Amperometric Detection of Quantal Catecholamine Secretion from

resolve spikes in amperometric current corresponding to ... of exocytosis is that release is quantal in that fusion of each vesicle with the plasma me...
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Anal. Chem. 2003, 75, 518-524

Amperometric Detection of Quantal Catecholamine Secretion from Individual Cells on Micromachined Silicon Chips Peng Chen,†,‡ Bai Xu,§ Natalya Tokranova,§ Xiaojun Feng,§ James Castracane,§ and Kevin D. Gillis*,†,‡,|,⊥

Departments of Biological Engineering, Electrical Engineering, and Physiology and Dalton Cardiovascular Research Center, University of MissourisColumbia, Research Park Drive, Columbia, Missouri, 65211, and School of Nanosciences and Nanoengineering, University at AlbanysSUNY, Albany, New York

We have fabricated electrochemical electrodes in picolitersized wells for measuring catecholamine release from individual cells with millisecond resolution. Each wellelectrode roughly conforms to the shape of the cell in order to capture a large fraction of released catecholamine with high time resolution. Using this device, we can resolve spikes in amperometric current corresponding to quantal catecholamine release via exocytosis. In addition, we have combined amperometric recording on the chip with patch-clamp recordings of membrane capacitance as an assay of exocytosis. A quantitative comparison of the two methods suggests that a large fraction of catecholamine release is oxidized on the surface of the wellelectrode. This technology has applications in cell-based biosensor development, high-throughput screening of drugs, and basic science investigations. Endocrine cells store hormone in thousands of membranedelimited vesicles. A rise in intracellular Ca2+ concentration triggers the fusion of vesicles with the outer membrane of the cell and release of hormone in a process called exocytosis. Release of neurotransmitter from nerve terminals also occurs by Ca2+triggered exocytosis of secretory vesicles. One of the hallmarks of exocytosis is that release is quantal in that fusion of each vesicle with the plasma membrane discharges a discrete packet of molecules. Measurement of quantal release provides detailed information about the mechanics and regulation of exocytosis (e.g., refs 1-7) and provides confidence that measured release * Corresponding author: (tel) (573) 884-8805; (fax) (573) 884-4232; (e-mail) [email protected]. † Department of Electrical Engineering, University of MissourisColumbia. ‡ Dalton Cardiovascular Research Center, University of MissourisColumbia. § University at AlbanysSUNY. | Department of Biological Engineering, University of MissourisColumbia. ⊥ Department of Physiology, University of MissourisColumbia. (1) Ales, E.; Tabares, L.; Poyato, J. M.; Valero, V.; Lindau, M.; Alvarez de Toledo, G. Nat. Cell Biol. 1999, 1, 40-44. (2) Chow, R. H.; von Ruden, L.; Neher, E. Nature 1992, 356, 60-63. (3) Jankowski, J. A.; Schroeder, T. J.; Holz, R. W.; Wightman, R. M. J. Biol. Chem. 1992, 267, 18329-18335. (4) Jankowski, J. A.; Schroeder, T. J.; Ciolkowski, E. L.; Wightman, R. M. J. Biol. Chem. 1993, 268, 14694-14700. (5) 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.

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is occurring via regulated exocytosis rather than by cell lysis. Automated and high-throughput assays of exocytosis have potential applications in both drug discovery and basic science. At a synapse, the postsynaptic cell can be used as an exquisitely sensitive biosensor for detecting quantal release from the presynaptic neuron. However, it can be difficult to sort out if modulation of synaptic transmission originates from changes in release from the presynaptic cell or changes in sensitivity of the postsynaptic cell. In addition, synaptic electrophysiology is a slow and laborintensive assay. Electrochemical detection of quantal secretion from individual cells using carbon-fiber microelectrodes has become popular in cases where electroactive substances such as catecholamines are released (see refs 8 and 9 for reviews). With this technique, a carbon fiber with a diameter of ∼10 µm is placed adjacent to a cell. Exocytosis of a vesicle near the surface of the electrode will produce a spike of current as the electroactive contents of the vesicle are oxidized on the electrode surface. One of the limitations of carbon-fiber amperometry is that only release from the fraction of the cell surface that is immediately adjacent to the electrode is recorded with high time resolution. Thus only 210-15%8 of total release is detected. In addition, assays using carbon-fiber electrodes are labor-intensive because the electrodes are individually fabricated and manually positioned adjacent to cells using a microscope and micromanipulators. Microfabrication techniques borrowed from the semiconductor industry offer the potential to build powerful devices for singlecell analysis that can be highly automated and mass produced at low unitary cost (see ref 11 for a review). For example, membrane currents from individual neurons have been indirectly measured using a field effect transistor.12 A number of groups are developing “patch-clamp on a chip” technology to directly measure the current (6) Wang, C. T.; Grishanin, R.; Earles, C. A.; Chang, P. Y.; Martin, T. F.; Chapman, E. R.; Jackson, M. B. Science 2001, 294, 1111-1115. (7) Fisher, R. J.; Pevsner, J.; Burgoyne, R. D. Science 2001, 291, 875-878. (8) Chow, R. H.; Ru ¨ den, L. v. In Single Channel Recording; Sakmann, B., Neher, E., Eds.; Plenum Press: New York, 1995; pp 245-275. (9) Travis, E. R.; Wightman, R. M. Annu. Rev. Biophys. Biomol. Struct. 1998, 27, 77-103. (10) Chan, S. A.; Smith, C. J. Physiol. 2001, 537, 871-885. (11) Cooper, J. M. Trends Biotechnol. 1999, 17, 226-230. (12) Vassanelli, S.; Fromherz, P. J. Neurosci. 1999, 19, 6767-6773. 10.1021/ac025802m CCC: $25.00

© 2003 American Chemical Society Published on Web 12/18/2002

Figure 1. Process flow for fabrication of the well-electrodes. The process is described in detail in the Experimental Section.

through the cell membrane.13-15 Cooper and colleagues have described a microfabricated electrochemical sensor for detecting purine release from individual heart cells16,17 whereas the Ewing laboratory has demonstrated electrochemical analysis in picoliter microvials.18 We have fabricated electrochemical electrodes in picoliter-sized wells using microsystems technology in order to measure quantal secretion from individual cells. The snug fit of the cell in the wellelectrode ensures that electroactive substances released from a large fraction of the surface area of the cell must diffuse only a short distance before they are oxidized on the surface of the electrode. The short diffusion distance is important to resolve quantal release events because an amperometric “spike” in current only occurs if the molecules released from an individual vesicle contact the electrode surface over a relatively short time interval. Using these devices, we have detected quantal release of catecholamine from adrenal chromaffin cells. In addition, we have simultaneously used patch-clamp techniques to measure changes in membrane capacitance as an independent assay of exocytosis. A quantitative comparison of the two techniques demonstrates that most of the catecholamine released from the cell is oxidized on the electrode surface. EXPERIMENTAL SECTION Process Flow. The process for fabricating the electrochemical devices is illustrated in Figure 1. (a) A 500-nm SiO2 layer was deposited by low-pressure chemical vapor deposition (LPCVD) on a 〈100〉 n-type Si wafer. (b) Following patterning of the SiO2, the microwells were created with an anisotropic etch using a 38% KOH solution at 80 °C. (c) Another SiO2 layer was deposited by plasma-enhanced chemical vapor deposition (PECVD) to insulate the bulk silicon substrate. (d) Following coating and patterning of photoresist, Cr (20 nm) and then Au (200 nm) were evaporated onto the substrate. After liftoff with photoresist remover, electrodes, conducting traces, and bonding pads were formed. (e) Deposition of an additional layer of SiO2 by PECVD insulates the conducting traces. (f) SiO2 was removed from the wells and bonding pads by buffered oxide etching. (13) Sigworth, F. J.; Klemic, K. G. Biophys. J 2002, 82, 2831-2832. (14) Klemic, K. G.; Klemic, J. F.; Reed, M. A.; Sigworth, F. J. Biosens. Bioelectron. 2002, 17, 597-604. (15) Fertig, N.; Blick, R. H.; Behrends, J. C. Biophys. J. 2002, 82, 3056-3062. (16) Bratten, C. D.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1997, 69, 253258. (17) Bratten, C. D.; Cobbold, P. H.; Cooper, J. M. Anal. Chem. 1998, 70, 11641170. (18) Clark, R. A.; Hietpas, P. B.; Ewing, A. G. Anal. Chem. 1997, 69, 259-263.

Chip Packaging and Testing. Wafers were diced into 8 × 8 mm chips and mounted in a 24-pin IC chip carrier. Poly(dimethylsiloxane) (PDMS; Dow Corning, Midland, MI) was applied around the edge of the chip in order to insulate the bonding wires and to confine the drop of solution containing cells to the middle of the chip. The appropriate pin of the chip carrier was connected to the headstage of an EPC-9 patch-clamp amplifier (HEKA, Lambrecht, Germany) for amperometric recording. In some experiments, a VA-10 amplifier (NPI electronics, Tamm, Germany) was used instead of an EPC-9. The drop of solution on top of the chip was grounded with a Ag/AgCl wire. A voltage of 0.65-0.7 V versus Ag/AgCl was applied to the active electrode, and the amperometric current was recorded using PULSE software (HEKA). Cells and well-electrodes were observed under an Olympus BX-50WI upright microscope with a 40× water immersion objective lens (0.8 NA). Carbon-fiber electrodes were purchased from ALA Scientific (Westbury, NY). Cells and Solutions. Adrenal chromaffin cells were isolated from bovine adrenal medullas as previously described,19 and ∼106 cells in 5 mL of culture media (Dulbecco’s Modified Eagles Medium supplemented with 10% fetal bovine serum) were placed in a 25-cm2 flask. Flasks were kept in a water-jacketed incubator at 37 °C with 5% CO2 for 1-3 days before use. Immediately before an experiment, the culture medium was removed and the cells were detached from the culture flask by vigorous washing with 2 mL of extracellular solution (defined below). A 70-µL aliquot of the cell suspension was then put on the chip for experimentation. The normal extracellular solution contained the following (in mM): 145 NaCl, 5 KCl, 1 CaCl2, 5 MgCl2, and 10 HEPES titrated to pH 7.2 with NaOH. For patch-clamp experiments, the extracellular solution contained 10 mM CaCl2. The “high-K+” solution used to stimulate exocytosis contained the following (in mM): 145 KCl, 10 CaCl2, 1 MgCl2, and 10 HEPES titrated to pH 7.2 with KOH. For patch-clamp experiments, the pipet solution contained the following (in mM): 150 cesium glutamate, 3 MgCl2, 3 Na4ATP, 0.2 EGTA, and 10 HEPES titrated to pH 7.2 with CsOH. All reagents were purchased from Sigma (St. Louis, MO). Electrophysiology. Patch-clamp electrodes were pulled from borosilicate glass and had a resistance of 3-5 MΩ. An EPC-9 patch-clamp amplifier was used. A sinusoidal voltage with an amplitude of 25 mV and a frequency of 1.5 kHz was applied at a dc potential of -70 mV. Pulse software (HEKA) was used to measure membrane capacitance using the sine+dc technique.20 RESULTS AND DISCUSSION I. Device Fabrication and Testing. We fabricated chips containing arrays of 16 well-electrodes using the process depicted in Figure 1 and described in detail in the Experimental Section. Each well was ∼9 µm deep and had square openings that were either 15 (volume, 0.79 pL), 20, 25, or 30 µm (volume, 5.15 pL) on a side. Figure 2A presents an electron micrograph of a portion of an electrode array whereas an expanded view of an individual well-electrode is depicted in Figure 2B. Amperometry (i.e., recording the faradaic current at a fixed potential) is generally the preferred technique for measuring quantal catecholamine release from individual cells because it offers maximal time resolution. In this case, it is important to (19) Zhou, Z.; Neher, E. J. Physiol. 1993, 469, 245-273. (20) Gillis, K. D. Pflugers Arch. 2000, 439, 655-664.

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Figure 3. Voltammograms measured in a solution containing 100 µM epinephrine and 0.5 M KCl. Squares indicate data obtained from a carbon fiber (5-µm diameter), triangles are average values from three well-electrodes with 15-µm openings, and circles are average values from five well-electrodes with 25-µm openings. The steadystate current was found by sequentially stepping the potential in 50100-mV increments every 30 s and sampling the amperometric current 20 s after the step. The background current in the absence of epinephrine was subtracted from the current in the presence of epinephrine. The currents are normalized to the theoretical limiting current calculated from eq 1 as described in the text.

Figure 2. Scanning electron micrographs of a well-electrode array. (A) Nine of 16 wells on the chip are depicted. The well openings are 15 µm and the spacing between wells is 100 µm. (B) Expanded view of a single well-electrode. Note the SiO2 layer insulates the Au conducting trace, but not the Au within the well.

choose a potential that is significantly positive so that virtually all of the catecholamine that contacts the electrode surface is oxidized. On the other hand, too large a potential can cause undesirable reactions on the electrode surface that obscure the reaction of interest. Therefore, we first measured voltammograms for our devices in the presence of a known concentration of epinephrine (100 µM in 0.5 M KCl). Figure 3 presents voltammograms obtained using normal pulse voltammetry for a commercial, carbon-fiber microelectrode (squares), and our well-electrodes with openings measuring 15 (triangles) and 25 µm (circles). As the potential becomes more positive, the fraction of catecholamine that is oxidized upon contact with the electrode surface increases toward 100% and the faradaic current reaches a limiting value (Ilim). The faradaic current displayed in Figure 3 is normalized to the theoretical value of Ilim calculated for each type of electrode from

Ilim ) KnFDCr

(1)

where K is a coefficient whose value depends on the geometry of the electrode, n is the valence of the reaction (2 for catecholamines), F is Faraday’s constant, D is the diffusion coefficient, C is the concentration, and r is the electrode radius. For a circular 520

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electrode located in an infinite plane, K ) 4,21 whereas K is ∼4.8 for insulated carbon-fiber electrodes prepared according to ref.22 Thus, using D ) 6 × 10-6 cm2/s for epinephrine, the theoretical value of Ilim for a carbon fiber with a diameter of 5 µm is 139 pA. If we assume the limiting current for the well-electrode is approximately equal to that of a circular disk with diameter equal to the opening size, then eq 1 predicts a current of 348 pA for the 15-µm well-electrode and 580 pA for the 25-µm well-electrode. Figure 3 demonstrates that the measured faradaic current for all three types of electrodes appears to approach a saturating value above ∼700 mV versus Ag/AgCl and the value of this saturating current is, in each case, within 12% of the predictions from eq 1. Unfortunately, the voltammogram could not be extended to potentials more positive than +750 mV for the well-electrodes because the background current (measured in the absence of epinephrine) becomes prohibitively large such that the current due to oxidation of epinephrine can no longer be adequately resolved. In addition, we observed the well-electrodes under a microscope during the measurements and observed the Au dissolving at potentials >) 800 mV. Thus we used a potential of +700 mV (and occasionally +650 mV) for amperometric recordings with the Au well-electrodes. In contrast, background currents in carbon-fiber microelectrodes are typically less than several pA at +800 mV. Nevertheless, the drift of the background current of the well-electrodes was quite slow and did not affect the ability to resolve quantal secretory events (see Figures 5 and 6). (21) Bard, A. J.; Faulkner, L. R. Electrochemical Methods: Fundamentals and Applications, 2nd ed.; Wiley & Sons: New York, 2001. (22) Schulte, A.; Chow, R. H. Anal. Chem. 1996, 68, 3054-3058.

(approximately equal to the elementary charge, 1.6 × 10-19 C), and B is the bandwidth of the recording. Using eq 2, the standard deviation of shot noise even for a dc current as large as 1 nA would only be ∼1 pA. The variance due to thermal noise can be described by

σI,t2 ) 4kTGB

Figure 4. Noise (standard deviation) of microelectrodes as a function of surface area. The noise was measured at 0 V vs Ag/AgCl, and the bandwidth of the recording was 3 kHz. The filled circle was measured for a carbon-fiber microelectrode, and the open circles are well-electrodes with openings of 15, 20, and 30 µm. The dashed line is the least-squares fit of the data constrained to go through the origin and has a slope of 0.04 pA/µm2.

Next, we compared the noise of our well-electrodes with carbon-fiber microelectrodes in normal extracellular solution without catecholamine. The current noise (standard deviation) for a 15-µm opening well-electrode was typically 14.5 pA for a 3-kHz bandwidth, whereas an 8-µm diameter carbon-fiber microelectrode has a typical noise of only 1.5 pA under the same conditions. The noise did not depend on voltage over the range of 0 to +700 mV (data not shown). The difference in current noise cannot be explained by thermal or shot noise. The variance due to shot noise can be described by the following equation: 2

σI,shot ) 2IdcqB

(2)

where Idc is the average current, q is the effective carrier charge

(3)

where k is Boltzmann’s constant, T is absolute temperature, and G is the source conductance of the signal, i.e., the slope of the I-V relationship in the absence of catecholamine depicted in Figure 3. Even if we assume a source conductance as large as 100 nS, eq 3 predicts a standard deviation for thermal noise of only 2.2 pA. Instead, the noise of microelectrodes is dominated by the double-layer capacitance of the active electrode surface. This is because the relatively small input voltage noise of the amplifier can result in a large current noise if it is loaded by a large capacitance.23 The active (uninsulated) electrode surface area of our 15-µm well-electrodes is ∼500 µm2, 10-fold greater than that of a 8-µm-diameter carbon-fiber electrode; therefore, the standard deviation noise per unit surface area is comparable for the two types of microelectrodes. Figure 4 is a plot of current noise versus surface area for a carbon-fiber microelectrode and our wellelectrodes with opening of 15, 20, and 30 µm. Note that the relationship is approximately linear with a best-fit slope of 0.04 pA/µm2 (dashed line). II. Detection of Single-Vesicle Fusion Events. We placed a 70-µL drop of solution containing ∼3 × 104 bovine adrenal chromaffin cells on the chip. The cells settled out of the drop at random locations on the chip, occasionally falling into one of the well-electrodes. If none of the well-electrodes contained a cell, then a cell was manipulated into a well with gentle blowing of extracellular solution from an application pipet manipulated near

Figure 5. Quantal release of catecholamines from a single cell measured in a well-electrode. (A) Photograph of a chromaffin cell in a wellelectrode with an opening of 15 µm. Note that an application pipet is visible to the right of the cell. (B) Amperometric trace depicting release of catecholamines in response to local application of a solution containing 4 mM carbamylcholine. Each spike in current represents the oxidation of the contents of a single secretory granule. The electrode potential was 0.7 V vs Ag/AgCl. (C) Response of a different cell to local application of the “high-K+” solution defined in the Experimental Section. (D) Histogram of 152 spike areas compiled from six cells stimulated with the high-K+ solution.

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Figure 6. Well-electrodes capturing a large fraction of catecholamine released from the cell. (A) Schematic depiction of a recording in a well-electrode. “P” denotes the patch-clamp pipet attached to the cell in the whole-cell recording configuration. A1 represents the voltage-clamp amplifier (I-V converter) used to measure the well-electrode current with the voltage fixed to +700 mV relative to a Ag/AgCl electrode placed in the bath solution. A2 is the patch-clamp amplifier used to record the cell membrane current in response to a variable pipet command potential (Vp). The patch-clamp recording allows direct stimulation of the cell via membrane depolarization and measures increases in membrane capacitance (Cm) that result from fusion of secretory granules with the plasma membrane. (B) Recording from a cell in a well-electrode. Two depolarizing pulses, 100 ms in duration from -70 to +10 and +15 mV are applied. The resulting current due to Ca2+ influx is depicted in the bottom trace (ICa). Ca2+ influx leads to exocytosis, and increases in Cm (middle trace) and faradaic current (IF) recorded amperometrically in the well-electrode as catecholamine is oxidized on the electrode surface (upper trace). The integral of IF (QF) is also plotted in the middle trace and is scaled to the equivalent vesicle content by assuming 3 × 106 catecholamine molecules/vesicle (∼1 pC). Cm is scaled by assuming a single vesicle increases Cm by 1.3 fF. (C) Recording from a cell using a 10-µm-diameter carbon-fiber electrode. Conditions are identical to those in (B). Note that IF and QF are much smaller than observed in the well-electrode despite a greater amount of stimulated exocytosis (∆Cm). (D) Summary of results (mean ( SEM) with the number of cells given by the n value in the figure. The dashed line indicates the expected ratio of 0.75 pC/fF if 100% of released catecholamine is oxidized on the electrode surface.

the chip surface. A cell in a well-electrode is depicted in Figure 5A. The cells were stimulated to secrete by gently blowing extracellular solution containing 4 mM carbamylcholine (CCH) from an application pipet located ∼5 µm from the cell. CCH opens nicotinic acetylcholine-gated ion channels and depolarizes the cell membrane. Membrane depolarization, in turn, opens voltage-gated Ca2+ channels, and the resulting influx of Ca2+ triggers exocytosis. Figure 5B presents an amperometric recording from a wellelectrode, typical of five experiments, illustrating quantal release events that are similar in amplitude, time course, and area as previously reported.3-5 The spike area is proportional to the amount of catecholamine detected per release event such that 1 pC of charge corresponds to ∼5.2 amol of catecholamine. The average spike area for CCH stimulation was 2.98 pC with a standard deviation of 2.16 pC (n ) 35). In a separate set of experiments, secretion was triggered by applying a “high-K+” solution to depolarize the cell. Figure 5C presents a sample trace, representative of 13 cells, illustrating K+triggered quantal secretion. In these experiments, the spike area 522 Analytical Chemistry, Vol. 75, No. 3, February 1, 2003

was 2.27 ( 1.87 pC (n ) 152). Figure 5D presents a histogram of spike areas for this set of experiments. The distribution of spike areas is similar to that reported for experiments using carbonfiber microelectrodes.3,5 As explained previously, the noise of our measurements is somewhat higher than that those of carbon-fiber microelectrodes, so it is likely that we missed some of the smaller quantal events. This likely explains why our mean spike area is somewhat larger than the range of 1-2 pC that is typically reported for carbon-fiber microelectrodes (e.g., refs 2, 3, 5, and 24). We also saw quantal events in response to direct voltage-clamp depolarization in whole-cell patch-clamp recordings of cells in wells (Figure 6B). In contrast, amperometric spikes were not observed while control extracellular solution was blown on the cells as the cells manipulated into the well-electrodes (data not shown) or while the cells were held at -70 mV during whole-cell patch-clamp recording (Figure 6B,C). III. Well-Electrodes Capture a Large Fraction of Released Catecholamines. We performed whole-cell patch-clamp recordings on cells in well-electrodes in order to estimate the fraction

of released catecholamines that is oxidized on the Au surface. The recording configuration is schematically depicted in Figure 6A. The potential across the cell membrane is under direct voltageclamp control during these recordings, and the current due to Ca2+ influx (ICa) elicited by depolarizing pulses is recorded. In addition, the capacitance of the cell membrane (Cm) can be measured by applying a sinusoidal voltage and analyzing the resulting sinusoidal current (see ref 25 for a review). Exocytosis increases Cm because vesicle fusion increases the surface area of the cell membrane. Figure 6B,C presents sample experiments depicting changes in Cm, ICa, and amperometric current (IF) in response to a pair of depolarizing pulses. The integral of the amperometric current (QF), which is proportional to the number of molecules oxidized on the surface of the microelectrode, is also indicated in the figure as a dashed line. Note that ICa and the increase in Cm are similar for experiments using the well-electrode (Figure 6B) and the carbon-fiber microelectrode (Figure 6C), but the amount of oxidized catecholamine (QF) is much larger for the wellelectrode. Figure 6D summarizes the results from these experiments and indicates that a ∼4-fold larger fraction of released catecholamine (expressed as the normalized ratio QF/∆Cm) is captured by the well-electrode compared to the carbon-fiber microelectrode. Note that the amperometric current of Figure 6B does not show clearly resolvable spikes such as depicted in Figures 5 and 6C. This likely results from diffusional broadening of spikes because the cell is not directly adjacent to the electrode. The fraction of released catecholamine that can be clearly resolved as quantal events critically depends on how tightly the cell fits into the well-electrode. In the experiments of Figure 6B, the cell is suspended in the well-electrode by the patch-clamp pipet whereas the cells in the experiments of Figure 5 are directly adjacent to the electrode because they are seated in the bottom of the well. A crude estimate of the absolute fraction of released catecholamine that is oxidized on the surface of the well-electrode can be made. The estimate of the mean increase in Cm elicited by fusion of an individual chromaffin granule ranges from about 1.3 26 to 2.7 fF27 whereas individual amperometric spikes have a mean charge that is generally reported to be between 1 and 2 pC (e.g., refs 2, 3, 5, and 24). Recordings using the cell-attached patchclamp configuration can resolve unitary increases in Cm in response to fusion of individual vesicles into the patch of membrane beneath the pipet. In addition, individual amperometric spikes can be correlated with unitary Cm increases if a carbonfiber microelectrode is placed inside the patch-clamp pipet. “Patchamperometry” recordings of this type indicate that the mean ratio of amperometric charge to Cm increase (QF/∆Cm) is ∼0.75 pC/ fF under conditions where essentially all of the catecholamine released into the pipet is oxidized by the carbon-fiber microelec(23) Sigworth, F. J. In Single-Channel Recording, 2nd ed.; Neher, B. S. a. E., Ed.; Plenum Press: New York, 1995; pp 95-127. (24) Albillos, A.; Dernick, G.; Horstmann, H.; Almers, W.; Alvarez de Toledo, G.; Lindau, M. Nature 1997, 389, 509-512. (25) Gillis, K. D. In Single-channel recording, 2nd ed.; Sakmann, B., Neher, E., Eds.; Plenum Press: New York, 1995; pp 155-197. (26) Moser, T.; Neher, E. Proc. Natl. Acad. Sci. U.S.A. 1997, 94, 6735-6740. (27) Chow, R. H.; Klingauf, J.; Heinemann, C.; Zucker, R. S. Neuron 1996, 16, 369-376.

trode.1 In this case, our average value of 0.60 pC/fF indicates that roughly 80% of catecholamine released over the entire surface of the cell is oxidized on the surface of the well-electrode. In contrast, this analysis suggests that our best recordings with carbon-fiber electrodes (Figure 6) capture ∼18% of released catecholamine. This value is consistent with previous estimates of 210-15%8 capture and a geometric calculation that a 10-µm-diameter fiber covers 15% of the surface area of a cell with diameter of 13 µm. Decreases in membrane surface area by endocytosis sometimes follow the increases in surface area by exocytosis. Thus, Cm measurements actually report the difference between the rates of exocytosis and endocytosis. Rapid endocytosis is generally not observed in response to mild depolarizing stimuli such as used in the experiments depicted in Figure 6.28 In addition, slow (“compensatory”) endocytosis is usually only observed in the “perforated patch” configuration, not in the whole-cell recording configuration.28 Nevertheless, there is a possibility that a slow rate of endocytosis is masked by simultaneous exocytosis. In this case, the comparison of Cm and QF presented above will tend to overestimate the fraction of catecholamine release that is captured by an electrochemical electrode. Endocytosis should have little or no effect on the comparison between the carbon fiber and well-electrode because the experiments are performed under the same conditions. As noted previously, we believe that the simplest and most plausible explanation for why the faradaic current of Figure 6B does not show clearly resolved spikes is the relatively large distance that catecholamines must diffuse before contacting the electrode surface when the cell is suspended in the well by a patchclamp pipet. However, another possibility is that the well-electrode is capturing oxidizable substances that are released slowly or via processes other than exocytosis in response to membrane depolarization that are not resolved in carbon-fiber experiments. If this were the case, then this will also lead to an overestimation of the fraction of catecholamine that is captured by the well-electrode. CONCLUSION Our well-electrodes achieve high sensitivity and time resolution by effectively concentrating released hormone in picoliter-sized vials. In addition, the well-electrodes capture a much larger fraction of released catecholamine than carbon-fiber microelectrodes. Since release of catecholamines is not uniform across the cell surface,29 the well-electrode should give a more representative measure of total cell secretion than carbon-fiber microelectrodes. Also, the microchip approach is highly scalable and has the potential to be automated, whereas carbon-fiber electrodes must be manually fabricated and manipulated to the cell surface. It is likely that using carbon rather than Au as the electrode material could reduce the offset current of the device. In addition, a method for automatically positioning cells within the wellelectrodes is necessary in order for microdevices of this type to (28) Smith, C.; Neher, E. J. Cell. Biol. 1997, 139, 885-894. (29) Robinson, I. M.; Finnegan, J. M.; Monck, J. R.; Wightman, R. M.; Fernandez, J. M. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 2474-2478.

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be truly practical for high-throughput assays. We are currently testing a second-generation design for tackling this problem.

reading of the manuscript. This work was supported by the National Science Foundation “XYZ on a chip” program (BES 0089018).

ACKNOWLEDGMENT

Received for review May 24, 2002. Accepted October 17, 2002.

We thank Xiuzhi Tang for preparing chromaffin cells, Zaichun Feng for helpful discussions, and Tzyh-Chang Hwang for critical

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AC025802M