A ~ I ctwm. . 1001, 83, 1589-1594 (13) Baba, Y.; I m , K.; NakasMo, F.; Matsumoto, M.; Goto, M. N@pon Kamku K a W 1987, 8 , 1823-1825. (14) Rlckelton, W. A.: Robertson, A. J. h4iner. Mstan. I.focess. 1987, 4 ,
7-10. (15) Baba, Y.; Ueda, T.; Inoue, K. Sdvent €xtr. Ion Ex&. 1988, 4 , 1223-1231. (18) , . Baba. Y.: Umezaki. Y.: Ueda.. T.:. Inoue. K. 8ulJ. Chem. Soc. J m . 1986; 59, 3835-3839.. (17) Baba, Y.; Oshima, M.; Inoue, K. 8M. Chem. Soc. Jpn. 1988, 59, 3829-3833. (18) Baba, Y.: Umezaki, Y.; Inoue, K. J . Chem. Eng. Jpn. 1986, 19, 27-30. (19) Cattrall, R. W.; Martln, A. R.; Trlbuzb, S. J . I w . Nud. Chem. 1978, 40. 887-890. (20) Rickelton. W. A.; Boyle, R. J . Sep. S d . Technd. 1988, 23, 1227-1 250. (21) Attlyat, A. S.; Kadry, A. M.; Badawy, M. A,: Hanna, H. R.; Ibrahim, Y. A.; Christian, G. D. Electroenaljlsls 1990, 2, 119-125.
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(22) Buck, R. P. I€€€ Trans. E k . Lkwices ED-29 1982, 108-115. (23) Buck,R. P. J . chem.Soc., Farady Trans. 1 1986, 82, 1189-1178. (24) Florido, A.; Daunert, S.; Bachas, L. G. Ektroenalysls 1991, 3 , 177- 182. (25) Thompson, E. 0. E.; Fisher, W. K. Aust. J . 8/01. Scl. 1978, 31. 433-442. (28) Diirselen, L. F. J.; Wegman, D.: May, K.; Oesch, U.;Simon, W. Anal. Chem. 1988, 60, 1455-1458.
RECEIVED for review November 5,1990. Revised manuscript received March 22,1991. Accepted March 26,1991. This work was supported by grants from the National Science Foundation (DMR-9000782), from the NATO Scientific Affairs Division (CRG 890610), and from the Spanish Commission for Research and Development, CICYT (MAT 88-752).
Etched Carbon-Fiber Electrodes as Amperometric Detectors of Catecholamine Secretion from Isolated Biological Cells Kirk T. Kawagoe, Jeffrey A. Jankowski, and R. Mark Wightman* Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Voltametric electrodes with mlcrometer dhnenslons have been fahkated from carbon flkn, etched to a conical shape and Insulated with poly(oxyphenylene) foUowlng literature procedures. The rewitant electrode has a tip radius In the micron range. The response of this electrode is compared to a carbon-flber electrode prepared by seallng a carbon-fiber electrode In a glass pipet (electrode rrrdlur > 3.5 pm). W M e the etched electrodes dld not folkw electrochemical theory as w d l as the glaswncas8d electrode, the etched electrode was found to be suttable for the amperometrlc measurement of the secretion of catecholamlnesfrom Isolated bovine adrenal cells. Comparable resuits are only obtained when the two different electrodes are placed 1 pm from the cell surface. When the etched ebclmde b placed furlhef away, less secretion b observed, because of dmgkn and accompanying dllutlon.
INTRODUCTION Electrodes of very small size are particularly useful to probe concentration inhomogeneites on a microscopic scale ( I ) . Submicrometer size electrodes, suitable for such measurementa, have been prepared by a variety of techniques: the use of Wollaston wires (2), electrochemical etching of carbon fibers (3, 4 ) or noble-metal wires (5-7), and the pyrolytic deposition of thin films of carbon on the interior of quartz pipets (8). In addition, several methods have been demonstrated to insulate the sides of these electrodes to provide electrodes whose exposed dimensions are in the micrometer range (4-10). These electrodes have been used in applications such as scanning electrochemical microscopy (11-13) and intracellular measurements (4,141. In this laboratory we have been interested in the use of microelectrodes to monitor the release of catecholamines from biological cells. In recent experiments we have shown that electrodes prepared from carbon fibers insulated in a glass
*Towhom correspondence should be addressed. 0003-2700/91/0383-1589$02.50/0
capillary (effective radius -6 Nm) can be used to measure release from individual cells grown in culture (15). The cells employed, a primary culture of bovine adrenal medullary cells, secrete norepinephrine and epinephrine in response to depolarization of the cell membrane or activation of nicotinic receptors found on the cell surface. The release of catecholamines observed from these cells appears as a series of sharp, irregular, concentration spikes. These observations are particularly interesting because they are consistent with the exocytotic theory of secretion (16). According to this hypothesis, substances to be secreted are stored in intracellular vesicles and are released by fusion of the vesicle with the cellular membrane and extrusion of its contents into the extracellular space. A wide variety of cell types, including neurons, are thought to use this mechanism. The use of microelectrodes at single cells provides the first method to directly monitor exocytosis in real time. Bovine adrenal medullary cells contain vesicles that have a mean diameter of 400 nm (17). Vesicles are estimated to contain an average 5-10 am01 of catecholamine (18). Thus, coulometric detection of the contents of a single vesicle should result in a charge of 1-2 pC, which is consistent with our measurements of catecholamine spikes made with beveled carbon-fiber electrodes placed adjacent to cells (15). However, because transport of the catecholamines from the cell surface to the detecting electrode is expected to be a diffusion-controlled process, the observed amplitude and time course would be expected to be determined in part by the distance between the cell and the carbon-fiber electrode. Furthermore, the size of the detecting surface could also affect the amount detected. Thus, although smaller electrodes would appear to be useful in such applications, their response may differ from larger electrodes. In this paper we contrast the measurements of catecholamine release made with different-sized microelectrodes. Measurements made with carbon fibers sealed in glass capillaries, as in our previous work (19, 21),are compared to responses measured with smaller electrodes prepared by the method of Josowicz et al. (10). The apparent radius of each 0 I991 American Chemical Society
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of the electrodes was determined from steady-state voltammograms of ferricyanide (22). In addition to characterization of these electrodes in the measurement of the release of catecholamines, we have examined some of the properties of etched carbon fibers with tip diameters in the micron range. EXPERIMENTAL SECTION Apparatus. Voltammetric measurements employed a locally constructed two-electrode system employing a Burr-Brown 104 CM operational amplifier as current transducer or a commercial potentiostat (EI-400, Ensman Instruments, Bloomington, IN). The gain settings were calibrated with precision resistors and a digital voltmeter specified with a 0.1% accuracy (Model 8010A, John Fluke Mfg. Co., Mountlake Terrace, WA). A sodium-saturated calomel reference electrode (SSCE) was used throughout. Electrode characterization was done in a flow injection system (21)with the working electrode positioned in the flow stream outlet (flow rate = 1mL/min). The flow ww stopped for measurements in the amperometric mode. Electrode Construction. Glass-encased carbon-fiber electrodes were constructed by sealing single carbon fibers into pulled-glass capillaries (19). Carbon fibers with nominal radii of 2.6,3.5, and 5 pm were used (types T650/42, T300, and P55, respectively, Amoco Performance Products, Greenville, SC). The electrodes were polished at an angle of approximately 45O on a micropipet beveler (Sutter Instrument Co., Novato, CA). Carbon electrodes of smaller size were prepared with 3.5-pm radius carbon fibers that were electrochemically etched. Fibers were inserted into microfilament soft-glass capillaries (A-M Systems, Inc., Everett, WA), and the glass was tapered around the fiber with a micropipet puller (Narishige, Tokyo, Japan). The tip of the capillary was trimmed with a scalpel so that approximately 1 mm of the carbon fiber was exposed. The tips were sealed with epoxy (Epon 828 with 15% m-phenylenediamine by weight, Miller-Stephenson Chemical Co., Inc., Danbury, CT); excess epoxy was removed from the carbon fiber by rinsing with hot acetone. The electrodes were left overnight at room temperature and cured for 1h at 100 OC. Electrical connection was made by back-filling the capillary with colloidal graphite (Polysciences, Inc., Warrington, PA) and inserting a chrome1wire. The connection was dried for 15 min at 150 OC. The protruding fiber was etched electrochemically in a solution containing 0.5 mM K2Cr20,and 5 M H2SO4 (3). The etching solution was suspended in a small platinum loop, and the fiber was positioned into the solution by a micromanipulator with the aid of an optical microscope. A 60-Hz sine wave, 6-15,,V was applied between the electrode and the platinum loop. The taper of the etched fiber can be controlled by adjustment of its position in the drop. The electrodes were then rinsed in distilled water and dried for 1 h at 150 OC. An insulating layer of poly(oxyphenylene) was applied to the etched carbon surface by electrochemical deposition. The deposition cell contained 0.23 M 2-allylphenol,0.4 M allylamine, and 0.23 M 2-butoxyethanol in 1:l (v:v) water-methanol and a platinum cathode (area = 1.25 cm2) (10). A total of 20-30 etched carbon fibers connected in parallel served as the anode. Deposition was accomplished at a potential of +4 V for 2 min. The electrodes were then rinsed with distilled water and cured for 2-4 h at 150 "C. Insulation was removed from the tip of the etched electrode by polishing on the micropipet beveler at an angle of approximately 45O. With this device, a thin f h of water is spread over the polishing surface for lubrication. To determine when the tip had made contact with the polishing surface, an ohmmeter was used to monitor the resistance between the polishing wheel and the electrode as the electrode was lowered with a micromanipulator in 20-100pm increments. Contact with the surface was indicated by a decrease in resistance. The ohmmeter was then dmnnected, and the electrode was polished for 1-2 min. In some experiments, the carbon electrodes were coated with perfluorinated ionomer by dip-coating the electrodes in a 10% Nafion solution for 15 min followed by hot air-drying at 65 "C for 15 min. The electrodes were allowed to dry overnight at room temperature before use. Cultured Cells. Bovine adrenal medullary cells were prepared and cultured as described previously (15). The cells are spherical in shape with a radius of 10 pm, and during culture, the cells
adhere to the base of the plate. All measurements were made on the stage of an inverted-stage microscope (Axiovert 35, Zeias, Eastern Microscope, Raleigh, NC). Microelectrodes were positioned adjacent to a cell with either a Burleigh Inchworm (Fkhers, NY) or a Kopf Model 640 (Tujunga, CA) micropositioner, both of which are capable of 1-pm resolution. The cell surface was found by bringing the electrode in contact with the cell 80 that its membrane was visually deformed. The electrode was then retracted to the desired measurement position. Release of catecholamines was elicited by using a 3-s pressure ejection of 100 pM nicotine solutionsfrom the tip of a micropipet. The pressure ejection device (Piwpritzer, General Valve Corp., FaMeld, NJ) allowed precise timing and pressure control of the ejected volumea. The ejection rate was approximately 3 nL/s. The current was monitored in the amperometricmode, E = 650 mV vs SSCE, with a resolution of 5 ms/point. Spikes,as indicated by current changea with a SIN > 3, were analyzed for the 10-8 period after the initiation of catecholamine release. The sensitivity of the electrodes was tested both before and after the experiment by using freshly prepared 10 pM catecholamine solutions. The electrodes retained approximately 60% of their sensitivity after measurements at the cells. Reagents. The electrochemistry of dopamine and ascorbate was examined in a buffer containing60 mM NazHpOland30 mh4 NaH#04, adjacent to pH = 7.4 with 4 M NaOH. Dopamine and ascorbate solutions were prepared from 10 mM and 100 mM (in 0.1 M HC104)stock solutions, respectively, by dilution. Fresh solutions were prepared for each electrode. RESULTS AND DISCUSSION Steady-State Voltammetry at Micro Carbon Electrodes. To determine the apparent radius of the exposed tip of the glass encased and etched carbon electrodes, amperometric measurements (dc potential) were made in 0.5 M KCl solutions (pH 3.0) of Fe(CN)& (1 mM), corrected for background current. The steady-state current was used to calculate the radius from the previously measured diEfusion coefficient for this system (22). For the glass-encased electrodes, the determined radius is larger than the fiber radius because of the elliptical tip surface that results from the beveling procedure. We have previously shown that radii determined in this way are consistent with the average of the major and minor radii of the elliptical surfaces determined from the arw measured by electron microscopy (19). For the etched carbon-fiber electrodes, the determined radii were consistent with the reduction in size seen with optical microscopy. It has been shown that as the size of the insulation material surrounding the disk approaches the dimension of the disk,larger currents may occur (23). However, for the glass-encased and etched electrodes (disk radii less than 1.5pm and insulation thickness of 1 pm) described here, the effect on the current will be less than 10%. Representative voltammograms for the reduction of Fe(CNIG3-at etched electrodes and glass-encased carbon fibers are shown in Figure 1along with voltammograms simulated (24)by using the determined radii. For the glass-encased fiber, good agreement is seen at both scan rates when finite electron-transfer rates are assumed. In contrast, an acceptable fit is only obtained at slow scan rates with the etched electrode. The low current at high scan rates may reflect inert portions of the surface formed during the electrochemical etching of the surface (25). Characteristics of Etched Electrodes. Electron micrographs of etched carbon-fiber electrodes coated with poly(oxyphenylene) are similar to those reported previously (IO). In a typical electrode the fiber extends 50-150 pm beyond the glass tip and 50-100 pm of the fiber is etched into a conical shape. The thickness of the polymeric insulation is estimated to be 1 pm for the deposition time employed. The electrode is elliptical in shape as a result of the polishing procedure. The insulating properties of the poly(oxypheny1ene) coating were determined with cyclic voltammetry at 200 V s-l in
ANALYTICAL CHEMISTRY, VOL. 63, NO. 15, AUGUST 1, lQQl 1591
Table I. Regression Data for Dopamine Calibration Curves at Carbon-Fiber Electrodesa fiber type
determined radius, pm
pA/pM
slope,
intercept, pA
r
P55 T300 T650
8.3 4.7 3.5 0.4
2.22 1.57 0.98 0.15
0.017 0.03 -0.02 0.09
0.999 0.999
T
1.OOO etched 0.9863 OThe electrode radii were determined from amperometric measurements in K3Fe(CN)@Dopamine concentrations tested were 0.05-1.0 pM. I I
3
250 0 E (mVva SSCE)
500 0 E (mV M SSCE)
-50(
Fbun 1. Simulated (circles)and experimental (solid line) voltammograms at carbon electrodes for the reduction of 1 mM ferricyanide in 0.5 M KCI, pH 3.0: (A, B) Etched caftxmfiber microelectrode (r = 1.3 pm); (C, D) T300 carbon-fiber electrode (r = 4.71 pm). Scan rates are 0.05 V s-l (A, C) and 200 V s-' (B, D). Simulation parameters empkyed were k o = 0.1 cm s-', D = 7.2 X l p c d s-', and nominal scan rate and electrode radlus. 1000
E
1
;
500
1
250
0
,
,I 0 -400 (mV vs SSCE) .
I
800 400
E (mV vs SSCE) E Figure 2. Cyclic vottammograms of dopamine recorded at etched carbon-fiber electrode~s: (A) scan rate, 50 mV s-l;radius, 1.4 pm; concentratbn, 100 pM; (B) scan rate, 200 V s-'; radius, 2.1 pm; concentratlon, 10 pM; background subtracted.
solutions containing 10 mM ferricyanide. Before the electrode was beveled, faradaic current was not observed, although charging current was measured. The apparent capacitance of the deposited film determined from this current was less than 1 pF cm-2 based on the area of the cone. After beveling, the apparent capacitance of the electrodes, based on the electrochemically determined area of the exposed tip, ranges from 70-220 p F cm-2. In contrast, the apparent capacitance for beveled electrodes with single column fibers sealed in glass was determined to be 55 p F cm-2. Thus, it appears that the capacitance of the encapsulated region of the etched fiber contributes to the overall background current. It was found that the stability of the electrode was dependent on the initial potential. At an initial potential of -0.4 V, the background current increased with time, suggesting remove1 of the insulation material. However, at an initial potential of 0.0 V, this did not occur. Electrochemistry of Catecholamines at Micro Carbon Electrodes. Voltammograms of dopamine recorded at etched microelectrodes at two different scan rates are shown in Figure 2. The voltammogram recorded at 50 mV s-l shows sigmoidal behavior, as expected for a microelectrode, and the maximal current is that expected for freely diffusing dopamine. However, at 200 V s-* the voltammogram has the characteristics of an adsorbed species; the current is approximately 10 times larger than expeded from digital simulations (24)using the kinetic constants for catechol oxidation reported previously (261, and the shape of the voltammogram is sharply peaked. Similar behavior has been reported for beveled carbon-fiber electrodes (20). Although adsorption is evident, the kinetics of adsorption and desorption are sufficiently rapid that the electrode response reaches a plateau within 300 ms after ex-
500 0 (mV vs SSCE)
5 10 Time (seconds)
15
Figure 3. (A) Background-subtractedvoltammograms of 10 pM dopamine (-) and 1 mM ascorbate (- - -) recorded at a Nafion-coated, etched electrode (r = 1.3 pm, 200 V s-l). (B) Time response of dopamine and ascorbate. Voltammograms were recorded every 0.1 s, and the cvrent, averaged over the window indicated by the dashed horizontal lines in (A), was plotted as a function of time. Recordings were made with a feedback resistor in the current transducer of lo' fl and a 0.1-ms time constant.
posure to a bolus of dopamine in the flow injection system. Since the faradaic current is directly proportional to the radius under conditions of convergent diffusion, small electrodes might be expected to be less capable of detecting low concentrations. Therefore, the sensitivity of carbon electrodes of several sizes were evaluated in the amperometric mode for dopamine over the concentration range 50 nM to 1.0pM. The results of calibration curves for four different carbon electrodes are given in Table I. In all cases the calibration curves are linear,and the slopes of the calibration curves are proportional to the determined radii. Noise in the measurements was independent of the electrode radius and had a value of 12 fA. This value is equivalent to the Johnson noise (27) of the feedback resistor in the current transducer (1 gQ, 10-Hz band-pass, amplified 100-fold by the subsequent gain stage). Thus, the detection limits are inversely proportional to the electrode radius, and with the smallest electrode, concentrations less than 250 nM could not be determined. Etched Electrodes with Perfluorinated Ion-Exchange Coatings. Voltammetric electrodes for in vivo use are often coated with Ndion (28) This material attenuates the signal from ascorbate and other anionic interferences and protects the surface from deterioration. The responses of a Ndioncoated, etched microelectrode to a 10 pM bolus of dopamine and a 1.0 mM bolus of ascorbate are shown in Figure 3. Voltammograms were recorded at 200 V s-l and repeated every 100 ms (20). The symmetrical shape of the backgroundsubtracted voltammogram for dopamine again indicates adsorption, and the measured current is approximately 6-fold higher than expected for a diffusion-controlled process. At this scan rate the voltammogram for ascorbate is featureless. As shown, the ascorbate current is attenuated by the film and the current at the peak potential for dopamine is 370 f 150 times greater than for an equimolar concentration of ascorbate. Mixtures of ascorbate and dopamine were found to give additive currents at this scan rate. The time response to the
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Figure 4. Light micrograph (phase-contrast) of a bovine adrenal medullary cell with etched and glass-encased carbon-fiber electrodes placed adjacent to it. Magnification is 450X.
bolus of ascorbate is more rapid than for dopamine, which is indicative of the low diffusion coefficient for dopamine in the polymer (21). When the coated electrodes are used in an amperometric mode, the diffusion layer exceeds the dimensions of the polymer film. Under these conditions, the electrode can be responsive to ascorbate (AA) because electrogenerated dopamine o-quinone (DOQ) can oxidize ascorbate resulting in an EC catalytic reaction:
-2e.
DA
2H+
DOQ
DOQ + AA L-.DA + DHA This reaction occurs because the rate of electrochemical oxidation of ascorbate, a compound that is thermodynamically easier to oxidize than dopamine, is slow (29-32). As a result of this reaction, regenerated dopamine can return to the electrode,resulting in an enhanced current. The enhancement is not seen at fast scan rates because the time scale of the experiment is too short for the reaction to occur (31). The enhancement of current at slow scan rates decreases with the electrode radius because it is more difficult for the regenerated material to return to the surface of smaller-sized electrodes (32). The effect of this reaction at Nafion-coated electrodes was examined by determining the current a t 400 mV in solutions of 1mM ascorbate and 10 pM dopamine and by comparing this with those measured in solutions containing only the individual species. The solution composition was altered by the use of a flow injection apparatus, and measurements were made 10 s after exposure to fresh solutions. Currents were corrected by measurements in buffer alone, and measurements were made in triplicate. When the catalytic current is expressed as the ratio of the current in the mixture (corrected for the current from the ascorbate solution) to that measured in the dopamine solution, it is found the effect of catalysis is almost nonexistent at the Nafion-coated carbon electrodes of micron dimensions (ratio = 1.16 f 0.17 for four electrodes with r = 1.7 f 0.4 pm) and is relatively small at the Ndion-coated glass-encased, carbon-fiber electrodes (ratio = 2.32 f 0.30 for four electrodes with r = 6.9 f 0.6 pm). Electrodes Positioned Adjacent to Single Cells. Figure 4 shows both an etched and glass-encased carbon-fiber microelectrode positioned near an isolated bovine adrenal medullary cell. The smaller size of the etched electrodes allows for easier positioning of the electrodes because of the smaller size of the insulating material. Although the electrochemical
Figure 5. Amperometric detection at isolated bovine adrenal medullary cells detected at glass-encased (A-C) and etched (D-F) carbon-fiber electrodes at 1 (A, D), 5 (B, E), and 10 p m (C, F). Measurements at each position were made simultaneously with the large and small electrode. Release of catecholamines was induced by a 3-s,100 pM nicotine exposure applied at 1.25 s.
radii of the electrodesdiffer by a factor of =4-5, the insulation thickness differs by =lo. For release measurements, the electrodes are positioned somewhat differently than shown in the micrograph. The electrode is placed so that its beveled surface is parallel to the cell surface. In this position, the glass-encased electrode blocks the view of the cell. Retraction of the electrode is done so that the reported distances are those from the beveled surface to the cell. Detection of Release from Single Cells. Exposure of a cell to 100 pM nicotine via the pressure ejection system leads to release of catecholamines that can be detected with a microelectrode placed adjacent to the cell (15). Nafion is not used in these experiments because the concentration of ascorbate is much lower than that of the catecholamines and also because the slow diffusion of catecholamines in the film distorts the temporal resolution (33). A typical amperometric measurement with the glass-encased, carbon-fiber (P-55) electrode 1pm away from a single cell is shown in Figure 5A. When nicotine is pressure-ejected onto the cell, the oxidation current is seen to increase, and to have a large number of sharp spikes. Fast-scan cyclic voltammetry demonstrates the current spikes are due to catecholamines (33). The spikes appear to be the direct detection of the vesicular release of catecholamines into the extracellular solution. Shown in panels B and C of Figure 5 are measurements obtained at the same cell and with the same stimulus, but with the electrode placed a t 5 and 10 pm, respectively, from the cell surface. When the electrode is placed further away, the spikes appear to become smaller in amplitude. Figure 5D-F shows measurements made with an etched electrode a t distances from the cell identical with those in Figure 5A-C. Each measurement was made simultaneously with the adjacent trace at the large electrode to control for differences in cellular response. While the measured responses are similar with the electrodes placed 1 pm from the cell surface, the frequency of spikes (number per second) is seen to drastically decrease with the smaller electrode. Characteristics of Spikes. To further characterize the spikes with respect to electrode position and size, the results from measurements at several cells have been combined. The
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0
2
4
6
8
Etched Glass Encased
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Distance (/."
Flguro 8. The effect of distance on the medlan spike amplitude (A), medlan area (B), and average frequency (C) at two different electrode types. Measurements were made with each electrode at 1 (n = 4), 3 (n = 6). 5 (n = 4), and 10 F m (n = 3 cells). Error bars Indicate the standard error of the mean.
features that have been determined are the median spike amplitude and area and the mean frequency. The median was used because the type of distribution of the spikes has not yet been determined; it is not Gaussian but is skewed to higher values. The distribution will depend on the range of amounts in the vesicles and distortion caused by partial and unresolved multiple spike detection. The results are summarized in the graphs shown in Figure 6 for measurements at different distances from the cell with the two different types of electrodes employed. First consider the data recorded at the larger electrode. Consistent with the examples shown in Figure 5, the height of the spikes measured with the glass-encased carbon-fiber electrode is found to decrease with increasing distance between the electrode and cell. If the spikes are indeed from the extrusion of vesicle contents following fusion of the vesicle with the cell surface, this is expected behavior. The mean vesicle diameter is -400 nm, and thus, the origin of the concentration pulse can be considered as an instantaneous small source. As this packet of molecules diffuses toward the electrode, the concentration pulse spatially broadens by diffusion. When the electrode is far from the cell surface, this results in lower heights of the observed spikes. Such behavior has been predicted earlier for diffusional processes (34). The area of the spikes measured in the amperometric mode has the units of charge and is, therefore, a direct measure of the number of molecules detected. The areas remain constant with the larger electrode over the range of distances examined because the decrease in height of the spikes is accompanied by an increase in spike width. The relatively large size of the sensing surface and insulation material act to confine the secreted catecholamines to the region between the cell and electrode, thus facilitating detection. This effect is related to the effect of the size of the probe insulator observed in scanning electrochemical microscopy at insulators (35). The frequency of spikes, which is taken to be the frequency of vesicular fusion events on the portion of the cell surface sampled by the electrode, also does not change significantly as the electrode-cell spacing is altered. The area of spikes measured with the etched electrode 1 pm from the cell are the same as that with the larger electrode. As this electrode is moved further away, the median area and the frequency of the spikes decrease, unlike the results obtained at the large electrode. The amplitude decreases with distance, at both electrodes. The latter result suggests that the spikes of small amplitude may become buried in the
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background noise leading to the observed decrease in frequency. Furthermore, with the smaller electrode at distances far from the cell surface, only a portion of the secreted e c a u s ethe molecules are less confiied molecules are detected b in the space between the cell and the electrode, as with the glass-encased electrode. Thus, both the size of the electrode and the insulator play a role in the capture of diffusing speciea.
CONCLUSIONS Etched microelectrodes with poly(oxypheny1ene) insulation do not show ideal electrochemical behavior. The electrochemically determined radii depend on the scan rate, and the electrode adsorbs catecholamines. Because of amplifier noise, detection limits are higher with smaller radii electrodes. However, when used in the amperometric mode to detect secretion of catecholamines from isolated cells, this electrode provides information identical with that of glass-encased carbon-fiber electrodes when placed directly adjacent to the cell. In the amperometric mode, adsorption is not a concern because adsorbed molecules are immediately electrolyzed. Because of the overall reduction in size of the etched electrodes, they are advantageous with respect to positioning near small objects where chemical inhomogeneities may exist. ACKNOWLEDGMENT The aid of David L. Leszczyszyn in the initial stages of this work is gratefully acknowledged. We thank J. Janata for providing us with a preprint concerning electrode fabrication. Registry No. Dopamine, 51-61-6. LITERATURE CITED Wightman, R. M.; Wlpf, D. 0. E&traenel)accel Chnism;Marcel Dekker: New York, 1989; Voi. 15, pp 267-353. Fieischmann, M.; Lasserre, F.; Robinson, J.; Swan, D. J . E/ectroanal. Chem. InterfacialElectroctiem. 1984, 177, 97-114. ArmsWongJames, M.; Fox, K.; Millar, J. J . Newoscl. Methods 1980, 2. 431-432. Meulemans, A.; Poulain, 8.; Baux, G.; Tauc, L.; Henzel, D. Anal. Chem. 1986, 58, 2088-2091. Penner, R. M.; Heben, M. J.; Lewis, N. S. Anal. Chem. 1989, 61, 1630- 1636. Penner, R. M.; Heben. M. J.; Longin, T. L.; Lewis, N. S. Sckrnce 1990, 250. 1118-1121. Pendley, B. D.; Abruna, H. D. Anal. Chem. 1990, 62. 782-784. Saraceno, R. A.; Ewing, A. G. J . Electroanel. Chem. InterfacialEketrochem. 1988, 257, 83-93. Potje-Kamloth, K.; Janata, P.; Janata, J.; JosowIcz, M. Sens. ActuatWS 1989. 18, 415-425. Potje-Kamloth. K.; Janata, J.; Josowicz, M. Ber. Bumsen-Gss. phvs. Chem. 1989, 93. 1480-1485. Bard, A. J.; Fan, F A ; Kwak, J.; Leo, 0. Anal. Chem. 1989, 61, 132- 136. Bard, A. J.: Denauit, G.; Lee. C.: Mandler. D.; Wbf, W. 0. Acc. Chem. Res. 1990, 23,357-363. Engstrom, R. C.; Pharr, C. N. Anal. Chem. 1989, 61, 1099A-1104A. Chien, J. B.; Wallingford, R. A.: Ewing. A. G. J .." 1990, 54, 633-638. ... - - -. Leszczyszyn, D. J.; Jankowski, J. A.; Viveros, 0. H.; Diiiberto, E. J., Jr.; Near, J. A.; Wightman, R. M. J . Blol. Chem. 1990, 285, 14736-14737. Vabrta, F.; Fesce, R.; Grohovaz, F.; Haimann, C.; Hurlbut, W. P.; Iezzl, N.; Torri Tareiii, F.; Villa, A,; Caccareiii, 8. N6woscksnce (Oxford) 1990, 35. 477-489. Coupland, R. E. Netwe 1968. 217. 384-388. Wlnkler, M.; Westhead, E. Neuroscience (Oxford) 1980, 5 , 1803- 1823. Kelly, R. S.; Wightman, R. M. Anal. Chlm. Acta 1988, 187, 79-87. Baur, J. E.; Kristensen, E. W.; May, L. J.; Wiedemann, D. J.; Wightman, R. M. Anal. Chem. 1988, 60, 1268-1272. Krlstensen, E. W.: Kuhr, W. 0.; Wightman, R. M. Anal. Chem. 1987, 59, 1752-1757. Baur. J. E.; Wightman, R. M. J . Electroanal. Chem. InterfacialElect r o d " . , in press. ShouD. D.:Szabo, A. J . Electroanal. Chem. InterfaclelElecbochem. 1984; 160. 27-31. Michael, A. C.; Wightman, R. M.; Amatore, C. A. J . Electfwnal. Chem. InterfacialE k t r d " . 1989. 276. 33-45. Kovach. P. M.; Deakin. M. R.: Wightma'n, R. M. J . phys. Chem. 1986, 90. 4812-4617. Deakin, M. R.; Kovach. P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1986, 58, 1474-1480. Howell, J. 0.; Kuhr, W. G.; Ensman, R. E.; Wightman. R. M. J . E&boana/. Chem. Interfaclel Elecm3?8m. 1988, 209, 77-90. Wightman, R. M.; May, L. J.; Michael, A. C. Anal. Chem. 1988, 80, 769A-779A.
1594
Anal. Chem. 1991, 63,1594-1599
(29) Stutts, K. J.; WlgMmen, R. M. Anel. Chum. 1989, 54, 1576-1579. (30) Wbert, M. 8.; Curan, D. J. AM/. C h m . 1986, 58. 1028-1032. (31) Stamford. J. A. Anal. Chum. 1986. 58. 1033-1036,
(35) Kwak, J.; Berd, A. J. Anal. Chem. 1989, 61, 1221-1227.
Determination of Trace Metals in Crude Oil by Inductively Coupled Plasma Mass Spectrometry with Microemulsion Sample Introduction Charles J. Lord, I11 Phillips Petroleum Company, Research and Development, Bartlesuille, Oklahoma 74004
A new technique Is doscrlb8.d for Introducing crude dk directly into an Inducthrdy coupkd plasma mass spwtmmder. Thk technique k based on the formation of ollin-water mi-c and greatly sbnpWe8 the determination d trace metals In 011. The advantages of the mkroemuldon ICPMS method Include rapid daectbn, ease of sample preparatlon, long-term sample stablltty, and low detection #mik. Accuacyandprechbnareon~orckrdf3%. olr that contain metals In the concentration range of 0.1 to several hundred parts per m#kn can be analyzed tarthdy. The upper end of thb range can be extended by dmpb aqueous dliutlon of the m k r o e " Became the sample rdutkns are composed prhnarlly of water, there Is no clrrbon bulldup based on the mass spectrometer Interface. Other p e t " materlals such as gasollnes, dlesel fuels, luklcallng olk, residual oils, and asphaltenes can ako b analyzed with thls methodology.
INTRODUCTION There is a need in the petroleum industry to quantify trace metal concentrations in a wide variety of organic materials. Because of its multielement capability and low detection limits, ICPMS (inductively coupled plasma mass spectrometry) is an attractive technique for this application. In aqueous solutions, ICPMS detection limits for most elements are in the sub parts per billion range (1). Theoretically, these low limits of detection should also be attainable for organic solutions. Unfortunately, a number of technical difficulties exist that complicate the direct analysis of organics and degrade the ICP mass spectrometer performance. The difficulties associated with the analysis of carbonaceous materials are discussed in the following paragraphs. It is well documented that the introduction of organic solvents into an ICP can dramatically alter the physical properties of the discharge (2-7). The significance of this alteration can range from minor changes in analyte signal intensities to total extinction of the plasma. Substantially higher energy is required to completely dissociate organic solvent vapors when compared to an equivalent quantity of water vapor (8). As a result of this endothermic process, volatile organic solvents create a plasma cooling effect that can cause unpredictable variations in analyte signal intensities. 0003-2700/9 110363-1594$02.50/0
Complex interrelationships exist between solvent vapor properties, aerosol transport efficiencies, ICP temperature profiles, and the distribution of analyt.8 species in the plasma (7,9). At the present time, there is no theoretical basis for accurately predicting the analytical consequences produced by these solvent-related effects. Although the papers cited above deal with optical ICP measurements, an analogous situation applies to ICPMS analyses. The accuracy and detection limits of the ICP mass spectrometer are severely degraded when high concentrations of organic solvent are injected into the plasma. The aerosols and vapors generated from these solvents can lower the axial temperature in the plasma and thereby suppress analyte ion production. The introduction of organics into the plasma also leads to the formation of carbon-containing ions such as C2+, C02+,and Arc+. When a quadrupole mass analyzer is employed, these polyatomic ions create interferences in the elemental mass spectrum and can seriously compromise trace level analyaea of certain elements. In addition, organic solvents produce sooty carbon deposits that rapidly foul and plug the orifices of the metal cones that serve as the interface between the argon plasma and the mass spectrometer. This orifice fouling phenomenon dramatically attenuates the ion count rates. In light of these complications, it is clear that substantial benefits can be obtained by limiting the input of organic carbon to the plasma. The traditional technique for eliminating the problems associated with organic matrices is to wet ash the sample prior to analysis by using an acid digestion procedure. The resulting solution is an aqueous matrix that is compatible with standard ICPMS inetrumentation (IO). The disadvantages of the acid digestion method are (a) the procedure is time consuming and severely limits the rate of sample throughput; (b) trace metal contamination from the acid reagents, digestion vessels, and airborne particulates can jeopardize the accuracy of the analytical resultq (c) certain elements such as barium and lead can form insoluble precipitates when sulfuric acid is used to digest the samples; and (d) quantitative recovery is not guaranteed for elementa such as mercury, boron, and selenium, which can be volatilized from the hot acid solutions. The direct dilution of petroleum based materials with an organic solvent is an attractive sample preparation procedure because it is rapid and simple. It can be easily automated and there are no element volatility or precipitation reactions to contend with. Unfortunately, the problems associated with 63 1991 Amerlcan Chemlcal Society
