelectrode diameter, 30 µm) and the diagrammatic cross section of the MEA biochip (excluding the parts of connector pads, wire bonding, and the printing circuit board), respectively. Cell Culture on MEA Biochip Chamber. PC12 cells were grown in Ham’s F12 medium supplemented with 25 mM HEPES, 10% FBS, and 1% p/s. All cells were propagated in a humidified incubator at 37 °C with 5% CO2. MEA biochips were sterilized with 70% (v/v) ethanol for 10 min and then with a 45-min exposure to UV light in a clean hood. After sterilization, the chamber surface was coated with 200 µL of collagen solution (a 10× dilution of collagen stock solution with 30% ethanol) for 12-16 h and then washed with Ham’s F12 medium. An 800-µL sample of trypsinized PC12 cell suspension (∼105 cells/mL) was subsequently plated on the collagen-coated MEA biochip chamber. The MEA biochips plated with PC12 cells were placed in Petri dishes (100 mm × 20 mm style, Corning Inc., Corning, NY) and incubated in a humidified incubator at 37 °C with 5% CO2 for 24-48 h. The morphology of cells on the biochip after incubation was observed under a microscope (Axiotech 100, Carl Zeiss). Electrochemical Experiments on MEA Biochip. Electrochemical experiments were performed by using a CHI 1030 electrochemical workstation (CH instruments Inc.) in a threeelectrode arrangement. Any three MEA biochip microelectrodes served as the three electrodes (i.e., working, counter, and reference electrodes) in the three-electrode arrangement. The electrochemical workstation possesses eight working electrode channels. Therefore, eight microelectrodes can be used as working electrodes simultaneously, and all the working electrodes on one MEA biochip share the same counter and reference electrodes. Multiplexing will be required if all the microelectrodes in the array are to be connected. In this research, only one to

two working electrodes were used at one time, as the focus of this research was to develop a detection technique for a drug effect study. Once the technique is developed, it would be easily to extend it to multiple working electrodes. All the electrochemical experiments were performed at room temperature (∼25 °C). Electrochemical Characterization of MEA Biochip. A MEA biochip before and after collagen coating was electrochemically characterized by using K3[Fe(CN)6] and DA as probes. Cyclic voltammetry (CV) was performed for K3[Fe(CN)6], while amperometry and CV were performed for DA. Both the amperometric detection limit (signal-to-noise ratio, S/N ) 3) and working curve of DA at the biochip microelectrodes were determined using DA standards at an applied potential of +0.3 V versus biochip microelectrode. Amperometric Detection of the K+-Stimulated DA Release by Using MEA Biochip. Amperometry was used to detect the K+-stimulated DA release from PC12 cells grown on a MEA biochip. The detailed detection procedures for MN9D cells were reported previously.29 The detection of K+-stimulated DA release from PC12 cells on a MEA biochip is similar to that from MN9D cells. Briefly, PC12 cells cultured on a biochip in BSS solution with or without L-dopa were washed with BSS solution 4 times and then kept in a 240-µL BSS solution. With the oxidation current being monitored by amperometry at an applied potential of +0.3 V versus the biochip microelectrode, the PC12 cells were multistimulated by multiple (4-9 times) injections 120 mM KCl solution through the fine tip of a glass pipet into the PC12 cell culture at a flow rate of 300 µL min-1 and a volume of 20 µL/injection. Detection of Drug Effect on the K+-Stimulated DA Release by Using MEA Biochip. To study the effect of drugs, including L-dopa and reserpine, on the K+-stimulated DA release, the second and even the third detections of the K+-stimulated DA release of the same preparation were performed. The first detection of the K+-stimulated DA release was in blank BSS, and the results obtained from the first detection were taken as the baseline. Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

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For L-dopa, the second detection was performed in BSS after incubating the cell culture in L-dopa and washing the cell culture with BSS. The incubation condition is briefly described as follows. After the first detection of the K+-stimulated DA release in BSS, PC12 cells on MEA biochips were washed with BSS and incubated in 100 µM L-dopa solution (supported by BSS) in a humidified incubator at 37 °C with 5% CO2 for 45 min. The negative control of the L-dopa effect was the second detection of the K+-stimulated DA release from the cell culture without L-dopa incubation. For reserpine, the second detection was conducted in 1 µM reserpine solution after incubating the cell culture in the reserpine solution in air at room temperature. The 1 µM reserpine solution was prepared by diluting reserpine stock with BSS. The incubation time was the time period from reserpine introduction to the first injection of 120 mM KCl solution. In some cases, a third detection in BSS was performed after the second detection in reserpine. Negative control for the effect of reserpine was performed in either blank BSS or DMSO:BSS (1:1000) solution. To study the effect of nomifensine on the K+-stimulated DA exocytosis, only one detection was performed for each cell culture preparation, and a 60 µM nomifensine solution was applied to a naive cell culture in the first detection. The 60 µM nomifensine solution was prepared by diluting nomifensine stock with BSS. The injection of 120 mM KCl solution was started at the amperometric running time of ∼5 min. Negative control for the effect of nomifensine was the K+-stimulated DA exocytosis in DMSO/BSS (1.5:1000) solution. RESULTS AND DISCUSSION Electrochemical Characterization of MEA Biochip. The CVs at a MEA biochip microelectrode (30 µm in diameter) before and after collagen coating are shown in Figure 2. The CVs in 5 mM K3[Fe(CN)6] solution at the microelectrode with and without collagen coating exhibited a typical sigmoidal shape (Figure 2A). Upon collagen coating, the formal redox potential of K3[Fe(CN)6] was unchanged (at -0.178 V), but the redox peak current decreased from 0.835 to 0.116 µA. Similarly, the oxidation peak current of a 50 µM DA solution was reduced from 15.17 to 8.09 nA upon collagen coating (Figure 2B). In addition, collagen coating shifted the oxidation peak potential of the 50 µM DA solution positively from +0.251 to +0.313 V. Obviously, the collagen coating membrane inhibits redox molecules from accessing the gold microelectrodes and consequently inhibits their electrochemical reactions. Although the electrooxidation of DA is inhibited by the collagen membrane, the CVs at microelectrodes still can sense the concentration change of DA. Figure 2C shows the CVs in different concentrations of DA at the collagen-coated MEA biochip microelectrode. With the increase of DA concentration from 10 to 50 µM, the oxidation peak current of DA increased from 3.64 to 8.09 nA, while the oxidation peak potential increased from +0.256 to +0.313 V. Based on the values of the oxidation peak potential, a potential of +0.3 V versus biochip microelectrode was applied for all the amperometric detections at collagen coated MEA microelectrodes. A calibration curve of DA concentration versus DA oxidation current (Figure S-1 in the Supporting Information), derived from an amperometric curve (at +0.3 V vs biochip microelectrode) in response to the injections of concentrated DA standards at a collagen-coated microelectrode (30 µm in diameter) showed the 6350 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

Figure 2. (A) Cyclic voltammograms in 5 mM K3[Fe(CN)6] solution supported by 1 M KCl at a MEA biochip microelectrode of 30-µm diameter before (dashed line) and after (solid line) collagen coating. (B) Cyclic voltammograms in 50 µM DA solution supported by PBS at a MEA biochip microelectrode of 30-µm diameter before (dashed line) and after (solid line) collagen coating. (C) Cyclic voltammograms in PBS (dotted line), 10 (solid line) and 50 µM (dashed line) DA solution supported by PBS at a collagen-coated MEA biochip microelectrode of 30-µm diameter. Potential scan rate (A-C), 0.1 V s-1.

following: the linear regression coefficient of this calibration curve was 0.9924; the amperometric detection limit (low limit) of DA

Figure 3. Microscopic picture of PC12 cells grown on a microelectrode array biochip (microelectrode diameter, 30 µm).

was 0.14 µM; and the detection sensitivity was 0.082 nA µM-1. The calibration condition performed did not reach the DA detection high limit, and the linearity was up to over 7.3 µM. These values of detection limits and sensitivity could unambiguously meet the detection requirements of the K+-stimulated DA exocytosis from PC12 cells. As illustrated in the following section, the typical extracellular DA concentration of PC12 cells was ∼1 µM, well within the linear range of DA amperometric response. PC12 Cell Culture on MEA Biochip Chamber. PC12 cells, established from rat adrenal pheochromocytoma, adhere poorly to plastic and tend to grow in small clusters. Upon coating MEA biochips with collagen, PC12 cells were tightly attached to the chip surface within 4 h after cell plating. Figure 3 shows the microscopic picture of PC12 cells grown on a MEA biochip (12 h after cell plating). Without exposure to nerve growth factor, 8090% of the chip chamber surface was covered by undifferentiated PC12 cells. The attached PC12 cells can withstand the processes of DA exocytosis detection. No cell detachment was observed after the detection. Detection of the K+-Stimulated DA Release from PC12 Cells. PC12 cells can synthesize catecholamine DA and NE and store, release, and take up these neurotransmitters in a manner similar to sympathetic neurons.30 It is known that an elevated extracellular K+ level causes the depolarization of dopaminergic cells and so triggers exocytosis by opening voltage-sensitive Na+ channels, which causes the subsequent opening of voltagesensitive Ca2+ channels.7,31 The opening of Ca2+ channels allows a rapid increase in the intracellular Ca2+ concentration to a level sufficient to trigger the mobilization of catecholamine-containing vesicles to the plasma membrane for exocytosis.7 Figure 4 shows the amperometric response of PC12 cells to multiple injections of 120 mM KCl solution. Amperometric curves a-c in Figure 4 represent the first, the second, and the third detection of the same preparation, respectively. Upon KCl stimulation, PC12 cell membrane is depolarized, causing the fusion of catecholamine vesicles with cell membrane and the release of catecholamine to the extracellular space. The extracellular DA is electrooxidized at the (30) Sombers, L. A.; Ewing, A. G. In Electroanalytical methods for biological materials; Brajter-Toth, A., Chambers, J. Q., Eds.; Marcel Dekker: New York, 2002; pp 279-327. (31) Stallcup, W. B. J. Physiol. (London) 1979, 286, 525-540.

Figure 4. Amperometric responses of PC12 cells in responding to multiple injections of 120 mM KCl solution at a biochip microelectrode of 30-µm diameter (+0.3 V vs biochip microelectrode). Amperometric curves a-c represent the first, second, and third detection, respectively. Arrows position the injection time point. Injection flow rate, 300 µL min-1; volume per injection, 20 µL.

MEA biochip microelectrode (+0.3 V vs biochip microelectrode); thus, a rapid rise of DA oxidation current is temporally recorded. In the spectrum of the first detection (spectrum a), immediately after each KCl injection, a strong peak of DA release was detected by the biochip microelectrode. The ascending phase of the peaks may be mainly from the DA exocytosis, and the descending phase may be mainly caused by DA re-uptake by DAT. DAT, located on the PC12 cell membrane, is responsible for terminating DA transmission by rapid re-uptake of DA into presynaptic terminals.32 When the extracellular DA concentration is high, DAT re-uptakes the extracellular DA into cytosol, leading to a gradually decrease of recorded DA oxidation current. In addition to the DA re-uptake, a diffusion of released DA should also contribute to the descending phase, but not to a large extent. This conclusion is deduced from the spectra b and c, where the amplitudes of the peak descending were very small. Compared to the first series of detection, the amplitudes of both the ascending and descending portions of the response peaks (with the exception of the ascending leg of the first peak in the second detection) were much smaller in the second and third detection. The decreased ascending amplitudes reflect the gradual depletion of the DA store, and the reduced descending amplitudes suggest the gradual desensitization of DAT under the high extracellular DA concentrations. To make the in situ detection of the drug effect on DA exocytosis high-throughput, fast and simple, two parameters, the peak current of the first KCl injection (Max1) and the steady current after the fourth KCl injection (St4), were defined and measured. The values of Max1 and St4 in the first detection were normalized as 1, when the second and even the third detection were performed. The values of Max1 and St4 in the second and third detection were compared to those obtained in the first detection and normalized for comparison. This data treatment can eliminate the tedious experimental procedure of DA concentration calibration and obviate the influence of different preparations of (32) Chen, N.; Reith, M. E. A. Eur. J. Pharmacol. 2000, 405 (1-3), 329-339.

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Figure 5. (A) Amperometric responses of PC12 cells in responding to multiple injections of 120 mM KCl solution. All the first detections were in blank BSS, and the second detection was in blank BSS (a), and 1 µM reserpine after incubation for 20 (b) or 30 min (c) in this reserpine solution, at biochip microelectrodes (+0.3 V vs biochip microelectrode). Arrows position the injection time point. Injection flow rate, 300 µL min-1; volume per injection, 20 µL. (B) Normalized extracellular DA level of Max1 (black bar) and St4 (gray bar) in the second detections, with or without application of reserpine. Error bars represent mean ( SEM of mean normalized levels of DA release (n ) 5-7). Values marked with * are statistically difference with p < 0.01; ** indicates a significant difference with p < 0.001 vs BSS control (Student’s t-test). (C) Normalized extracellular DA level of Max1 (black bar) and St4 (gray bar) in the second and third detection, with (panel b) or without (panel a) application of drug reserpine. Error bars represent mean ( SEM of mean normalized levels of DA release (n ) 5-7). Values marked with * are statistically difference with p < 0.01; ** indicates a significant difference with p < 0.001 vs the third detection in blank BSS after the second detection in reserpine (Student’s t-test).

cell culture, of different biochips, and of different biochip microelectrodes on the results of detection. Figure 5B shows that the normalized DA levels of Max1 and St4 in the second detection were 0.916 ( 0.097 and 1.062 ( 0.106 (mean ( SEM), respectively. Both of these values are ∼1, suggesting a counteraction of reduced exocytosis and reduced re-uptake in the second detection. The normalized DA levels of Max1 and St4 in the third series of detection were 0.496 ( 0.064 and 0.605 ( 0.052 (mean ( SEM), respectively (Figure 5C), suggesting a significant depletion of DA stores after the first and second detection. These statistic data will be used as controls to study the effect of drugs, such as reserpine and L-dopa, on the K+-stimulated DA exocytosis. The extracellular DA concentration of Max1 in spectrum a of the Figure 4 was calibrated to be 1.06 µM, by using a DA calibration curve. This DA concentration is well within the linear range of DA amperometric response, indicating that the collagencoated MEA biochip is suitable for the detection of K+-stimulated DA release from PC12 cells in this design. 6352 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

Effect of a VMAT Inhibitor, Reserpine, on the Level of K+Stimulated DA Release. Reserpine is a VMAT inhibitor. VMATs locate in the membranes of two distinct classes of regulated secretory vesicles: small synaptic vesicles and large dense core vesicles.1 They translocate monoamines (i.e., catecholamines, serotonin, and histamine) from the cytoplasm into secretory vesicles of endocrine cells and neurons as an electrochemical antiporter of protons and monoamines by using proton electrochemical gradient.33,34 VMAT inhibitors displace catecholamine from neurotransmitter vesicles and, therefore, deplete catecholamine storage. Two homologous, but distinct VMAT genes, VMAT1 and VMAT2, have been cloned from rat, bovine, and human adrenal glands.34 Reserpine is an inhibitor of both VMAT1 and VMAT2, although its inhibition to VMAT2-mediated transport is (33) Nirenberg, M. J.; Liu, Y. J.; Peter, D.; Edwards, R. H.; Pickel, V. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 8773-8777. (34) Henry, J. P.; Botton, D.; Sagne, C.; Isambert, M. F.; Desnos, C.; Blanchard, V.; Raisman-Vozari, R.; Krejci, E.; Massoulie, J.; Gasnier, B. J Exp. Biol. 1994 196, 251-262.

Figure 6. (A) Amperometric spectra of KCl stimulated responses of PC12 cells in the first detection for naive PC12 cells and the second detection after incubation in L-dopa (both in blank BSS solution) at a biochip microelectrode of 60-µm diameter (+0.3 V vs biochip microelectrode). Arrows position the injection time point. Injection flow rate, 300 µL min-1; volume per injection, 20 µL. (B) Normalized extracellular DA level of Max1 (black bar) and St4 (gray bar) in the second detection after L-dopa incubation. Control is the second detection in BSS without L-dopa incubation. Values marked with * are statistically difference with p < 0.01; ** indicates a significant difference with p < 0.001 vs control (Student’s t-test).

slightly more potent than to VMAT1-mediated transport.35 PC12 cells express the neurosecretory VMAT subtype VMAT1 and show preferential expression of VMAT1 on large dense core vesicles.36 Pharmacological manipulation of PC12 cells with reserpine has been found to cause the reduction of vesicular volume and the depletion of DA stores.9 Figure 5 also shows the effect of reserpine on the K+-stimulated DA release, detected by MEA biochips. In Figure 5A, the amperometric spectra of the second detection in blank BSS (a) and 1 µM reserpine after incubation in this reserpine solution for 20 (b) and 30 min (c) are illustrated. Incubation in 1 µM reserpine reduced the amplitude of both Max1 and St4, and the amount of reduction was increased with the increase of incubation time. As shown in Figure 5B, incubation in 1 µM reserpine for 20 min reduced both the Max1 and St4 to about half of those in the BSS control. Incubation in the reserpine solution for 30 min reduced the normalized DA level of Max1 and St4 to 0.115 ( 0.059 and 0.275 ( 0.098 (mean ( SEM), respectively. These results are consistent with those detected on single PC12 cells by using carbon fiber microelectrodes. Kozminski et al.4 reported that incubating PC12 cells in 1 µM reserpine for 10 and 30 min diminished the number of total release per stimulation to half and one-fourth of its initial value, respectively. To confirm that the reduction of K+-stimulated DA release detected by the MEA biochip is due to the effect of reserpine, control experiments of the second detection in DMSO/BSS (1:1000) (the vehicle used for reserpine administration) were performed. As shown in Figure 5B, incubation in this DMSO dilution for 30 min did not affect the K+-stimulated DA release, in comparison with the BSS control. The effect of reserpine is reversible, as show in the amperometric curve c of Figure 5A and in panel b of Figure 5C. After (35) Erickson, J. D.; Schafer, M. K. H.; Bonner, T. I.; Eiden, L. E.; Weihe, E. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 5166-5171. (36) Liu, Y.; Schweitzer, E. S.; Nirenberg, M. J.; Pickel, V. M.; Evans, C. J.; Edwards, R. H. J. Cell Biol. 1994, 1419-1433.

the second detection in reserpine, the K+-stimulated DA release was reversed in the third detection in blank BSS, indicating that DA in the cytosol can be rapidly packed into catecholamine vesicles by VMAT. Effect of L-Dopa Incubation on the Level of K+-Stimulated DA Release. In the cytosol of catecholamine neurons, tyrosine hydroxylase converts tyrosine to L-dopa, a precursor of DA, with the assistance of cofactors: molecular oxygen; ferrous ion; nicotinamide adenine dinucleotide cofactors (NADH or NADPH);37 and tetrahydrobiopterin.38 This is normally the rate-limiting step in catecholamine biosynthesis. In contrast, the cytosolic conversion of L-dopa to DA by aromatic L-amino acid decarboxylase with the assistance of pyridoxal phosphate cofactor is rapid. DA formed in the cytosol is actively transported from the cytoplasm into the vesicles by VMAT and then stored in the catecholamine vesicles. The effect of L-dopa on the K+-stimulated DA release is shown in Figure 6. Figure 6A illustrates the amperometric spectra of KClstimulated response of PC12 cells in the first detection for naive PC12 cells and the second detection after incubation in L-dopa. It was observed that both Max1 and St4 in the second detection for L-dopa-incubated PC12 cells were much stronger than those in the first detection. Figure 6B compares the statistic results of Max1 and St4 in the second detection for L-dopa-incubated PC12 cells with those of the control. Compared to the control, the normalized extracellular DA levels of Max1 and St4 significantly increased from 0.916 ( 0.097 to 2.226 ( 0.318, and from 1.062 ( 0.106 to 3.996 ( 0.327 (mean ( SEM), respectively. These results are consistent with that reported by Pothos et al. They reported that incubation of PC12 cells in 100 µM L-dopa at 37 °C for 1 h increased the K+-stimulated DA release (2-min stimulation) to 371% of the control.5 The significant increase of extracellular DA concentration is due to the conversion of L-dopa to DA in the cytosol, which (37) Kuhn, D. M.; Geddes, T. J. Brain Res. 2002, 933 (1), 85-89. (38) Kuhn, D. M.; Geddes, T. J. Mol. Pharmacol. 2003, 64 (4), 946-953.

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Figure 7. (A) Amperometric spectra of KCl stimulated responses of PC12 cells in the first detection in 60 µM nomifensine and DMSO/BSS (1.5:1000) at biochip microelectrodes of 60-µm diameter (+0.3 V vs biochip microelectrode). Arrows position the injection time point. Injection flow rate, 300 µL min-1; volume per injection, 20 µL. (B) Ratio of Max1 to St4 in the first amperometric detection of K+-stimulated DA release in 60 µM nomifensine and the control solution of DMSO/BSS (1.5:1000). Values marked with ** indicates a significant difference with p < 0.001 vs control (Student’s t-test).

subsequently leads to the increase of DA store in the catecholamine vesicles. Colliver et al.9 found that the increase of DA store upon L-dopa incubation was due to increased vesicular volume, while the DA concentration inside the vesicles remained unchanged. Consequently, upon KCl stimulation, the fusion of catecholamine vesicles of increased vesicular volume with cell membrane leads to an increased release of DA into extracellular space. Effect of Nomifensine on the Level of K+-Stimulated DA Release. Nomifensine is a DAT inhibitor. The function of DAT is to terminate DA transmission by the rapid re-uptake of DA into presynaptic terminals.32 By blocking the re-uptake of DA, nomifensine can increase the extracellular DA concentration. However, comparisons of the K+-stimulated DA release in the second detection in nomifensine with that in the BSS control did not reveal a statistically significant effect of nomifensine on the increase of extracellular DA level (p >0.05, Student’s t-test) (data not shown). When nomifensine concentration was increased to 80 µM, a significant decrease of extracellular DA level of Max1 was detected (data not shown). This decrease might be due to the toxicity of nomifensine, as the K+-stimulated DA release in the second detection in DMSO/BSS (2:1000) (the vehicle used for 80 µM nomifensine administration) was similar to the BSS control (data not shown). However, the toxicity test by counting the percentage of viable cells with trypan blue staining and hemocytometer demonstrated that incubating PC12 cells in 80 µM nomifensine for 20 min was nontoxic to cells, as shown in Table S-1 in the Supporting Information. These results indicate that the toxicity test by measuring DA release using MEA biochips may be more sensitive than that with trypan blue staining and hemocytometer. The insignificant effect of nomifensine, detected by comparing the K+-stimulated DA release in the second detection in nomifensine with that in BSS control, was not unexpected considering that DAT may have been desensitized to some extent in the first detection. Therefore, an analytical method different from that used for reserpine and L-dopa was developed for nomifensine. In this 6354 Analytical Chemistry, Vol. 78, No. 18, September 15, 2006

method, nomifensine was applied in the first detection of K+stimulated DA release, and the ratio of St4 to Max1 (St4/Max1) was compared to that of the control. Figure 7 demonstrates the effect of 60 µM nomifensine on the K+-stimulated DA release. Upon nomifensine administration, the value of St4/Max1 was significantly increased from 0.181 ( 0.024 of the control to 0.426 ( 0.045 (mean ( SEM). The result indicates that this method is especially useful and valid for detecting the effect of DAT inhibitors on the K+-stimulated DA release. In addition, this result supports the assumption that the value of St4 is a combinational result of DA exocytosis and DA re-uptake, while the value of Max1 reflects mainly the DA exocytosis. CONCLUSIONS The study attempted to develop rapid and high-throughput analytical methods for real-time detection of the drug effect on DA exocytosis from PC12 cells by using MEA biochips. First, in this study, electrochemical techniques were used to temporally detect the release of K+-stimulated DA exocytosis from biochip grown PC12 cells. From the temporally recorded amperometric spectra of the K+-stimulated DA release, two parameters, Max1 and St4, representing the peak current responding to the first KCl injection and the steady current responding to the fourth KCl injection, respectively, were defined. These two parameters turned out to be very useful, yet simple in defining the K+-stimulated DA release, yielding a wealth of information regarding amplitude and temporal aspects of the DA release from PC12 cells. Second, by defining these two parameters, analytical methods for detecting the effects of three kinds of drugs, including a VMAT inhibitor (reserpine), a DA precursor (L-dopa), and a DAT inhibitor (nomifensine), were successfully developed. Only one or two working electrodes were used simultaneously in this study. The next step of this research should be extending the number of working electrodes used at one time, first to 8, and finally up to 23 with the help of multiplexing. The detection methods developed in this study are novel, yet useful, and the data analysis for the effect of drugs is simple. The

analytical methods may provide a high-throughput, in vitro drug screening approach for the DA-related psychiatric disorders. Based on the approach used in this research, in situ detection of the release of other neurotransmitters and endogenous intracellular molecules, such as glutamate, ATP, and insulin, may be developed.

024-112 to F.-S. S., and by the Agency for Science, Technology and Research (A*STAR) to Y.C.

ACKNOWLEDGMENT This work was supported by Academic Research Grants of the National University of Singapore R-398-000-006-112 and R-398-000-

Received for review January 4, 2006. Accepted July 14, 2006.

SUPPORTING INFORMATION AVAILABLE Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

AC060018D

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