Compact Microelectrode Array System: Tool for in Situ Monitoring of

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Anal. Chem. 2008, 80, 1133-1140

Compact Microelectrode Array System: Tool for in Situ Monitoring of Drug Effects on Neurotransmitter Release from Neural Cells Yu Chen,*,† Chunxian Guo,‡ Layhar Lim,† Serchoong Cheong,† Qingxin Zhang,† Kumcheong Tang,† and Julien Reboud†

Institute of Microelectronics, 11 Science Park Road, Singapore 117685, and School of Chemical and School of Chemical and Biomedical Engineering, Nanyang Technological University, Singapore

This paper presents a compact microelectrode array (MEA) system, to study potassium ion-induced dopamine release from PC12 neural cells, without relying on a micromanipulator and a microscope. The MEA chip was integrated with a custom-made “test jig”, which provides a robust electrical interfacing tool between the microchip and the macroenvironment, together with a potentiostat and a microfluidic syringe pump. This integrated system significantly simplifies the operation procedures, enhances sensing performance, and reduces fabrication costs. The achieved detection limit for dopamine is 3.8 × 10-2 µM (signal/noise, S/N ) 3) and the dopamine linear calibration range is up to 7.39 ( 0.06 µM (mean ( SE). The effects of the extracelluar matrix collagen coating of the microelectrodes on dopamine sensing behaviors, as well as the influences of K+ and L-3,4digydroxyphenylalanine concentrations and incubation times on dopamine release, were extensively studied. The results show that our system is well suited for biologists to study chemical release from living cells as well as drug effects on secreting cells. The current system also shows a potential for further improvements toward a multichip array system for drug screening applications. Drug screening at the cellular level has been attracting noticeable attention recently. An in-depth understanding of the dynamic relationships among chemical and molecular events, together with the genomics and proteomics in living cells, will be greatly helpful to effectively pursue drug discovery. The potential new therapies must be evaluated within the context of the living cell in order to measure biological behavior and function. However, the bottleneck in this area is the lack of readily available cellular response analysis systems, which would allow fast, sensitive, and quantitative analyses, while using lower amounts of reagents. Microfabricated devices with cell culture capability and integrated sensors present a high potential to meet these requirements. More particularly, they provide integrated platforms to perform studies on living cells by optical or electrical means in * To whom correspondence should be addressed. E-mail: ime.a-star.edu.sg. † Institute of Microelectronics. ‡ Nanyang Technological University. 10.1021/ac071182j CCC: $40.75 Published on Web 01/15/2008

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a controlled environment to reveal temporal changes within the cells or their constituents, while enabling automatic data collection. Drug screening on cell models is usually based on cellular assays on two-dimensional monolayers, often using well-described cell lines. These assays can be performed efficiently in highthroughput screening systems. Cultured cells transduce and transmit a variety of chemical and physical signals, through the production of specific substances and proteins. These cellular signals can be used as parameters to monitor chemical information to build drug efficacy profiles in vitro.1 Recently, microfluidics has been shown to be an attractive approach to create a stable and uniform microenvironment for cell growth in microchips.2-5 It is well-known that neurotransmitters are important for brain functions and the central nervous system. An abnormal concentration of dopamine, one of the neurotransmitters, is related to Parkinson’s disease.6 Monitoring dopamine release and studying drug effects at the cellular level are particularly interesting to neuroscientists and in drug discovery. There are two types of techniques used for the detection of dopamine exocytosis from neural cells, which could be categorized based on their sample collection mode as “static” and “dynamic”. In the static mode, the dopamine originating from exocytosis or from lysis of the cells is collected and analyzed by HPLC6-8 or capillary electrophoresis,9 integrated with optical or electrochemical sensors. This method provides averages and static and steady-state results. To the contrary, in the dynamic mode, the dopamine exocytosis is detected in situ and in real-time. In previous works, external microelectrodes were often used.6,8,10-12 This setup requires (1) Haruyama, T. Adv. Drug Delivery Rev. 2002, 1, 1-9. (2) Thie´baud, P.; Lauer, L.; Knoll, W.; Offenha¨user, A. Biosens. Bioelectron. 2002, 17, 87-93. (3) Andersson, H.; Berg, A. V. D. Lab Chip 2004, 4, 98-103. (4) Walker, G. M.; Zeringue, H. C.; Beebe, D. J. Lab Chip 2004, 4, 91- 107. (5) Hung, P. J.; Lee, P. J.; Sabounchi, P.; Aghdam, N.; Lin, R.; Lee, L. P. Lab Chip 2005, 5, 44-48. (6) Gao, F.; Xu, M.; Wang, L.; Shi, G.; Zhang, W.; Jin, L.; Jin, J. Talanta 2001, 55, 329- 336. (7) Eugenia, B. S.; Saleh, A. R.; Victoria, T.; Hassia, B.; Arie, G.; Michal, L.; Philip, L. J. Pharmacol. Exp. Ther. 2002, 301 (3), 953-962. (8) Zhang, W.; Gao, X.; Xie, Y. F.; Jin, S.; Ai, L.; Jin J. J. Chromatogr., B 2003, 785, 327- 336. (9) Wang, Z.; Yeung, E. S. Pure Appl. Chem. 2001, 73 (10), 1599- 1611. (10) Kiyatkin, E. A.; Kiyatkin, D. E.; Rebec, G. V. Neuroscience 2000, 98 (4), 729-741. (11) Kozminski, K. D.; Gutman, D. A.; Davila, V.; Sulzer, D.; Ewing, A. G. Anal. Chem. 1998, 70, 3123-3130.

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Figure 1. (A) Overview of the compact microelectrode array system. (B) Overview of the testing jig. (C) Microchip design-1, 22 small disk working electrodes with 60-µm diameter, one large disk counter electrode in the center and one ring reference electrode in the center. This design was used in this research. (D) Microchip design-2, 24 small disk electrodes with 60-µm diameter, one of them used as counter electrode and one of them used as reference electrode. This design was used in our previous research.13-15

locating the electrodes very close to the cells or tissues. Conventionally, the operation has to be done under the microscope, which is inconvenient, time-consuming, and may injure cells with the sharp external electrodes. To provide a simple and reliable tool for in situ, real-time detection of neurotransmitter exocytosis at the cellular level, our group developed a microfabricated siliconbased biochip.13-15 A microelectrode array (MEA) was integrated on the microchip and an electrochemical amperometry technique was applied. Neural cells cultured on the chip were stimulated by potassium ions, and the induced dopamine exocytosis was temporally detected in situ by the microelectrodes. The effects of drugs on the dopamine release and uptake could be monitored as well. The MEA used in our previous work13-15 needed wire bonding to its printing circuit board (PCB), and soldered connectors were required to link it with the electrochemical workstation. The cost and time needed for those stages were not compatible with such application, as drug screening requires a large number of arrays. To reduce fabrication costs, increase the chip reliability and sensitivity, and improve the detection limit, a compact testing system is presented. It comprises an MEA chip, a cell culture chamber, an electrical connection socket, a potentiostat, and a microfluidic syringe pump. The latter “test jig” (Figure 1) allows (12) Venton, B. J.; Wightman, R. M. Anal. Chem. 2003, 75 (19), 414A-421A. (13) 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. (14) Cui, H. F.; Ye, J. S.; Chen, Y.; Chong, S. C.; Sheu, F. S. Anal. Chem. 2006, 78, 6347-6355. (15) Chen, Y.; Cui, H. F.; Ye, J. S.; Chong, S. C.; Lim, T. M.; Sheu, F. S.; Cheong, H. W. Proc. SPIE 2005, 6112, 611260C-1-11.

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inserting the chip into a predefined housing without prolonged operation time. We also report further modifications of the microchip, optimization of the collagen coating process and storage condition to achieve higher sensitivity and lower detection limit of dopamine. The conventional techniques used to trigger dopamine release include ionophores,10,16 electrical stimulation,17-19 and high concentration of K+19,20 in either cells, tissues, or animals. Raising the external K+ concentration is very efficient and easy to achieve; hence it is widely applied.9 Both steady-state calibration and peak ratio approaches were applied in our previous research.13-15 Using a local K+ injection in the vicinity of the dopaminergic cells, thus needing a micromanipulator and a syringe pump, triggered dopamine release. To simplify and streamline handling operations, the compact system developed enables the use of bulk injections of K+. The steady-state calibration method is more suitable here, since it allows lower constraints in the spatial control of the K+ injection. The effects of different drugs on dopamine release from PC12 cells were the major focus of our previous research.13-15 However, doses and incubation times of drugs significantly affect dopamine release, through different processes including drug and dopamine diffusion and intracellular dopamine synthesis. This calls (16) Bowyer, J.; Weiner, N. J. Pharmacol. Exp. Ther. 1990, 254 (2), 664-670. (17) Palij, P.; Bull, D. R.; Sheehan, M. J.; Millar, J.; Stamford, J.; Kruk, Z. L.; Humphrey, P. P. A. Brain Res. 1990, 509, 172-174. (18) Schulte, D.; Callado, L. F.; Davidson, C.; Phillips, P. E. M.; Roewer, N.; Esch, J. S. A.; Stamford, J. A. Br. J. Anaesth. 2000, 84 (2), 250-253. (19) Stamford, J. A.; Justice, J. B. Anal. Chem. 1996, 68, 359A-366A. (20) Bowyer, J. F.; Newport, G. D.; Lipe, G. W.; Frame, L. T. J. Pharmacol. Exp. Ther. 1992, 261 (1), 72-80.

for a detailed study of these effects on dopamine release, addressed in the research presented here. The dopaminergic cell line PC12 was used as cell model, and L-3,4-digydroxyphenylalanine (L-DOPA; dopamine precursor) as a drug. L-DOPA is the most effective and frequently prescribed therapy for controlling the symptoms of Parkinson’s disease. No other drug was used in this research since a multidrug study had been reported before.13-15 EXPERIMENTAL SECTION Chemicals. 3,4-Dihydroxyphenethylamine (dopamine), L-3,4digydroxyphenylalanine (L-DOPA), 4(2-hydroxyethyl)piperazinel-ethanesulfonic acid (HEPES), sodium chloride, potassium chloride, calcium chloride, magnesium chloride, sodium hydrogen phosphate, potassium dihydrogen phosphate, D-glucose, phosphatebuffered saline solution (PBS, pH 7.4), collagen type I (Catalog No. C9791), and acetic acid were purchased from Sigma. F-12K nutrient mixture (Kaifhn’s modification, Catalog No. 21127-022), fetal bovine serum (FBS), penicillin-streptomycin, and horse serum (heat activated) were from Invitrogen. PC12 cells were from ATCC and were maintained following the supplier’s protocol. Chemical Solution Preparation. Two types of balanced salt solutions (BSS) were used for the experiments. Type one of BSS (BSS1) contained 138 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES; and type two of BSS (BSS2) contained 0.5 mM NaCl, 140 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM D-glucose, and 10 mM HEPES. BSS1 was used to dilute dopamine and L-DOPA solutions and served as the base medium for PC12 cell dopamine release experiment. BSS2 was used to stimulate the release of dopamine from PC12 cells. PBS contained 0.14 M NaCl, 2.68 mM KCl, 10.14 mM Na2HPO4, and 1.76 mM KH2PO4. It was used to wash the chips and to dilute the collagen solution. The pH of all those solutions was adjusted to 7.4 by using 1 M NaOH. The 2 mg/mL collagen stock solution was prepared by dissolving 2 mg of collagen I powder in 1 mL of 0.2% acetic acid in DI water by stirring for 1 h at room temperature. The solution was then filtered using 0.45-µm cellulose acetate filters. The collagen stock solution was stored at 4 °C until it was needed. The collagen solution could be further diluted in PBS, to be used to coat the chips to promote PC12 cell adhesion. The 527.2 µM dopamine and 1 mM L-DOPA stock solutions were prepared daily in nitrogen-bubbled BSS1 solution and could be further diluted to various concentrations in BSS1 to perform sensor calibration and drug dosage tests. Microchip Design and Fabrication. The MEA was fabricated from two 8-in.-diameter silicon wafers, which were ∼1.5 mm thick with a chip size of 1 cm × 1 cm. The microchip consists of 22 small Au disk working electrodes, one Au ring reference electrode, and one larger Au disk counter electrode. Each individual electrode was leaded out to its pad. The working electrodes had a diameter of 60 µm with a spacing of 160 µm. The microchip fabrication process was presented in our previous publication.14 Finally, a rectangular polycarbonate chamber was taped on the microchip. The chamber created was 0.5 cm (width) × 0.4 cm (length) × 0.5 cm (height) with volume capacity of 100 µL. Prior to testing, the microfabricated chip was swapped with 70% ethanol and exposed to UV light in the biosafety cabinet for 1 h. The 100 µL of diluted collagen solution (5 µg/cm2) was loaded into the microchip. The microchip was then sealed with a biocompatible membrane to prevent evaporation and incubated at 37 °C

overnight. Before PC12 cells were seeded onto the collagen-coated microchip, the excess collagen solution was removed and the chip was rinsed extensively with sterile PBS. Compact Testing System. The compact testing system was designed and manufactured as shown in Figure 1A. It included three major parts: (1) the test jig (Figure 1B), incorporating 24 contact probes spaced evenly by 500 µm, contacted the electrode pad on the microchip directly to enable the electrical connection, thereby avoiding the need of PCB for the microchip packaging; (2) the potentiostat from CH Instruments (CHI 1232 electrochemical analyzer) provided the electrochemcial monitoring function for microchip characterization and dopamine release detection; (3) the microfluidic syringe pump (KDS) was assembled into this compact system, to perform K+ injections. The microchip (Figure 1C, D) was loaded on the test jig. Cell Culture on the Microchip. F-12K nutrient mixture (Kaighn’s modification) mixed with 15% horse serum (heat inactivated), 1% penicillin-streptomycin, and 2.5% FBS was used for PC12 cell culture. The 5 × 104 cells in a 100 µL of culture media were seeded onto the microchip. It was then sealed with a biocompatible, gas-permeable tape (Jereiner Bio-1; 676050 plate sealer-breath seal) and incubated at 37 °C in a 5% CO2 atmosphere for 5 h. In these conditions, the cells would reach 90% confluence. Electrochemical Characterization of Microelectrodes for Dopamine. Cyclic voltammetry was used for the microelectrode electrochemical characterization of dopamine at 0.6 µΜ. The larger Au disk electrode in the center of the array was used as counter electrode, and the larger Au ring electrode was used as reference electrode, while the remaining small electrodes were used as working electrodes (Figure 1C, design-1). The voltage scan from 0.6 to -0.3 V was carried out with 0.05 V/s scan speed to get a full cyclic voltammetry picture. It was found that +0.3 V was the dopamine oxidation potential on the collagen-coated Au electrode. Therefore, +0.3 V was used for the dopamine amperometry detection. All the tests were done at room temperature. BSS1 solution was used as a base solution to perform the dopamine standard calibration. The differences between the two steady-state currents, before and after dopamine injection, was defined as the sensor‘s response and plotted against dopamine concentration to draw the dopamine standard calibration. The low dopamine concentration detection limit was defined by a signal/ noise (S/N) ratio of 3. Characterization of the Collagen-Coating Process. The collagen-coating process was evaluated by studying its impact on the sensor’s sensitivity; its reproducibility, and the sensing stability of the collagen-coated microelectrodes. The sensor’s sensitivity was defined by the slope of the dopamine calibration curve (current difference/concentration). The collagen coating density on the chip was within the range of 0-20 µg/cm2. The sensors’ sensitivities were recorded for both electrodes with and without collagen. The ratio between them was as an indicator used for evaluation of the collagen-coating effects on sensing sensitivity. Two electrodes from each chip and a total of four chips were tested. Each electrode was coated with collagen only once. The collagen-coating process reproducibility was studied by using four chips. The collagen-coating process was repeated five times for each chip by applying the same amount of collagen. Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

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Before each coating, the chip was soaked in isopropyl alcohol for 2 h, followed by DI water rinses. The dopamine-sensing sensitivities of one electrode, with and without collagen, was recorded against the number of collagen coatings it had withstand. The ratio of the sensitivities with and without collagen was used as reproducibility index for evaluation. The collagen-coated microchips were stored at 4 °C until used. The dopamine calibration was repeated for 43 days at a few days’ intervals. The ratio of the sensitivities between the testing day and the first day was used as the stability index for evaluation. Two microchips were used for this study, and four electrodes of each chip were tested. Detection of K+-Stimulated Dopamine Release from PC12 Cells. After the cells had been attached to the chip surface and reached 90% confluence, the 55 µL of culture medium was gently pippetted out of the chip and then the PC12 cells were washed four times with 20 µL of BSS1 solution. The amperometry detection was carried out at +0.3 V. Once the base current reached a steady state, 10 µL of BSS2 solution (with a higher K+ concentration) was injected by a syringe pump at the rate of 44.15 mL/h to stimulate the release of dopamine. A total of five injections were done at 100-s intervals or longer depending on the dopamine quantity released at each stage. After the first five K+ stimulations, the medium containing the PC12 exocytosis material was removed, the chip was washed with BSS1 solution, and the next release test could be started immediately. The dopamine release test for the cells without L-DOPA treatment was used as a control run, to serve as a reference for the results from the tests with L-DOPA, which followed. To quantify the dopamine release from the PC12 cells under various conditions, a standard calibration was performed for each electrode after each cell release test. All measurements were done at room temperature. Each condition was repeated three times, and two electrodes of each chip were monitored simultaneously per test. The mean of these total six data and the standard mean error were presented. L-DOPA Effects on Dopamine Release from PC12 Cells. Before the L-DOPA treatment, two consecutive dopamine release tests under control conditions were carried out on the 90% confluent adherent PC12 cells. The same chip containing the same cells was then incubated with L-DOPA from 50 to 150 µM for 20-60 min at 37 °C. Immediately after the incubation, the dopamine release tests on the L-DOPA pretreated PC12 cells were started. The experimental procedures were the same as that of the control run. Each condition was repeated three times, and two electrodes of each chip were monitored simultaneously per test. The mean of these total six data and the standard mean error were presented. RESULTS AND DISCUSSIONS Electrochemical Characterization of Dopamine. Cyclic voltammetry scans using collagen-coated microelectrodes were carried out with 0.6 µM dopamine, and results are shown in Figure 2. Based on this test, +0.3 V was selected as dopamine oxidation potential for amperometry testing. The sensitivity of the microelectrode for the chip design-1 (Figure 1C) coated with collagen was 1.17 × 10-11 ( 0.17 × 10-11 A/µM (mean ( SE), which was less than the sensitivity of uncoated electrodes, at 24.3 ( 2.4% (mean ( SE). These results are based on the optimized collagen 1136 Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

Figure 2. Cyclic voltammetry response to 0.6 µM dopamine, scan speed 0.05 V/s and voltage scan from 0.6 to -0.3 V. Design-1 was used.

coating condition, enhancing cell attachment while significantly maintaining sensor sensitivity. Compared to the previous microchip design (Figure 1D), using working electrodes of the same diameter but smaller reference and a counter electrodes, the sensor sensitivity was increased two to four times. The lowest dopamine concentration detectable was 3.8 × 10-2 µM (S/N ) 3) for design-1 microchips (Figure 1C) coated with collagen. The dopamine linear calibration range was up to 7.39 ( 0.06 µM (mean ( SE), 10 times the one obtained with uncoated electrodes. This may be attributed to an increase in the dopamine diffusion layer thickness due to the collagen coating. The diffusion control could be the sensing mechanism of this type of sensors.21 Study of the Collagen Coating Process. . In order to identify the critical parameters of the collagen coating process, as well as maintain the optimal conditions on the collagen-coated electrodes, to achieve a better sensing performance, an in-depth understanding of the effects of the collagen coating process on the sensor behavior was necessary. The effects of the collagen density on the sensor sensitivity, the sensing stability of collagen-coated chip, and the reproducibility of the collagen coating process were studied. Figure 3A presents the effects of the collagen density on the sensor sensitivity. The results show that the electrode sensitivity decreases when the collagen density increases. The higher the collagen density, the more collagen would be coated on the electrode surface, thus increasing the collagen thickness on the electrode and resisting dopamine diffusion. Hence, the effective dopamine concentration on the Au electrode surface was reduced. In this study, 5 µg/cm2 was selected as the optimal collagen density for the microchip. It achieved a sensitivity ratio of 77.0 ( 5.3% (mean ( SE) with minimum effects on the sensing sensitivity. Although lower concentrations show better sensitivity ratio (94.1 ( 0.47% (mean ( SE) for 1 µg/cm2, for example) poor cell attachment was obtained. Therefore, 5 µg/cm2 was used for all studies. The results of the sensing stability of collagen-coated microchip are shown in Figure 3B. The sensitivity of the collagen-coated electrodes after 35 days is 94.1 ( 5.1 (mean ( SE) and 77.5 ( 6.2% (mean ( SE) after 39 days. This good stability indicates that (21) Chen, Y.; Tan, T. C. AIChE J. 1995, 41 (4), 1025-1036.

Figure 3. (A) Effect of collagen density on electrode sensitivity to dopamine (two electrodes from each chip and a total of four chips were tested, n ) 8, mean ( SE). (B) Sensing stability of collagencoated electrodes (0, chip-1; [, chip-2) (two electrodes from each chip and a total of four chips were tested, n ) 8, mean ( SE). (C) Reproducibility of the sensitivity of collagen-coated electrodes. The ratio of the sensitivities between the testing day and the first day was defined as the reproducibility index (5 tests were done on each chip and a total of 4 chips were tested, n ) 20, mean ( SE).

the collagen coating process and storage conditions were appropriate. It also opens the possibility of using stored collagencoated microchips for cell-based assays, in compliance with mass production of the devices. To evaluate the reproducibility of the collagen coating process, the reproducibility index was used. The data were collected before and after each collagen coating. Results are shown in Figure 3C. Excellent reproducibility was obtained for each electrode, as shown by a standard mean error of the reproducibility index within a range of 0.8-2.3% (SE) for the five coating tests among four chips. However, the reproducibility index was within a range of 0.616 ( 0.008 to 0.896 ( 0.023 (mean ( SE) among four chips. This important variation among the chips indicates that individual calibration for every electrode from each chip is necessary to obtain an accurate quantitative result. Real-Time Monitoring of Dopamine Release from PC12 Cells. The real-time dopamine release responses from PC12 cells

Figure 4. Real-time amperometric response of K+-induced dopamine release from PC12 cells (A, without and, B, with 100 µM L-DOPA pretreatment at 37 °C for 1 h). +0.4 V was used for the amperometry detection. 5 × 104 cells in 100 µL were incubated on the chip for 5 h at 37 °C before release test. Measurement was conducted at room temperature (25 °C). Arrows indicate K+ injection time.

both with and without drug are shown in Figure 4. The arrows indicate the injection of the K+ stimulating solution. The results show that the current response increased significantly after four or five K+ injections. At these points, K+ concentration had been increased to above 80 mM. PC12 cells are known as dopaminergic cells, which contain catecholamine molecules.22,23 To further verify whether the current signal change was mainly due to dopamine released from PC12 cells, the cell culture medium after stimulation was analyzed by HPLC, as reported previously by Cui et al.13 Kumar et al.24 also reported that, after K+ stimulation, dopamine release was ∼11-fold higher than norepinephrine release. Therefore, only dopamine release was studied in this research. The effect of K+ injection on the stability of the baseline current (without cells attached on the electrodes) was also investigated (results not shown), confirming that the steady-state current did not increase significantly during K+ injection. The signal shape of the real-time dopamine release responses presented here is not identical to our previous reported results14 as the K+ stimulation approaches were different. Previously, the capillary used for K+ delivery was brought closer to the cells monitored with the (22) Chen, T. K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (23) Pothos, E.; Desmond, M.; Sulzer, D. J. Neurochem. 1996, 66, 629-636. (24) Kumar, G. K.; Overholt, J. L.; Bright, G. R.; Hui, K. Y.; Lu, H.; Gratzl, M.; Prabhakar, N. R. Am. J. Physiol. Cell Physiol. 1998, 274, 1592-1600.

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Figure 5. Effects of K+ concentration and number of repetition of the experiments on dopamine release. (A) Without L-DOPA pretreatment; dotted box, crosshatched box, 0, and 9 represent the first, second, third and fourth stimulations. The 5 × 104 cells in 100 µL were incubated on the chip for 5 h at 37 °C before release test. (Two electrodes simultaneously monitored each time and a total of three repeating tests, n ) 6, mean ( SE). (B) With pretreatment by 100 µM L-DOPA for 1 h at 37 °C; dotted box, crosshatched box, and 0 represent the first, second and third stimulations (two electrodes simultaneously monitored each time and a total of three repeated tests, n ) 6, mean ( SE). The 5 × 104 cells in 100 µL were incubated on the chip for 5 h at 37 °C before release test. Measurement was the conducted at room temperature (25 °C). Signals from four electrodes were collected for each condition. *p < 0.01.

Figure 6. Effect of L-DOPA on the dopamine release from PC12 cells. The 5 × 104 cells in 100 µL were incubated on the chip for 5 h at 37 °C before dopamine release test. The confluent PC12 cells were subjected to two continuous dopamine release tests under control run conditions (without L-DOPA first, without L-DOPA second). L-DOPA effects were tested subsequently. Cells were incubated with 100 µM L-DOPA at 37 °C for 1 h before the dopamine release experiments (with L-DOPA first, with L-DOPA second). Signals from two electrodes were collected for each condition. dotted box, crosshatched box, 0, and 9 represent 57.5, 71.3, 81.0, and 88.5 mM KCl, respectively (two electrodes simultaneously monitored each time and a total of three repeating tests, n ) 6, mean ( SE).

help of a microscope and micromanipulator. Once the K+ injection location was fixed, the dopamine release signal shape could be well repeated. Therefore, the peaks’ ratio was used as the sensor signal. However, in the compact system presented here, where a K+ bulk injection approach is used, the dopamine release response was slower due to the K+ ions’ diffusion, rendering the steadystate calibration approach more relevant. Effects of K+, L-DOPA, and Number of Repetitions of the Experiments on Dopamine Release from PC12 Cells. The effect of K+ concentration on the quantity of dopamine released from PC12 cells, both with and without L-DOPA pretreatment, was extensively studied. The results are presented in Figure 5. They clearly show that the higher the K+ concentration, the more dopamine is released out of the PC12 cells. This is consistent throughout the experiments, independent from L-DOPA pretreatment, and agrees with Almaraz et al.25 The highest dopamine (25) Almaraz, L.; Gonzalea, C.; Obeso, A. J. Physiol. 1986, 379, 293-307 293

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concentration secreted was found to be 3.36 ( 1.0 (mean ( SE; with L-DOPA first) and 2.33 ( 0.36 µM (mean ( SE; without L-DOPA first) after stimulation with 88.5 mM K+. The fact that the intracellular calcium (cytosolic Ca2+) ion concentration increased in K+-depolarized PC12 cells and GT1-7 neurons in Ca2+containing medium was experimentally demonstrated by Virgillio et al.26 and Javor et al.,27 respectively. And the cytosolic Ca2+dependent neutransmitter release from PC12 cells was further shown by Pozzan et al.28 These would explain the mechanism of the K+-triggered dopamine release from PC12 cells in a Ca2+containing medium, which may be due to a K+-induced increase in cytosolic Ca2+, consequently leading to dopamine release. Our testing results further confirm that complex local K+ injections can be replaced by the easily performed bulk injection when stimulating dopamine release from neural cells on the compact system. Moreover, it was found that the highest dopamine concentration was mostly obtained from the first or second release experiment, especially at higher K+ concentrations. It is gradually decreasing as the tests are repeated, independently of the L-DOPA treatment. This behavior can be explained by considering that dopamine is secreted from vesicles, which are fully loaded at the moment of the first release experiment. As the release is repeated, the intracellular dopamine content decreases; the dopamine intracellular synthesis is not fast enough to compensate the dopamine loss during the tests. Figure 6 shows L-DOPA effects on the dopamine quantity released from PC12 cells. The same confluent cell layer was first K+-stimulated without L-DOPA pretreatment and analyzed twice (without L-DOPA first, without L-DOPA second) and subsequently incubated with 100 µM L-DOPA for 1 h at 37 °C, K+ stimulated and analyzed twice (with L-DOPA first, with L-DOPA second). It can be found that the dopamine amount significantly increased (26) Javors, A. M.; King, S. T.; Chang, X. Y.; Klein, A. N.; Schenken, S. R. Brain Res. 1995, 694, 49-54. (27) Javors, A. M.; King, S. T.; Chang, X. Y.; Klein, A. N.; Schenken, S. R. Brain Res. 1995, 694, 49-54. (28) Pozzan, T.; Gatti, G.; Dozio, N.; Vicentini, L. M.; Meldolesi, J. J. Cell Biol. 1984, 99, 628-638.

Figure 7. (A) Effect of L-DOPA dosage on dopamine release. The 5 × 104 cells in 100 µL were incubated on the chip for 5 h at 37 °C and L-DOPA pretreatment was followed at 37 °C for 1 h. K+ concentration was 88.45 mM. All measurements were done at room temperature. 2 and 9 represent the first and second stimulations (two electrodes simultaneously monitored each time and a total of three repeating tests, n ) 6, mean ( SE). B: Effect of L-DOPA incubation time on dopamine release from PC12 cells. The 5 × 104 cells in 100 µL were incubated on the chip for 5 h at 37 °C and L-DOPA incubation at 100 µM concentration at 37 °C was followed. K+ concentration was 88.45 mM. Measurement was the conducted at room temperature (25 °C). 9 and 2 represent the first and second stimulations (two electrodes simultaneously monitored each time and a total of three repeating tests, n ) 6, mean ( SE).

from 0.21 ( 0.09 to 3.36 ( 1.01 µM (mean ( SE; KCl within 57.588.5 mM) after L-DOPA treatment (with L-DOPA first) compared to that without L-DOPA treatment (without L-DOPA second), which only increased from 0.13 ( 0.13 to 1.51 ( 0.59 µM (mean ( SE; KCl from 57.5 to 88.5 mM). It also shows that the highest quantity of dopamine was realeased during the first test, regardless of the L-DOPA pretreatment. Dopamine is formed by decarboxylation of L-DOPA, a precursor of dopamine. It could be converted to dopamine and stored in catecholamine-containing vesicles. Dopamine intracellular content is increased by L-DOPA as shown by Jin et al.28 Effects of L-DOPA Dosage and Incubation Time on Dopamine Release from PC12 Cells. The effects of L-DOPA dosage and incubation time were further studied. The results are shown in Figure 7A and B, respectively. The comparisons are presented by the ratio of the dopamine concentration released with L-DOPA pretreatment to that from the second release without L-DOPA (control run) test. It was found that the dopamine concentration released from the cells increased with the L-DOPA concentration (Figure 7A). After pretreatment with L-DOPA at concentrations ranging from 50 to 150 µM at 37 °C for 1 h and stimulation by a K+ concentration of 88.5 mM, PC12 cells released 2.80 ( 0.26 (mean ( SE) to 4.21 ( 0.26 (mean ( SE) (with L-DOPA first) times the amount of dopamine comparing with the control experiment and 1.86 ( 0.17 (mean ( SE) to 3.15 ( 0.01 (mean ( SE) times of dopamine in the second release experiment (with L-DOPA second). L-DOPA has been shown to induce an increase in the quantity of intracellular dopamine in a dose-dependent manner by Jin et al.29 Walkinshaw et al.30 and Pedrosa et al.31 have found that higher concentrations of L-DOPA (200 µM) lead to necrotic cell death. Therefore, L-DOPA concentrations ranging from 50 to 150 µM were selected for this study. (29) Jin, C. M.; Lee, J. J.; Yang, Y. J.; Kim, Y. K.; Ryu, S. Y.; Lee, M. K. Arch. Pharm. Res. 2007, 30 (8), 984-2007. (30) Walkinshaw, G.; Waters, C. M. J. Clin. Invest. 1995, 95, 2458-2464. (31) Pedrosa, R.; Soares-da-silva, P. Br. J. Pharmacol. 2002, 137, 13051313.

Moreover, the secreted quantity of dopamine increased with the duration of the incubation with L-DOPA, as shown in Figure 7B. PC12 cells were preincubated with 100 µM L-DOPA at 37 °C and stimulated by 88.5 mM K+; the ratio of the concentration of released dopamine with L-DOPA (the first release) and without L-DOPA (control run, the second release) increased from 1.38 ( 0.08 (mean ( SE) to 3.77 ( 0.31 (mean ( SE) once the L-DOPA incubation time increased from 20 to 60 min. However, the dopamine concentration ratio was found from 1.22 ( 0.14 (mean ( SE) to 2.30 ( 0.06 (mean ( SE) in the second release test with L-DOPA incubation time increasing, which is lower than that obtained from the first release test. If the comparison is made relative to the control run of the first release (without L-DOPA first), the similar effects can also be observed both for L-DOPA concentration and for incubation time variations. In PC12 cells, dopamine is synthesized through L-DOPA decarboxylation.28 Dopamine release mechanism involves a few steps, such as L-DOPA diffusion from the bulk solution to the inside of the cell, intracellular dopamine synthesis, and dopamine diffusion out of the cell to the bulk solution. These steps all are time dependent. The longer the L-DOPA incubation, the higher the L-DOPA intracellular concentration; this enables the synthesis of more dopamine. This consequently induces more dopamine released out of the cells. CONCLUSIONS This paper presents a compact microelectrode array system, which achieved in situ and real-time monitoring of the dopamine released from K+-stimulated PC12 cells. The multiprobe testing jig introduced allows users to load the chips easily and quickly. Microelectrode arrays represent a useful tool for in vitro, noninvasive, label-free, real-time and in situ drug screening, as demonstrated here by the analysis of the effect of a drug on the dopamine release by PC12 cells. A dopamine detection limit of 3.8 × 10-8 M (S/N ) 3) and a linear calibration range up to 7.39 ( 0.06 µM (mean ( SE) were obtained, which fully meet the requirements of this kind of study. The impacts of dosage, incubation time of the drug L-DOPA, and K+ concentration were extensively studied and the results are reported Analytical Chemistry, Vol. 80, No. 4, February 15, 2008

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for the first time. The detailed study of the ECM collagen coating process shows a good sensing stability and reproducibility, which would enable mass production. The compact system and microchip was demonstrated to be a very useful tool for biologists to study chemical release from living cells as well as drug effects at the cellular level. Further improvements to a multichip array system to fulfill the needs of drug screening applications are being considered.

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ACKNOWLEDGMENT The authors thank Miss Jong Ming Ching for the mask layout. The PC12 cell culture was guided by Dr. Hui-fang Cui and Associate Professor Fwu-shan Sheu of Biological Sciences Department in National University of Singapore. Received for review June 4, 2007. Accepted November 24, 2007. AC071182J