Anal. Chem. 2006, 78, 2521-2525
On-Chip Amperometric Measurement of Quantal Catecholamine Release Using Transparent Indium Tin Oxide Electrodes Xiuhua Sun† and Kevin D. Gillis*,†,‡,§
Dalton Cardiovascular Research Center, Department of Biological Engineering, and Department of Medical Pharmacology and Physiology, University of MissourisColumbia, Columbia, Missouri 65211
Carbon-fiber amperometry has been extensively used to monitor the time course of catecholamine release from cells as individual secretory granules discharge their contents during the process of quantal exocytosis, but microfabricated devices offer the promise of higher throughput. Here we report development of a microchip device that uses transparent indium tin oxide (ITO) electrodes to measure quantal exocytosis from cells in microfluidic channels. ITO films on a glass substrate were patterned as 20-µm-wide stripes using photolithography and wet etching and then coated with polylysine to facilitate cell adherence. Microfluidic channels (100 µm wide by 100 µm deep) were formed by molding poly(dimethylsiloxane) (PDMS) on photoresist and then reversibly sealing the PDMS slab to the ITO-glass substrate. Bovine adrenal chromaffin cells were loaded into the microfluidic channel and adhered to the ITO electrodes. Cells were stimulated to secrete by perfusing a depolarizing “high-K” solution while monitoring oxidation of catecholamines on the ITO electrode beneath the cell using amperometry. Amperometric spikes with charges ranging from 0.1 to 1.5 pC were recorded with a signalto-noise ratio comparable to that of carbon-fiber electrodes. Further development of this approach will enable high-throughput measurement of quantal catecholamine release simultaneously with optical cell measurements such as fluorescence. Many excitable cells, including neurons and endocrine cells, release transmitter molecules by a process called exocytosis. In this process, a rise of the intracellular Ca2+ concentration triggers the fusion of transmitter-laden vesicles with the cell membrane and release of vesicle contents into the extracellular space.1 Since transmitter is released in discrete vesicle packets, exocytosis is quantal in nature. In the case of endocrine cells, released hormone enters the circulation and is carried to distant tissues to exert its effects. * Corresponding author. Phone: (573) 884-8805. Fax: (573) 884-4232. E-mail:
[email protected]. † Dalton Cardiovascular Research Center. ‡ Department of Biological Engineering. § Department of Medical Pharmacology and Physiology. (1) Breckenridge, L. J.; Almers, W. Nature 1987, 328, 814-817. 10.1021/ac052037d CCC: $33.50 Published on Web 03/02/2006
© 2006 American Chemical Society
Release of the catecholamines dopamine, adrenaline, and noradrenaline from single vesicles can be detected electrochemically using a carbon-fiber electrode placed immediately adjacent to a cell.2-7 During amperometric recording, spikes of faradaic current serve as the signature of quantal exocytosis with each spike denoting the oxidation of the contents of an individual vesicles as it fuses to the surface membrane of the cell underneath the electrode. This powerful technique has become widely used to study the regulation of exocytosis because it allows details of the fusion process, such as the flux of catecholamine through the “fusion pore” that marks the initial contact between the vesicle and the surface membrane, to be monitored. Carbon-fiber amperometry, however, is a slow and labor-intensive technique for the following reasons: (1) electrodes are manually positioned adjacent to the cell surface using micromanipulators while observing the preparation under a microscope, (2) experiments are performed on one cell at a time, and (3) carbon-fiber electrodes are manually fabricated in small lots and need to be cut to expose a fresh surface or replaced frequently. An active research area is the application of microsystems technology to develop “lab-on-a-chip” devices for biophysical and biochemical analysis of cell function.8-13 Such devices offer the promise of high throughput, low unit cost due to mass production, low consumption of reagents, and exploitation of short length scales to achieve higher sensitivity and performance. Examples of cell assays performed on microchips include a disposable (2) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J., Jr.; Viveros, O. H. Proc. Natl. Acad. Sci. U. S. A. 1991, 88, 10754-10758. (3) Chen, T. K.; Luo, G.; Ewing, A. G. Anal. Chem. 1994, 66, 3031-3035. (4) Ciolkowski, E. L.’ Cooper, B. R.; Jankowski, J. A.; Jorgenson, J. W.; Wightman, R. M. J. Am. Chem. Soc. 1992, 114, 2815-2821. (5) Schroeder, T. J.; Jankowski, J. A.; Senyshyn, J.; Holz, R. W.; Wightman, R. M. J. Biol. Chem.1994, 269, 17215-17220. (6) Chow, R. H.; von Ruden, L.; Neher, E. Nature 1992, 356, 60-63. (7) Cans, A. S.; Wittenberg, N.; Eves, D.; Karlsson, R.; Karlsson, A.; Orwar, O.; Ewing, A. Anal. Chem. 2003, 75, 4168-4175. (8) Beebe, D. J.; Mensing, G. A.; Walker, G. M. Annu. Rev. Biomed. Eng. 2002, 4, 261-286. (9) Sia, S. K.; Whitesides, G. M. Electrophoresis 2003, 24, 3563-3576. (10) Su, J.; Bringer, M. R.; Ismagilov, R. F.; Mrksich, M. J. Am. Chem. Soc. 2005, 127, 7280-7281. (11) Fu, A. Y.; Spence, C.; Scherer, A.; Arnold, F. H.; Quake, S. R. Nat. Biotechnol. 1999, 17, 1109-1111. (12) Takayama, S.; Ostuni, E.; LeDuc, P.; Naruse, K.; Ingber, D. E.; Whitesides, G. M. Nature 2001, 411, 1016. (13) Inoue, I.; Wakamoto, Y.; Moriguchi, H.; Okano, K.; Yasuda, K. Lab Chip 2001, 1, 50-55.
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fluorescence-activated cell sorter,11 microchip systems for monitoring nitric oxide release from stimulated pulmonary endothelial cells14 or peritoneal macrophages,15 chips that assay gene expression in individual cells,16 and cell culture microdevices capable of conducting long-term, high-throughput cell-based assays.17 Microchip assays of quantal exocytosis have been introduced over the past few years and include a Pt array on glass,18 a carbonbased electrode on a silicon substrate,19 and work from our laboratory using cell-sized well electrodes consisting of Au electrodes patterned on a silicon substrate.20 A drawback of each of these studies is that they require manual manipulation of cells to place them over the electrodes. Another limitation is that these studies use opaque electrodes, so that it is not possible to optically monitor cells directly on top of the electrodes with an inverted microscope. Here we report patterning micrometer-scale transparent indium tin oxide (ITO) film electrodes on a glass substrate using photolithography and wet etching, to allow simultaneous observation of cells and amperometric detection of quantal exocytosis. ITO represents a material of choice because it exhibits excellent optical transparency, high electrical conductivity, and a wide electrochemical working window.21-23 The ITO-glass substrate was coated with polylysine to facilitate attachment of chromaffin cells to the surface of the electrodes. Cells were loaded onto the electrode through a microfluidic channel fabricated by molding poly(dimethylsiloxane) (PDMS) over photoresist in a soft lithographic process.24 Perfusion of cells with a high-K+ solution resulted in cell depolarization and spikes of oxidative current recorded amperometrically on the ITO electrodes. Analysis of the current spikes indicates that their total charge and time course are consistent with previous measurements of quantal exocytosis of catecholamines using carbon-fiber electrodes. EXPERIMENTAL SECTION ITO Electrode Patterning. Indium tin oxide (In2O3/SnO2)coated glass slides (75 × 25 × 1 mm, film thickness ∼150-300 Å) with a resistance of 70-100 Ω/square were obtained from Sigma (St. Louis, MO). The ITO film was patterned into 20-µmwide stripes terminating in 1 by 1 mm squares at the edge of the slide to allow connection to an amplifier. ITO was patterned following a photolithographic and wet-etching procedure adapted from Zhan et al.21 Briefly, a thick layer (∼10 µm) of AZP 4600 (14) Spence, D. M.; Torrence, N. J.; Kovarik, M. L.; Martin, R. S. Analyst 2004, 129, 995-1000. (15) Goto, M.; Sato, K.; Murakami, A.; Tokeshi, M.; Kitamori, T. Anal. Chem. 2005, 77, 2125-2131. (16) Burns, M. A.; Johnson, B. N.; Brahmasandra, S. N.; Handique, K.; Webster, J. R.; Krishnan, M.; Sammarco, T. S.; Man, P. M.; Jones, D.; Heldsinger, D.; Mastrangelo, C. H.; Burke, D. T. Science 1998, 282, 484-487. (17) Hung, P. J.; Lee, P. J.; Sabounchi, P.; Lin, R.; Lee, L. P. Biotechnol. Bioeng. 2005, 89, 1-8. (18) Dias, A. F. D., G.; Valerro, V.; Yong, M. G.; James, C. D.; Craighead, H. G.; Lindau, M. Nanotechnology 2002, 13, 285-289. (19) Parpura, V. Anal. Chem. 2005, 77, 681-686. (20) Chen, P.; Xu, B.; Tokranova, N.; Feng, X.; Castracane, J.; Gillis, K. D. Anal. Chem. 2003, 75, 518-524. (21) Zhan, W.; Alvarez, J.; Crooks, R. M. J. Am. Chem. Soc. 2002, 124, 1326513270. (22) Qiu, H.; Yan, J.; Sun, X.; Liu, J.; Cao, W.; Yang, X.; Wang, E. Anal. Chem. 2003, 75, 5435-5440. (23) Hayashi, K.; Iwasaki, Y.; Horiuchi, T.; Sunagawa, K.; Tate, A. Anal. Chem. 2005, 77, 5236-5242. (24) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984.
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Figure 1. Schematic depiction of the device. A patterned ITO electrode stripe is depicted in dark gray. The PDMS slab molded to contain a 100-µm-wide microfluidic channel is depicted in light gray. The working electrode is the portion of the 20-µm-wide ITO stripe that intersects the microfluidic channel. The Ag/AgCl reference electrode is placed in the outlet reservoir of the microfluidic channel. Dimensions are not to scale.
positive photoresist (Clariant Electronic Materials, Somerville, NJ) was spin-coated onto the glass slide at 3000 rpm and baked at 65 °C for 3 min and then 95 °C for 3 min on a hot plate. It was next exposed under UV light (KVB-30 exposure box, Kinesten, Taiwan) using a high-resolution (5080 dpi, Printing Service, University of IllinoissUrbana Champaign) transparency film as a photomask. The photoresist was then developed in the recommended developer solution. An acidic solution composed of 5% HNO3 and 20% HCl was used to etch the portion of the ITO that was not protected by photoresist. The slide with patterned ITO electrodes was cleaned with propanol and water, and then a drop of 0.0025% L-polylysine solution (Sigma) was applied to the electrode area. After 20 min, the slide was rinsed with distilled water and then dried. Integration of Electrodes into a Microfluidic Channel. The microfluidic chip consists of a PDMS (Sylgard 184, Dow Corning, Midland, MI) molded slab reversibly sealed to a glass substrate with patterned ITO film electrodes. The device is depicted in Figure 1. Microfluidic channels were formed by molding PDMS over photoresist patterned on a silicon wafer substrate using standard methods.24 Briefly, to form masters for PDMS device construction, a silicon wafer was coated with SU-8 2025 negative photoresist. After baking, another layer of SU-8 2025 was spincoated onto the wafer until the thickness of the photoresist reached ∼100 µm. The fully baked wafer was exposed to light through a transparency film that contained the channel features and then developed. The photomask pattern was laid out using Freehand software (Macromedia Inc. Orem, UT). A high ratio of PDMS monomer to the crossing-linking agent (20:1) was used to facilitate reversible sealing of the slab to the ITO-glass substrate. The prepolymer mixture was degassed under vacuum and poured onto the master. After 1 h of curing at 80 °C, the PDMS was peeled off the master leaving a 100 µm wide by 100 µm deep notch in the PDMS slab. The PDMS slab was then reversibly sealed to the glass-ITO substrate to complete the microfluidic channel. Two reservoirs were also molded into the PDMS slab, the inlet reservoir was used to introduce the cells and to perfuse cells with a solution that stimulates cell secretion, and the outlet reservoir was used to accommodate the ground/reference electrode. Cell Preparation and Solutions. Bovine adrenal chromaffin cells were prepared as described before.25 About 106 cells in 3 (25) Zhou, Z.; Neher, E. J. Physiol. 1993, 469, 245-273.
mL of culture media (Dulbecco’s modified Eagles medium supplemented with 10% (v/v) fetal bovine serum and 1% penicillin/ streptomycin) were seeded onto a 30-mm polystyrene culture dish. Cells were kept in a 37 °C incubator in a humidified environment with 5% CO2 and used 1-3 days after preparation. The standard bath solution for experiments consisted of 150 mM NaCl, 5 mM KCl, 5 mM CaCl2, 2 mM MgCl2, 10 mM glucose,and 10 mM HEPES titrated to pH 7.2 with NaOH. The “high-K+” solution used to stimulate exocytosis consisted of 55 mM NaCl, 100 mM KCl, 5 mM CaCl2, 2 mM MgCl2, 10 mM HEPES, and 10 mM glucose. All reagents were obtained from Sigma, unless otherwise stated. Immediately before use, cells were dissociated from the 30-mm dish using a vigorous wash with the standard bath solution to give a density of ∼106 cells/mL. A syringe pump (picoPlus, Harvard Apparatus. Boston, MA) was used to drive solutions through the microfluidic channel. Electrochemistry. Cyclic voltammetry and constant potential amperometry studies were performed using an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany), and PULSE software (HEKA), A Ag/AgCl wire was used as the ground/reference electrode. Cells and electrodes were observed under an inverted microscope (Nikon TMS, Tokyo, Japan). The analysis of spike widths and areas was performed using software kindly provided by Ricardo Borges (Universidad de La Laguna, Tenerife, Spain).26 RESULTS AND DISCUSSION Microfluidic Chip Design. Microchip devices to assay quantal exocytosis require that cells be placed within several micrometers of the working electrochemical electrode so that catecholamine released from a single vesicle diffuses to the detecting electrode over a short enough time interval so that a recognizable oxidative spike in current is generated. Other desirable features for such a device are a convenient method to exchange the solution surrounding the cell to stimulate exocytosis and a way to conveniently observe cells with a microscope during an experiment. Here we describe a device that implements all three of these features. The basic device consists of a microfluidic channel formed by reversibly sealing a micromolded PDMS slab to a glass slide. The glass slide serves as a substrate for an ITO film that has been patterned into one or more 20-µm-wide stripes that widens near the edge of the glass slide to allow connection to an amplifier. The microfluidic channel (100 µm wide by 100 µm tall) intersects the ITO stripe at a right angle so that the PDMS slab insulates most of the stripe but leaves a 20 by 100 µm working electrode at the intersection of the microfluidic channel and the ITO stripe (Figure 1, also see Spence et al.14). Note that this design does not require precise alignment of the PDMS slab with the glassITO substrate. Characteristics of the ITO Electrode. Figure 2 depicts cyclic voltammograms of the 20 µm by100 µm ITO electrode at a scan rate of 0.6 V/s. The light gray curve represents the background current response in a physiological bath solution (predominantly 150 mM NaCl; see Experimental Section) and is flat, featureless, and symmetric between ∼-0.25 and ∼+1.0 V, which indicates a wide and stable working window. The black curve is the current response when the bath solution includes 100 µM epinephrine, (26) Segura, F.; Brioso, M. A.; Gomez, J. F.; Machado, J. D.; Borges, R. J. Neurosci. Methods 2000, 103, 151-156.
Figure 2. Cyclic voltammetry of the PDMS insulated ITO electrode. The electrode area was 2000 µm2, and the scan rate was 0.6 V/s. The gray trace depicts the background recording from the physiological bath solution whereas the black trace depicts the response when 100 µM/L epinephrine is included in the bath solution. A clear oxidation wave is noted, but the reduction peak is absent.
Figure 3. Current noise of the ITO electrode as a function of electrode area. The standard deviation of the background current was measured at a bandwidth of 2.9 kHz while holding the electrode at 0.7 V versus Ag/AgCl (circles). The area of the working electrode was varied by using various widths of the PDMS microfluidic channel. The best-fit line has a slope of 1.4 fA/µm2.
Note that the epinephrine is oxidized beginning at ∼0.2 V, and the oxidation peak is at ∼0.5 V. A reduction peak is not clearly evident in the voltammogram, presumably due to cyclization reactions that are prominent at physiological pH.27 At potentials more positive than ∼0.8 V, the background current begins to increase, therefore, we use a potential of 0.7 V to measure catecholamine release from cells with amperometry. A similar or identical potential is used to detect catecholamine release from cells with carbon-fiber electrodes. At 0.7 V, the background current of the ITO electrodes in the physiological bath solution is typically
