Cylindrically Etched Carbon-Fiber Microelectrodes for Low-Noise

The most important sources of noise with disk-shaped carbon-fiber microelectrodes (CFMEs) are the exposed cut disk face of the fiber itself and the se...
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Anal. Chem. 1998, 70, 985-990

Cylindrically Etched Carbon-Fiber Microelectrodes for Low-Noise Amperometric Recording of Cellular Secretion Albert Schulte†,‡ and Robert H. Chow*,‡

Department of Molecular Biology of Neuronal Signaling, Max Planck Institute for Experimental Medicine, Hermann Rein Strasse 3, 37075 Goettingen, Germany, and Department of Physiology, University of Edinburgh Medical School, Teviot Place, Edinburgh EH8 9AG, Scotland

The most important sources of noise with disk-shaped carbon-fiber microelectrodes (CFMEs) are the exposed cut disk face of the fiber itself and the seal region between the carbon fiber and the applied insulating layer. To reduce noise and to fabricate simple, reproducible lownoise CFMEs, we sealed commercially available carbon fibers in pulled glass pipets and then we performed cylindrical etching of the fiber extending beyond the glass sheath, followed by insulation with anodic electrophoretic deposition of paint. The resulting CFMEs had electroactive carbon disks with radii as small as ∼0.5 µm. The noise of such electrodes was minimized by virtue of a design that ensures a good seal between the carbon fiber and its insulation and a reduced diameter of the exposed carbon. In contrast to CFMEs made of conically etched carbon fibers, cylindrically etched CFMEs offer the significant advantage that they can be easily reused: The cylindrically etched region extends over several hundreds of micrometers and, therefore, can be cut back repeatedly to expose a fresh carbon surface of uniform diameter. The low noise and small size of these electrodes make them ideal for the high-sensitivity measurements demanded in studies of single-vesicle transmitter release from secretory cells. Furthermore, the small cross-sectional diameter of the tips allows them to be used in restricted spaces, such as inside the tapering micrometer-diameter tips of melted and pulled glass microcapillaries (e.g., patch pipets).

are large and contain millions of molecules, permitting the resolution of fine kinetic details of single secretion events, for example, the trickling release of a few thousand molecules of transmitter out of individual secretory vesicles (the so-called “foot signal”) during the early stages of vesicle fusion to the cell surface membrane.2 The main goal of the work in this paper was to design a diskshaped CFME which is optimized for low-noise recordings of vesicular neurotransmitter release. We examine the relationship between the noise, the capacitance, and the radius of the electroactive carbon disk in CFMEs and show how the noise and capacitance decreases for smaller radii. Then we introduce a method for simple, reproducible fabrication of CFMEs featuring low noise, high sensitivity, and small size. The major advance is the use of cylindrical electrochemical etching to generate carbon fibers having radii of less than 1 µm. Several methods for etching carbon fibers have been previously published, including flame etching, electrochemical etching (in both acidic and in basic electrolytes), and argon ion beam etching.3-12 All these methods yield “cone-shaped” carbon-fiber tips, but the tapering tip shapes are often poorly reproducible. Furthermore, to expose a small disk surface with a conically etched carbon fiber, it is necessary to insulate the entire cone and then cut or bevel back the narrowed tip region. Repeated cutting or beveling of such an electrode leads to the exposure of progressively larger cross sections of carbon, which leads to progressively higher electrical noise.

Use of low-noise amplifiers and attention to the details of microelectrode manufacture will be needed to advance the electrochemical measurements of the minute signals arising from the detection of neurotransmitter or hormone secretion from animal cells. The source of such secretion is tiny soap-bubblelike vesicles inside the cell, which may contain as few as 5000 electroactive molecules.1 The pulselike release of the molecules from one of these vesicles can be monitored with a carbon-fiber microelectrode (CFME) and yields a redox charge integral that can be in the femtocoulomb range.1 In some cells, the vesicles

(2) Chow, R. H.; von Rueden, L.; Neher, E. Nature 1992, 356, 60-63. (3) Schulte, A. Ph.D. Thesis, University of Muenster, Muenster, Germany, 1993. (4) Besenhard, J. O. International Meeting on Ion-Selective Electrodes, Shanghai, 1985. (5) Besenhard, J. O.; Kurtze, A.; Sauter, R.; Josowics, M.; Liess, H. D.; Potje, K. Carbon ’86; Deutsche Keramische Gesellschaft: Bad Honnef, Germany, 1986; p 417. (6) Besenhard, J. O.; Schulte, A.; Schur, K.; Jannakoudakis, A. D. In Microelectrodes: Theory and Applications; Montenegro, M. I., et al., Eds.; Kluwer Acad. Publ.: Dordrecht, The Netherlands, 1991; pp 189-204. (7) Strein, T. G.; Ewing, A. G. Anal. Chem. 1992, 64, 1368-1373. (8) Armstrong-James, M.; Fox, K.; Millar, J. J. Neurosci. Methods 1980, 2, 433434. (9) Stamford, J. A. Anal. Chem. 1986, 58, 1033. (10) Meulemans, A.; Poulain, B.; Baux, G.; Tauc, L.; Henzel, D. Anal. Chem. 1986, 58, 2088-2091. (11) Abe, T.; Itaya, K.; Uchida, I. Chem. Lett. 1988, 399. (12) Zhang, X.; Zhang, W.; Zhou, X.; Ogorevc, B. Anal. Chem. 1996, 68, 33383343.

* Corresponding author: (phone) +44-131-650-3259; (fax) +44-131-650-6527; (e-mail) [email protected] or [email protected]. † Max Planck Institute for Experimental Medicine. ‡ University of Edinburgh Medical School. (1) Bruns, D.; Jahn, R. Nature 1995, 377, 62-65. S0003-2700(97)00934-7 CCC: $15.00 Published on Web 01/21/1998

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Cylindrical electrochemical etching, in contrast, yields carbon fibers having uniformly narrowed cross-sectional diameters. Such fibers can be insulated for use in CFMEs and offer the significant advantage, compared to conically etched CFMEs, that they can be cut repeatedly to expose a fresh carbon surface of nearly identical cross-sectional area. Cylindrically etched electrodes are suitable for voltammetric detection of very small amounts of electroactive substances (as in the case of vesicular transmitter release) or for studies performed in extremely minute volumes and restricted spaces, for example, the tapering micrometerdiameter tip of melted and pulled glass microcapillaries (e.g., patch pipets, as in patch amperometry13). EXPERIMENTAL SECTION Materials. Commercially available polyacrylonitrile (PAN)and mesophase pitch-based carbon fibers with circular cross sections were used to manufacture the CFMEs. The radius (mean ( SD), as determined by optical and electron microscopy, of the pitch-based P100S fibers (Amoco Performance Products, Inc., Greenville, SC) was 5.1 ( 0.4 µm; of the polyacrylonitrile (PAN)based T650 fibers (Amoco Performance Products, Inc., Greenville, SC), 2.7 ( 0.2 µm; and of PAN-based HM12 fibers (Sigri GmbH, Meitingen, Germany), 3.7 ( 0.3 µm. Prior to use, sizing compound was removed from the carbon fibers by either boiling them overnight in acetone in a Soxhlet extractor or heating them at ∼400 °C for 6 h. Single carbon fibers were attached to the ends of 0.5-mmdiameter copper wires using a conductive carbon paste (Electrodag 5513, Acheson Colloids, Scheemda, Netherland). Anodic electrophoretic deposition paint (EDP, Canguard, BASF Farben und Lacke GmbH, Muenster, Germany) was used for electrical insulation of the carbon filaments. Glass microcapillaries (borosilicate glass, type 7052, 1.15-mm i.d., 1.55-mm o.d., 7.5 cm long) were obtained from Garner Glass Co. (Claremont, CA). The two-component silicon elastomer Sylgard was obtained from Dow Corning (Midland, MI). K3Fe(CN)6, dopamine hydrochloride, Na2HPO4, NaH2PO4, KCl, NaCl, HCl, and NaOH were obtained from Sigma Chemical. All chemicals were of reagent-grade purity and used without further purification. Aqueous solutions were prepared using doubledistilled, deionized water. Electrochemical measurements of ferricyanide were performed with solutions of K3Fe(CN)6 in 0.5 M KCl at pH 3, to take advantage of the known diffusion coefficient for ferricyanide in these conditions.14 Dopamine was dissolved in a buffer containing 60 mM Na2HPO4, 30 mM NaH2PO4, and 100 mM NaCl, adjusted to pH 7.4 with 1 M NaOH. Apparatus. A horizontal glass micropipet puller (Model P-97, Sutter Instruments Co., Novato, CA) was used to pull the carbonfiber/glass capillary assemblies. Anodic electrophoretic deposition of paint was performed in a one-compartment cell at room temperature using a conventional laboratory dc power supply. A sweep/function generator (model 19, Wavetec, San Diego, CA) was used for electrochemical etching of the carbon fibers. Scanning electron microscopy (SEM) was performed on a Hitachi S4500II (Hitachi, Tokyo, Japan) and on a Cambridge Stereoscan (13) Albillos, A.; Dernick, G.; Horstmann, H.; Almers, W.; Alvarez de Toledo, G.; Lindau, M. Nature 1997, 389, 509-512. (14) Kawagoe, K. T.; Jankowski, J. A.; Wightman, R. M. Anal. Chem. 1991, 63, 1589-1594.

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250 (Leo Electron Microscopy Ltd., Cambridge, U.K.). All microelectrode experiments were performed at room temperature in a one-compartment electrochemical cell in a twoelectrode configuration. The CFMEs were mounted on the headstage of a computer-controlled patch-clamp amplifier (HEKA EPC-9, HEKA Elektronik, Lambrecht, Germany), located on a vibration-damping table in a Faraday cage. The EPC-9 allows highgain current measurements with low noise and high-frequency response. The bath reference/counter electrode was either a calomel reference electrode or a silver/silver chloride pellet. The voltage ramps used for the cyclic voltammograms were generated by the pulse generator system of the EPC-9 system, and data acquisition was performed with the built-in ITC-16 of the EPC-9 amplifier controlled by a Power Macintosh 8100/100 (Apple Computer GmbH, Ismaning, Germany) running the program Pulse (HEKA Elektronik). Analysis was performed with the program IGOR (Wavemetrix, Lake Oswego, OR), using programs written by us. The root-mean-square (rms) noise of the CFMEs was measured with the electrode tip above the bath and with the tip just immersed. The rms noise was directly read off the EPC-9 amplifier. The filter was an analogue four-pole low-pass bessel filter with variable, computer-controlled corner frequency, and the amplifier gain was 20 mV/pA, selecting for a 500-MΩ feedback resistor in the head stage current-to-voltage converter. The noise variance was calculated by squaring the noise, and the excess noise variance was calculated by subtracting the noise variance measured with the electrode held just above the bath from the variance measured with the tip just immersed. The corner frequency of the low-pass filter was routinely set to 3 kHz. The capacitance of the CFMEs was determined by applying 10-mV step depolarizations to the electrode, integrating the obtained current transients, and dividing by the amplitude of the voltage step. The capacitance attributable to the cut face of the electrode (“excess capacitance”) was calculated by subtracting the capacitance measured with the tip above the bath (due to the amplifier input and stray capacitances) from the capacitance with the tip just immersed. Microelectrode Construction. Figure 1 is a schematic showing the design of and stages in the manufacture of the CFMEs. A 3-cm-long bundle of the multifiber tow of the carbon fibers of choice was placed onto a clean sheet of white paper. An individual fiber was withdrawn from the bundle by attaching it to the tip of a dissecting needle covered with a tiny dab of paper glue. The filament was then attached to the end of a 5-cm-long piece of the copper wire using a small drop of carbon paste to create an electrically conductive junction, the paste was allowed to dry, and the fiber was scalpel-trimmed so that it extended only ∼1 cm beyond the end of the copper wire. The free end of the copper wire was then cannulated into a borosilicate glass tube until the carbon/copper junction was positioned approximately in the middle of the tube and the free end of the copper wire extended out one end of the capillary. The copper wire was fixed in this position relative to the glass tube with a drop of a quicksetting two-component epoxy glue. The epoxy glue was allowed to cure until firm. The carbon-fiber-containing capillaries were placed in the micropipet puller. Pulling resulted in two glass pipets, one of which was waste, and the other of which had the carbon fiber

Figure 1. Schematic of the design and steps in the fabrication of cylindrically etched CFMEs (not shown to scale): (A) design of the CFME; (B) electrode arrangement for EDP insulation; (C) cutting of the EDP-insulated carbon fibers; (D) electrode arrangement for etching.

protruding from its tip. The parameters of the pulling program (a series of heating and pulling steps) were chosen so that the glass at the pipet tip contracted around the carbon fiber to produce a water-tight carbon-to-glass seal (although a water-tight seal is more convenient, it is not essential; see below). The protruding fiber was trimmed to a length of ∼2 mm and carefully rinsed first with a stream of 70% ethanol and then with distilled water. At this stage, the basic CFME construction was complete. The remaining steps were insulation of the carbon fiber extending out of the glass tip or etching of the carbon fiber and then insulation. For CFMEs without etched tips (cylindrical etching is described below), the protruding carbon fiber was insulated with an organic polymer using the anodic electrophoretic deposition of paint, as previously published.15 Briefly, the pulled glass pipet/ carbon fiber assembly was mounted on a coarse micromanipulator and the copper wire extending out the back of the pipet was connected to a dc power supply. The carbon fiber was lowered into the EDP solution until the glass tip just entered (see Figure 1B). A constant voltage of 4 V was applied for 1-2 min between the carbon fiber (anode) and a platinum counter electrode (cathode). Coated (electropainted) carbon fibers were heat-cured (15) Schulte, A.; Chow, R. H. Anal. Chem. 1996, 68, 3054-3058.

at 194 °C for at least 2 min. Typically, the concentric EDP coating was very uniform along the entire length of the insulated carbon fiber and without any defects in the polymer/carbon-fiber seal. The thickness of the polymeric insulation was estimated to be ∼1 µm for the specified deposition time and voltage. Thus, the smallest possible overall tip dimension of electropainted CFMEs made from unetched T650 carbon fibers was found to be ∼7 µm in diameter. Note that, if the glass-carbon seal is not water-tight, then some EDP solution may creep into the glass tip during the insulation procedure. Heat curing may then lead to a small bubble at the glass tip which can cause defects in the insulation at this region. This was not a serious problem, as Sylgard was applied routinely to the glass tip region after heat curing of the EDP insulation to ensure the water-tight seal (See Figure 1A). The electrode was mounted horizontally on a piece of modeling clay on a glass microscope slide, and the glass tip region was visualized under a dissecting microscope. At the same time, a piece of fine-gauge wire was mounted horizontally on another slide with a piece of modeling clay, at the same height as the pipet tip and with a small hook at the end of the wire. A small drop of Sylgard was applied to the hook in the wire. This drop was visualized under the microscope, and the glass/carbon fiber junction of the pipet was maneuvered into the drop to apply a thin coating. Curing of the silicone elastomer was accomplished at 194 °C for 2-5 min. Prior to each experiment, the electrode was cut to reveal a fresh carbon surface (see Figure 1C). The electrode was positioned, using modeling clay, nearly parallel to the surface of a microscope slide onto which a piece of cellophane single-sided adhesive tape was attached. The electrode tip was pushed downward until the carbon fiber gently made contact with the tape/slide surface at a very low angle. Under microscopic visualization, the electropainted carbon fiber was transected perpendicular to the longitudinal axis using a hand-held curved scalpel blade. The cellophane tape served to reduce slippage of the blade on the microscope slide and to cushion the fiber during the transection. The cut end was inspected at high magnification for irregularities. The cut end was generally near-planar, but if not, the procedure was repeated. As the carbon fibers extended ∼2 mm out of the glass tips, recuttings were possible to generate fresh electrode surfaces for multiple measurements. Cylindrical Electrochemical Etching. To produce carbon fibers with radii smaller than those of commercially available fibers, we developed a procedure for cylindrical etching. The twoelectrode etching setup (see Figure 1D) consisted of the carbon fiber to be etched and a platinum counter electrode made from a piece of platinum foil that was partially rolled up to give a “U”shaped cross section (4 mm in diameter and 1 cm in length). The platinum counter electrode was positioned under a dissecting microscope, and the trough was filled with 0.01 N NaOH solution. The carbon fiber extending out from the glass tip of the electrode was maneuvered into the electrolyte solution, under microscopic observation, taking care not to immerse the glass-carbon junction. Both the platinum counter electrode and the carbon-fiber working electrode were connected to a function generator that applied a periodic square wave potential. We have investigated various settings and conditions for the etching (electrolytes, frequencies and amplitudes of the square wave, cathodic or anodic Analytical Chemistry, Vol. 70, No. 5, March 1, 1998

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offsets). The most uniform (“cylindrical”) etching was obtained in 0.01 N NaOH with a frequency of 45 Hz, an amplitude of 3.9 V, and with no cathodic or anodic offset. In these conditions, it took ∼4 min to etch the PAN-based carbon fibers to radii of ∼0.5 µm and 6-8 min to etch the pitch-based carbon fibers to radii of ∼2.5 µm. To help determine the treatment duration giving the desired degree of etching, visual inspection of the carbon fiber during trial runs of etching is recommended, as etching too long results in narrowing of the shaft to such an extent that the fiber spontaneously starts to bend and ultimately breaks. The cylindrically etched carbon fibers were then insulated using anodic deposition of paint, however, in a slightly different way from that described above for the unetched carbon fibers. Instead of applying the voltage of 4 V instantaneously, the voltage was manually increased slowly from 0 to 4 V over ∼20 s, always maintaining the current below 0.5 µA, and then held at 4 V for another 30 s. Limiting the maximal current was necessary to ensure only a thin layer of insulation and to avoid the formation of beads of EDP. After heat curing at 194 °C, the cylindrically etched, EDP-insulated CFMEs were treated with Sylgard as described above, to ensure a liquid-tight seal at the junction between the glass and the EDP-insulated carbon fiber, and then cut to expose the electroactive carbon disk. RESULTS AND DISCUSSION In a previous study we showed that the principal source of electrical noise in an electropainted 10-µm-diameter disk-shaped CFME is the exposed cut surface of the carbon fiber at the microelectrode tip.15 The insulated cylindrical surface of this CFME contributed to the total noise in proportion to the length immersed into solution; however, this noise was found to be negligible compared to that from the cut disk face of the 10-µmdiameter carbon fiber. We were motivated to quantify the relationship between the capacitance, the noise, and the radius of the electroactive carbon disk in disk-shaped CFMEs, to help us design lower noise CFMEs. Unetched Carbon Fibers. We started by examining CFMEs made from unetched carbon fibers. The commercially available carbon fibers with which we worked had radii (see materials) of 2.7, 3.7, and 5.1 µm. As shown in Figure 2A, the excess capacitance of unetched CFMEssillustrated as solid circlesswas approximately linearly proportional to the cross-sectional area of the fibers (see below, for further details of the unfilled symbols). The rms noise of uncut and cut CFMEs held above the bath solution was independent of the diameter of the carbon fiber and was found to be ∼360 fA at 3-kHz bandwidth (giving a noise variance of 0.13 × 10-24 A2). The rms noise of fully insulated (uncut) CFMEs did not change significantly upon immersion just into the bath, regardless of the carbon-fiber diameter. After cutting the tip, however, the total rms noise of immersed electrodes decreased dramatically with decreasing carbon-fiber radius. The solid symbols in Figure 2B represent the measured values of the unetched fibers as a function of the carbon-fiber radius. The excess noise variance of the unetched carbon fiber tips (calculated as indicated in Apparatus) also decreased when plotted against the square of r (solid symbols in Figure 2C). Cylindrically Etched Carbon Fibers. Given the marked reduction in the capacitance and noise of CFMEs obtained by using the commercially available carbon fibers of smaller diam988 Analytical Chemistry, Vol. 70, No. 5, March 1, 1998

Figure 2. (A) Excess capacitance of the cut face of CFMEs as a function of the carbon-fiber surface. Solid symbols, unetched CFMEs; hollow symbols, etched CFMEs; dashed line, linear fit. (B) Total rms noise vs carbon-fiber radius. The total noise decreases as the radius decreases, until the amplifier noise becomes predominant. Solid symbols, unetched CFMEs; hollow symbols, etched CFMEs; dashed curve, fit by hand. (C) Excess noise variance vs square of carbonfiber radius. Solid symbols, unetched CFMEs; hollow symbols, etched CFMEs. A line has been fitted to the initial four points, to emphasize the supralinear relationship between the excess variance and the surface area of the disk face of the CFME (each point in (A-C) is the mean of six or more measurements; ( bars indicate the standard deviation, except where the bars are obscured by the symbol).

eters, we introduced cylindrical electrochemical etching to obtain uniform narrowing of carbon fibers. Cylindrical etching as described in the Experimental Section was successful on all of the carbon-fiber types used in this study. However, the T650 was found to be the most convenient for making submicrometer-sized CFMEs because it etches more reproducibly, and because less etching is required to obtain micrometer and submicrometer tips. For etching, we used a dilute alkaline solution, which has the advantage that it minimizes intercalation damage of the highly oriented carbon fibers. Scanning electron micrographs of carbon fibers that have been etched but not insulated or else insulated with EDP are shown in Figures 3 and 4, respectively. Etching was generally uniform along the length of the carbon fiber exposed to the etching

Figure 3. Scanning electron micrographs of T650 carbon fibers, before and after etching: (A) unetched fiber; (B, C) cylindrically etched fiber at two magnifications.

Figure 5. Cyclic voltammograms using unetched and etched EDPinsulated CFMEs. The top panel shows cyclic voltammograms of potassium ferricyanide (1 mM), scan rate 0.1 V/s, using an unetched (A) or etched (B, C) CFMEs. The lower panel shows cyclic voltammograms of dopamine (100 µM), scan rate 0.1 V/s using an unetched (D) or an etched (E) CFME. (A) and (D) were both performed with the same CFME, which was made with an unetched T650 carbon fiber having a radius of ∼2.7 µm. (B) and (E) were both performed with the same CFME, which was made with an etched T650 carbon fiber having a radius of ∼1.6 µm. (C) was performed with the CFME shown in Figure 4, which has a radius of ∼0.6 µm. Figure 4. Scanning electron micrographs of cylindrically etched and electropainted CFMEs: (A) tapered region that arises at the air/water interface during etching; (B, C) the tip region at two magnifications.

solution (Figure 3). Note that the carbon-fiber surface was relatively smooth, with no cracks or pits, although there appear to be small carbon particles adhering to the surface in this electrograph. In a typical cylindrically etched CFME, the carbon fiber extends ∼2 mm beyond the glass tip and maintains its linear shape, showing that the mechanical strength of the carbon-fiber structure is reasonably well preserved. The radius of the etched T650 fiber in Figure 3 was estimated to be ∼0.3 µm. At the air/water interface, the carbon-fiber diameter tapered down from the original diameter of the unetched fiber (5 µm) to the narrower diameter of the more uniformly etched region. The tapering region for an EDP-insulated fiber is shown in Figure 4A. The etched, EDP-insulated region had an overall tip diameter (including insulation) of ∼1.4 µm. It is possible to make CFMEs having submicrometer diameters (not shown). The capacitance of the cut face of CFMEs made from cylindrically etched carbon fibers is illustrated as a function of the carbon-fiber surface in Figure 2A (unfilled symbols). As expected, the points for the etched and unetched carbon fibers fall on the same line (linear correlation coefficient r ) 0.990), yielding a specific capacitance of 27.1 µF/cm2. The noise and the excess noise decrease with decreasing carbon fiber radius; however, unlike excess capacitance, both appear to approach a lower asymptotic value (unfilled symbols in Figure 2B and C). The asymptotic lower limit in the total rms noise was ∼380 fA

rms, similar to the noise measured when the electrodes are held above the bath. Electrochemical Characterization. CFMEs as described above were subjected to cyclic voltammetry in 1 mM ferricyanide (Figure 5, top panel) and 100 µM dopamine (Figure 5, bottom panel). The i-E response at a scan rate of 0.1 V/s displayed the expected sigmoidal voltammetric wave for either ferricyanide reduction or dopamine oxidation. The obtained half-wave potentials for the reduction of ferricyanide (∼220 mV vs SSCE) and the oxidation of dopamine (∼150 mV vs SSCE) were found to be in accord with previously published values at other disk-shaped CFMEs made of etched carbon fibers.4,16 In theory, the steady-state diffusion-limited current (ilim) for disk-shaped microelectrodes is proportional to the radius r of the disk and the concentration of the electroactive species present in the electrolyte.17 In good agreement with theory, a plot of ilim vs r (not shown) is linear (linear correlation coefficient r ) 0.995). Repeated backcutting of the same CFME yielded ilim values that varied by about 5-20%. Cyclic voltammograms were measured at electropainted CFMEs made from unetched and etched T650 carbon fibers in solutions of ferricyanide (10-1000 µM) and dopamine (10-100 µM) to test the dependency of ilim on the concentration of the electroactive species. In all cases, ilim increased linearly with the concentration of the electroactive compound, and the slope of the corresponding calibration plots decreased as the diameter decreased. (16) Kelly, R. S.; Wightman, R. M. Anal. Chim. Acta 1986, 187, 79-87. (17) Heinze, J. Angew. Chem., Int. Ed. Engl. 1993, 32, 1268-1288.

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The cross-sectional morphology of PAN- and pitch-based carbon fibers, respectively, differs significantly. The T650 (PANbased) fibers have an “onion peel” arrangement of the graphitic layers, at least on the outside. In contrast, the P100S (pitch-based) carbon fibers have a radial orientation or “PanAm” texture of the graphitic ribbons. Neither the capacitance, nor the rms noise, nor the electrochemistry of ferricyanide and dopamine solutions differs significantly at disk-shaped CFMEs of the same size but made from carbon fibers of different precursors. Furthermore, the etching behavior is not noticably different for the different carbon fiber structures. CONCLUSION We have demonstrated a method for making carbon-fiber microelectrodes with cylindrically etched carbon fibers that are insulated with electrodeposition paint. These electrodes have significantly lower noise than electrodes having unetched tips, and unlike conically etched electrodes, they offer the advantage of being reusable by allowing repeated cutting. The electrochemical behavior of the electrodes is highly reproducible, yielding sigmoidal cyclic voltammograms with well-defined half-wave potentials. Although we limited our present results to the fabrication of electrodes having total tip diameters (including insulation) down to ∼1 µm, smaller diameters can be obtained. However, there is a limit to the gains achieved through etching the fibers to smaller diameters. In terms of the signal-to-noise ratio for measurements of single-vesicle exocytosis, the optimal situation is obtained when the diameter of the electrode tip is similar to that of the vesicles releasing the transmitter.18 The diameter of chromaffin cell vesicles is ∼300 nm,19 while that of synaptic vesicles is ∼50-100 nm.1 When the tip is much smaller than the vesicle diameter, the Coulombic efficiency (ability to “capture” the released molecules) is dramatically decreased; and when the tip is larger, the excess carbon surface area adds more noise, but does not enhance the detection of released molecules. (18) Schroeder, T. J.; Jankowski, J. A.; Senyshyn, J.; Holz, R. W.; Wighman, R. M. J. Biol. Chem. 1994, 269, 17215-17220. (19) Parsons, T. D.; Coorssen, J. R.; Horstmann, H.; Almers, W. Neuron 1995, 15, 1085-1096. (20) Sherman-Gold, R., Ed. The Axon Guide for Electrophysiology & Biophysics: Laboratory Techniques; Axon Instruments, Inc.: Foster City, CA, 1993; Chapter 3, pp 25-80.

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There is an additional consideration: the noise of the amplifier being used. The total noise in measurements includes contributions from both the electrode and the amplifier. We have shown that reductions in the carbon-fiber tip diameter lead to measurable decreases in noise for radii down to ∼0.5 µm (Figure 2A). Further size reductions do not significantly decrease the total noise, as the amplifier noise becomes predominant. Our measurements were performed with a resistive-feedback amplifier. Use of a capacitive-feedback current amplifier should make it possible to obtain further noise reductions and allow working with electrodes of smaller radii.20 Small tip diameters are an advantage, not only because they confer low noise but also because they allow the use of carbon fibers in restricted environments, such as the inside of a patch pipet. Recently carbon-fiber microelectrodes were cannulated inside patch pipets for “patch amperometry”, the simultaneous measurement of membrane capacitance and amperometric currents during single-vesicle exocytosis.13 The development of a simple method for producing CFMEs with micrometer and submicrometer tips will further this and other applications that involve low-noise electrochemical measurements in confined spaces. ACKNOWLEDGMENT We thank Prof. Walter Stu¨hmer and Prof. Erwin Neher for support and helpful discussions; Drs. Mike Shipston, Dieter Bruns, and Corey Smith for critical comments on the manuscript; the Hiltrup Plant of BASF Farben und Lacke GmbH for supplying the electrodeposition paint; and the Microelectronics Imaging and Analysis Centre (MIAC) and the Science Faculty EM Facility at the University of Edinburgh for help with preparing the scanning electron micrographs. R.H.C. was supported partly by a Howard Hughes Medical Institute Physician Postdoctoral Fellowship and the Wellcome Trust. A.S. is a recipient of a European Community Training and Mobility Research (TMR) fellowship and was partly funded by a Max Planck Fellowship.

Received for review August 26, 1997. Accepted December 2, 1997. AC970934E