Anal. Chem. 1996, 68, 3054-3058
A Simple Method for Insulating Carbon-Fiber Microelectrodes Using Anodic Electrophoretic Deposition of Paint 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
We describe a simple method for the insulation of carbonfiber microelectrodes (CFMEs). Using the technique of anodic electrophoretic deposition of paint (EDP), we deposited thin and uniform films of electrodeposition paint onto 10-µm-diameter carbon fibers. The polymer films were then heat cured, leading to an electrically insulating coat. The insulated carbon fibers were transected perpendicular to their axes to expose a 10-µm carbon disk and characterized using conventional electrochemical methods and scanning electron microscopy. As expected, cyclic voltammograms measured with electropainted CFMEs in solutions containing ferricyanide displayed a sigmoidal response without hysteresis. The insulating films had a low dielectric constant, resulting in low capacitance. In addition, the film thickness could be controlled simply by varying such deposition parameters as the applied voltage and the duration of treatment. Electrical noise of the transected fibers was determined principally by the cut surface of the fiber, not by the exposed insulated surfaces. Compared to electropolymerization of phenol compounds, another electrochemical method for insulating CFMEs, insulation by anodic EDP has the very significant advantages of greater simplicity, much shorter processing time, and much lower toxicity of the reagents involved, important considerations for those who work with the procedure on a daily basis. Furthermore, electrodeposition paints are commercially available and have long shelf lives. During the last decade, voltammetric measurements with electrodes having diameters of less than about 50 µm (i.e., microelectrodes) have become routine in electrochemistry.1 Microelectrodes of various geometries, sizes, and electrode materials have been developed.2,3 For studies in aqueous solutions, the most commonly used electrode material is carbon, particularly for in vivo electrochemical analysis in biological tissue.1,4,5 High†
Max Planck Institute for Experimental Medicine. University of Edinburgh Medical School. (1) Heinze, J. Angew. Chem., Int. Ed. Engl. 1993, 32, 1268-1288. (2) Rolison, D. R. In Ultramicroelectrodes; Fleischmann, M., Pons, S., Rolison, D. R., Schmidt, P. P., Eds.; Datatech Systems, Scientific Publishers Div.: Morganton, NC, 1987; pp 65-106. (3) Wightman, R. M.; Wipf, D. O. In Electroanalytical Chemistry: A Series of Advances; Bard, A. J., Ed.; Marcell Dekker: New York, 1989; Vol. 15, pp 268-353. (4) Kawagoe, K. T.; Zimmermann, J. B.; Wightman, R. M. J. Neurosci. Methods 1993, 48, 225-240. ‡
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modulus carbon fibers (“graphite fibers”), having dimensions of only a few micrometers, are often preferred, because they are mechanically rigid, highly conducting, and easy to handle compared to carbon of other forms or metal wires of similar size. In addition, carbon fibers are cheap and readily available in a range of diameters, rigidity, and cross-sectional morphology. Insulation of the carbon fibers is an important step in the fabrication of disk-shaped carbon-fiber microelectrodes (CFMEs). Many published procedures are based on sealing single strands of carbon fibers into tapered glass capillaries using epoxy resins or nonconducting waxes.6 An alternative approach involves inserting the fibers into thin plastic tubes that are then melted and pulled to seal the fibers into the molten plastic.7 Radio frequency sputtering or chemical vapor deposition techniques have also been used, for example, to insulate carbon fibers with silica coatings.8,9 While these approaches have been used successfully, all are relatively time consuming or require training, skill, and/or highly specialized equipment. In addition, the control of the thickness of the insulation and, therefore, of the overall dimension of the microelectrode is not simple for carbon fibers sealed in glass or plastic. Very thick insulation at the tip of the microelectrodes may interfere, for instance, with the positioning of the tip in microenvironments. To circumvent the problems mentioned above, anodic electropolymerization with phenol compounds was introduced for insulating CFMEs.10 The anodization of a carbon fiber in methanolic solutions of o-allyphenol leads to in situ formation of a thin and uniform coating of poly(oxyphenylene) over the entire exposed surface of the carbon-fiber substrate. Upon subsequent heat curing, the deposit is transformed into a tightly adherent and highly resistive insulation. A very important feature of electropolymerization is that the thickness of the insulation can be controlled within certain limits simply by varying such parameters as the deposition voltage, the current density during deposition, the concentration of monomer, and the deposition time. In fact, for this reason, the anodic electropolymerization of (5) O’Neill, R. D. Analyst 1994, 119, 767-779. (6) Kelly, R. S.; Wightman, R. M. Anal. Chim. Acta 1986, 187, 79-87. (7) Chow, R. H.; von Ru ¨ den, L. In Single Channel Recording, 2nd ed.; Sakmann, B., Neher E., Eds.; Plenum Press: New York, 1995; pp 245-275. (8) Abe, T.; Itaya, K.; Uchida, I. Chem. Lett. 1988, 399-402. (9) Zhao, G.; Giolando, D. M.; Kirchhoff, J. R. Anal. Chem. 1995, 67, 25922598. (10) Potje-Kamloth, K.; Janata, J.; Josowicz, M. Ber. Bunsen-Ges. Phys. Chem. 1990, 93, 1480-1485. (11) Strein, T. G.; Ewing, A. G. Anal. Chem. 1992, 64, 1368-1373. (12) Schulte, A. Ph.D. Thesis, University of Mu ¨ nster, Mu ¨ nster, Germany, 1993. S0003-2700(96)00210-7 CCC: $12.00
© 1996 American Chemical Society
o-allylphenol was used recently to insulate the submicrometer tips of etched carbon fibers with ultrathin poly(phenylene oxide) films.11,12 Here, a new electrochemical approach for insulating carbon fibers is presented, using the anodic electrophoretic deposition of paint (EDP). EDP was introduced in industry to provide corrosion-resistant priming coats to electrically conducting bulk articles such as food tins and car bodies.13,14 Electrodeposition paint is commercially available in anodic and cathodic forms. The anodic paint consists of an aqueous dispersion of poly(acryliccarboxylic acid) resins of micellar structure. The water-soluble form of the polymer is negatively charged, due to the presence of carboxlate groups. Acidification leads to neutralization of the carboxylate groups and precipitation of the micelles. In an electrochemical cell, negatively charged micelles are electrophoretically attracted to the anode. Hydrolysis of water at the anode leads to the local production of protons and precipitation of the micelles as a thin, uniform, and tightly adherent polymer film on the surface of the electrode (the anode is then “electropainted”). Upon heat curing, this coat becomes electrically insulating and relatively chemically inertsproperties that have been used to advantage in the fabrication of insulated scanning tunneling microscopy (STM) tips.12,15 In this paper, we demonstrate that anodic electrophoretic painting is a convenient method for insulating CFMEs. We examine the basic properties of insulated CFMEs using standard electrochemical methods (voltammetry, amperometry, capacitance, and noise measurements) and scanning electron microscopy. In addition, we show that electropainted CFMEs can be used to detect catecholamine release from single secretory granules in bovine adrenal chromaffin cells. Compared to electropolymerization of phenol compounds, insulation of CFMEs by anodic EDP has the very significant advantages of greater simplicity, much shorter processing time, and much lower toxicity of the reagents involved, important considerations for those who work with the procedure on a daily basis. Furthermore, electrodeposition paints are commercially available and have long shelf lives. EXPERIMENTAL SECTION I. Materials and Reagents. High-modulus carbon fibers made from mesophase pitch (Thornell P100S, Amoco Performance Products, Inc., Greenville, SC) were boiled in acetone for 8 h using a Soxhlet extractor to remove sizing compounds. The radii of P100S carbon fibers measured by SEM were found to be 5.1 ( 0.4 µm (mean ( SD; n ) 25). Single filaments of the carbon fibers were connected electrically and mechanically to insulated 0.5-mm copper wires using a small drop of conductive carbon paste (Electrodag 5513, Acheson Colloids, Scheemda, The Netherlands). The electrodeposition paint was Glassophor ZQ 84-3211 (BASF Lacke u. Farben AG, Muenster, Germany). All chemicals were of reagent grade purity and were used without further purification. K3[Fe(CN)]6 was obtained from Aldrich Chemical. KCl, HCl, Na2SO4, NaCl, CaCl2, MgCl2, NaOH, and Hepes were obtained from Sigma Chemical. The aqueous solutions were prepared using doubly distilled and deionized (13) Industrial Electrochemistry; Pletcher, D., Walsh, F. C., Eds.; Chapmann and Hall: London, 1990. (14) Beck, F. Electrochim. Acta 1988, 33, 838-850. (15) Mao, B. W.; Ye, J. H.; Zhuo, X. D.; Mu, J. Q.; Fen, Z. D.; Tian, Z. W. Ultramicroscopy 1992, 42-44, 464-467.
water. Voltammograms and chronoamperometric measurements were recorded in freshly prepared nitrogen-saturated solutions of 1 mM ferricyanide in 0.5 M KCl at pH 3, to take advantage of the known diffusion coefficient of ferricyanide in these conditions.16 The capacitance and noise measurements were performed at room temperature in a solution containing 140 mM NaCl, 2.8 mM KCI, 2 mM CaCl2, 1 mM MgCl2, and 10 Hepes. The pH of this “rat Ringer solution” was adjusted to 7.2 using 1 M NaOH. II. Instrumentation. Anodic electrophoretic deposition of paint was performed in a one-compartment cell at room temperature by applying a constant voltage from a conventional constantvoltage laboratory power supply between a platinum electrode (cathode) and the carbon-fiber substrate (anode). All microelectrode experiments (voltammetry, chronoamperometry, capacitance, and noise measurements) were performed at room temperature in a one-compartment electrochemical cell using a two-electrode configuration. The CFMEs (working electrodes) were mounted on the headstage of a computercontrolled patch-clamp amplifier (HEKA EPC-9, HEKA Elektronik, Lambrecht, Germany), located on a vibration-damping table in a Faraday cage. The EPC-9 patch-clamp amplifier allows high-gain measurements with low noise and high frequency response. The bath reference/counter electrode was either a calomel reference electrode (SCE) 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 aquisition was performed with the built-in ITC-16 of the EPC-9 amplifier, controlled with the program Pulse (HEKA Elektronik). The computer was a Macintosh Quadra 800 (Apple Computer GmbH, Ismaning, Germany). Analysis was performed in the program IGOR (Wavemetrix, Lake Oswego, OR) using programs written by us. The root-mean-square (rms) noise of the electrodes was directly read off the EPC-9 amplifier. The filter was an 8-pole bessel with a corner frequency of 3 kHz, and the amplifier gain was 50 mV/pA. For analysis, the rms readings were converted to noise variance and plotted as a function of the capacitance. The capacitance of each electrode was determined by integrating the current transients in response to 10-mV step depolarizations and dividing by the amplitude of the voltage step. The scanning electron microscopy (SEM) was carried out on a Camscan microscope (Cambridge Scanning Co. Ltd., Cambridge, UK). To improve the quality of the SEM images, the CFMEs were coated prior to the SEM investigations with ultrathin platinum films using a plasma coater. III. Fabrication of Disk-Shaped Carbon-Fiber Microelectrodes. Single P100S filaments were attached to the stripped end of 5-cm-long pieces of insulated copper wire using a small drop of conductive carbon paste, and the carbon paste was allowed to dry. The attached carbon fibers were then cut back to a length of about 1 cm. The carbon fibers were insulated using the anodic EDP procedure with Glassophor ZQ 84-3211. The cell consisted of a two-loop platinum spiral (cathode) surrounding the carbonfiber substrate, which served as the anode. A constant voltage either of 5 or 20 V was applied between these two electrodes for 4 min to induce electrophoretic deposition of paint. The current throughout the procedure was monitored with a multimeter and typically decreased rapidly from values greater than 5-10 µA down (16) Kawagoe, K. T.; Jankowski, J. A.; Wightman, R. M. Anal. Chem. 1991, 63, 1589-1594.
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to values of about 0.5 µA after 4 min. Coated carbon fibers were cured at 194 °C for 5 min. For uniform deposition of insulation, it was found empirically that the junction between the carbon fiber and copper wire should be kept out of the electrodeposition paint. If coating of the junction was desired, the carbon fiber alone was first treated, and then, after baking to cure the first coating, the electrophoretic painting was repeated with the junction immersed. For complete insulation of the junction, it was necessary to repeat the painting/curing cycles several times, as heat curing leads to shrinkage of the coating and sometimes to disruption of the insulation over the irregular cut end of the copper wire. To expose a disk-shaped electroactive surface area, the insulated carbon fiber was transected with a scalpel blade under a dissecting microscope. The copper wire with the electropainted carbon fiber was fixed with modeling clay onto a microscope specimen slide. It was important to place the carbon fiber nearly in parallel with and in contact with the glass surface. Cutting was carried out with a fresh surgical scalpel blade. The cut end was examined at a higher magnification, and only if it looked smooth was it used for measurements. RESULTS AND DISCUSSION I. Scanning Electron Microscopy. Two important characteristics for the insulation of CFMEs are the integrity and the thickness of the coating. Figure 1A shows a scanning electron micrograph of a P100S carbon fiber to which the electrodeposition paint was applied at 20 V for 4 min. The insulating film was reproducibly uniform along the entire length of the carbon fiber, with no gaps, beading, or other defects in the polymer/carbon fiber seal. Figure 1B shows the scanning electron micrograph of the scalpel-transected tip of the same carbon fiber. The interface between the insulating electropaint and the exposed electrochemically active carbon-fiber surface is clearly visible, allowing one to estimate the thickness of the coating. The concentric coating had an average thickness of about 6 µm; hence, the total tip diameter of such a microelectrode is typically about 22 µm. Thinner coatings can be obtained by decreasing the applied voltage during the painting procedure. For example, the average thickness of insulation was about 1.5 µm for electrodeposition at 5 V for 4 min (not shown). The smaller total tip diameter is an important feature for measurements such as those of secretion from single cultured animal cells (see section IV, below). II. Steady-State Voltammetry and Chronoamperometry. Unless stated otherwise, all measurements presented below were performed on electrodes for which the electrodeposition painting was applied at 5 V for 4 min. To test the integrity of the insulation, carbon fibers that had been electropainted but not cut were studied voltammetrically in solutions containing 1 mM ferricyanide in 0.5 M KCl at pH 3.0. No faradaic current could be detected (not shown), indicating that no electroactive surface of the carbon fiber was exposed. After scalpel-cutting, cyclic voltammograms (scan rate of 100 mV/s) measured with electropainted CFMEs displayed a sigmoidal response, with no significant hysteresis (see Figure 2). The half-wave potential for the reduction of ferricyanide was about 220 mV vs SCE, consistent with previous measurements on ferricyanide with other CFMEs.16 The steady-state limiting currents (ilim) measured by voltammetry and chronoamperometry (Estep ) -350 mV vs SCE; tstep ) 20 s) were nearly identical in solutions containing 1 mM ferricyanide. For the electropainted 10-µm3056
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Figure 1. (A, top) Scanning electron micrograph of an electropainted 10-µm-diameter carbon fiber, insulated by anodic EDP at 20 V for 4 min. (B, bottom) Close-up view of the scalpel-transected tip of the same carbon fiber.
Figure 2. Cyclic voltammogram for the reduction of ferricyanide at an electropainted CFME, insulated using anodic EDP at 5 V for 4 min (1 mM ferricyanide in 0.5 M KCl at pH 3; v ) 100 mV/s).
diameter CFMEs, the limiting current was 1.92 ( 0.17 nA (mean ( SD; n ) 21), the variations in magnitude arising presumably due to slightly differing diameters of the carbon fibers and irregularities in the cut surfaces. The determination of ilim allowed us to calculate the apparent radii of electropainted CFMEs. The steady-state diffusion-limited current obtained from a voltammogram at a microelectrode with either spherical, hemispherical, or disk geometry is given by the following equation:1
ilim ) KnFDCr
(1)
K is a geometry-dependent coefficient, r is the radius of the microelectrode, and the other terms have their usual electro-
chemical meanings. Calculation of the size of a particular microelectrode with eq 1 is possible only if the value of K is known. Theoretical analysis has revealed that, for an ideal spherical diffusion model around a microelectrode, K ) 4π, and for a hemispherical diffusion model, K ) 2π.1 For disk-shaped planar microelectrodes, digital simulation studies have shown that K is determined by the relative dimensions of the electroactive area (r) and the thickness (d) of the insulation.17 For a disk-shaped microelectrode embedded in an infinite plane (d . r), K ) 4, whereas for a thinly insulated disk-shaped microelectrode (d f 0), values larger than 4 are predicted. These theoretical calculations were consistent with experimental findings described by Kelly and Wightman, where K was found to be 4.14 ( 0.36 at beveled 10-µm carbon-fiber microelectrodes insulated with thin glass/epoxy coatings.6 To calculate the value of K for the electropainted CFMEs, we took advantage of a study recently published by Zhao et al.,9 who derived an empirical equation that fit their data on silica-coated CFMEs. The equation, modified to incorporate our symbols, is as follows:
K ) 4 + 1.516[r/(r + d)]2.342
(2)
In the present study, r ≈ 5.1 µm and d ≈ 1.5 µm (see above) if the electropaint is applied at 5 V for 4 min to a P100S carbon fiber. With these values, for electropainted CFMEs, K ) 4.83. Using eq 1 with K ) 4.83 and D ) 7.2 × 10-6 cm2/s, the expected steady-state limiting current for the reduction of 1 mM ferricyande is calculated to be 1.73 nA, assuming a smooth planar electrode surface. The measured steady-state limiting current obtained by cyclic voltammetry and chronoamperometry is about 10% higher (1.92 nA), perhaps reflecting irregularities in the scalpel-cut end, which increase the surface area (see Figure 1B). III. Capacitance and Noise Measurements. Fully insulated carbon fibers were immersed to various depths into Ringer solution, and the capacitance and noise were monitored. With the fiber tip just above the bath, typical capacitance and rms noise values were 1.80 pF and 94 fA, respectively (at 3 kHz and 50 mV/ pA gain). The capacitance value here reflects the total input capacitance of the amplifier plus the capacitance due to the electrode. The noise value in air compares favorably to that of normal patch pipets. Upon immersing the fully insulated tip 2 mm into the bath, the capacitance increased typically to 3.6 pF, and the rms noise was 150 fA. These values are to be compared with values obtained at the cleaved carbon-fiber microelectrode also immersed 2 mm into the bath. The capacitance values for the transected fibers were found to be about 20 pF, and the rms noise increased typically to about 1500 fA. These values indicate that the noise and capacitance of the cut carbon fibers were dominated by those of the exposed carbon. To determine the dielectric constant of the electrodeposition paint, the capacitance of fully insulated P100S carbon fibers immersed in rat Ringer solutions was measured as a function of the submersion depth of the fibers, yielding a linear plot. From the slope, the specific capacitance was found to be 0.85 ( 0.12 pF/mm (mean ( SD; n ) 13). The total capacitance for the immersed carbon fibers can be described by the equation for a cylindrical capacitor: (17) Shoup, D.; Szabo, A. J. Electroanal. Chem. 1984, 160, 27-31.
Figure 3. Amperometric detection of the quantal adrenaline release due to the fusion of single bovine chromaffin cell secretory vesicles. Detection was performed with an electropainted CFME (insulated at 5 V for 4 min) held at + 650 mV vs Ag/AgCl.
C ) 2π0L/ln[(r + d)/r]
(3)
Here, 0 is the permittivity of free space (8.854 × 10-12 C/V‚m), is the dielectric constant, and L is the immersion depth of the electrode; r and d have the same definitions as above. The dielectric constant of the electrodeposition paint was calculated to be 3.94. For comparison, quartz has a dielectric constant of 3.8 and polypropylene 2.1. Poly(oxyphenylene) insulation has an of 14.10 The noise variance of fully insulated carbon fibers was linearly proportional to the capacitance, with a slope of 7500 ( 900 fA2/ pF (mean ( SD; n)13). In comparison, the noise variance of quartz patch pipets was nearly 10 times less at 750 ( 150 fA2/pF (mean ( SD; n ) 4). This indicates that, while the electrodeposition paint has a very low dielectric constant, it has a high loss factor, which leads to the noise being significantly larger than that of quartz pipets of similar capacitance. However, as noted above, the noise in cut CFMEs originated primarily from the exposed carbon surface, not from the insulated region. IV. Application to Measurements of Single-Vesicle Transmitter Release. One area in which CFMEs have found elegant application is in the recording of quantal transmitter release from animal secretory cells. Certain animal cells release readily oxidizable transmitters. For example, adrenal chromaffin cells secrete the catecholamines epinephrine and norepinephrine,18,19 mast cells discharge serotonin,20 and sympathetic neurons release norepinephrine.21 The transmitter is stored in tiny vesicles and discharged at the cell surface in discrete multimolecular packets called quanta. For recording, the cells are placed in a physiological electrolyte solution (essentially 150 mM NaCl), and a CFME tip is placed near the cell surface with an appropriate dc potential applied to it (with a Ag/AgCl counter electrode). The quantal release of transmitter is detected as spiking oxidative current transients. Of critical importance for such an application is that the microelectrode tip diameter not be larger than the dimension of (18) Wightman, R. M.; Jankowski, J. A.; Kennedy, R. T.; Kawagoe, K. T.; Schroeder, T. J.; Leszczyszyn, D. J.; Near, J. A.; Diliberto, E. J.; Viveros, O. H. Proc. Natl. Acad. Sci. U.S.A. 1991, 88, 10754-10758. (19) Chow, R. H.; von Rueden, L.; Neher, E. Nature 1992, 356, 60-63. (20) Alvarez de Toledo, G.; Fernandez-Chacon, R.; Fernandez, J. M. Nature 1993, 363, 554-558. (21) Zhou, Z.; Misler, S. Proc. Natl. Acad. Sci.U.S.A. 1995, 92, 6938-6942.
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the single cell. CFMEs insulated using the deposition procedure described above are sufficiently thin to be positioned near single secretory cells of 12-15-µm diameter. Figure 3 shows signals due to the fusion of single adrenal chromaffin cell secretory vesicles. Detection of the released catecholamine was made using an electropainted CFME in amperometric mode, with a dc voltage of 650 mV. The cell was mechanically stimulated to secrete by prodding the tip of the CFME against the cell membrane. The shape of the signals and the integral of the charge correspond well to those previously reported in the literature.18,19 Thus, anodic electrophoretic deposition of paint is a rapid and simple method for the insulation of CFMEs. The thickness of the insulation allows maneuvering of the microelectrode tips into microenvironments, such as the vicinity of a cultured animal cell. Of note, this method should be particularly advantageous when the application calls for long, thin, insulated carbon fibers. For example, work in our laboratory is currently underway to investigate combining the techniques of patch-clamp and carbonfiber amperometry, with the CFME located inside the patch-pipette microcapillary. With such an arrangement, secretion originating from a confined patch on the cell surface can be studied. This
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work will be described in future publications. In addition, EDP insulation is nonbulky and thus can be used to insulate the finely tipped probes used for in situ electrochemical scanning tunnelling microscopy.12 ACKNOWLEDGMENT We thank Prof. Dr. Erwin Neher, Prof. Dr. Walter Stu¨hmer, and Prof. Dr. Ju¨rgen Otto Besenhard for support and helpful discussions, Frauke Friedlein and Michael Pilot for cell preparation, Dr. Karl. Schur for obtaining the scanning electron microscope images presented in this paper, and the Hiltrup Plant of BASF Paints and Coatings for supplying the electrodeposition paint. A.S. was partially supported by a Max Planck Fellowship. R.H.C. was supported by a Howard Hughes Medical Institute Postdoctoral Fellowship and a fellowship from the Alexander von Humboldt Foundation. Received for review March 1, 1996. Accepted June 10, 1996.X AC960210N X
Abstract published in Advance ACS Abstracts, July 15, 1996.
