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Anal. Chem. 1987, 59, 1863-1867
1:150 for ethanol and acetaldehyde, respectively). In a previous paper (5) this problem was solved by using a diode array spectrophotometer, allowing for application of dilution and amplification methods (11). In this paper the problem was solved by using the optimum working conditions for determination of acetaldehyde and by circulating the plug for determination of ethanol through a very long and wide reactor (rJ. This reactor (621 cm long, 0.7 mm i.d.1 exerts two recognizable effects: (a) It provides a longer residence time for the ethanol plug, whose peak appears after that of acetaldehyde has attained the base line. (b) It dramatically dilutes ethanol in plug VI, making possible the use of sample dilution suitable for acetaldehyde (1:150) in the simultaneous determination. With this manifold, a calibration curve for acetaldehyde was run between 0.9 and 15.0 pg/mL: absorbance = 0.012 (f0.008) 0.0272 (fO.0001) [acetaldehyde]; r2 = 0.996; the RSD being *0.4% (11 determinations of 7.0 pg/mL). For ethanol, the experimental points were ajusted to a logarithmic curve in the range 0.025%-0.800% (v/v): log absorbance = 0.825 (f0.005) 0.509 (fO.009) log [ethanol]. The sampling frequency was 46 h-l. The recovery of both species in different types of wines was assayed by adding 0.1% of ethanol + 3.5 pg/mL of acetaldehyde and 0.2% ethanol + 7.0 pg/mL of acetaldehyde to the samples. From the experimental data were calculated a mean recovery of 100.4% and 100.1%, and an average deviation from 100% of f2.370 and f1.370 for ethanol and acetaldehyde, respectively (see Table IV).
+
+
CONCLUSIONS A comparative study between methods involving dissolved ( 5 ) and immobilized enzymes (see Table V) leads to the following conclusions: (a) Precision, expressed as RSD, for the use of immobilized enzymes is equal to of higher than with dissolved ones. (b) The use of immobilized enzymes dramatically widens the linear range of the calibration curve, increasing the ratio between the lower and upper limit at least 2-3-fold with respect to that achieved with dissolved enzymes. The determination limit for ethanol is similar in both methods; yet, while in the individual determination of acetaldehyde the lower determination limit corresponds to methods with im-
mobilized enzymes, in the simultaneous determination the lower determination limit is achieved in the method with dissolved enzymes. This apparent contradiction can be clarified by considering that the use in the last case of a diode array detector, which allows application of a method for amplification of the analytical signal, results in increased sensitivity and a lower determination limit (11). (c) Enzyme consumption per analysis is considerably lower (between 7and 16-fold) for immobilized enzymes. (d) The recovery is better when enzymes are immobilized and so is the stability and convenience. (e) The sampling frequency is only slightly higher for dissolved enzymes. All these arguments testify to the suitability of the proposed methods for use in enological control laboratories.
ACKNOWLEDGMENT We gratefully acknowledge “Gonzdez Byass” for providing samples of wine. Registry No. EtOH, 64-17-5; acetaldehyde, 75-07-0; alcohol dehydrogenase, 9031-72-5; aldehyde dehydrogenase, 9028-88-0. LITERATURE CITED Yao, T. J . Flow Injection Anal. 1985, 2(2), 115. Ribereau-Gayon, J.; Peynaud, E.; Sudrau, P.; Riberau, P. Trait6 d ’ E n ologie. Sciences Techniques do Vin; Tome I. Dunod: paris, 1976. Lizaro, F.; Luque de Castro; M. D., Valclrcel, M. Enologia Enotecnica , in press. Worsfold, P. J.; Ruzicka, J.; Hansen, E. H. Anal. Chim. Acta 1981, 106, 1309. Lizaro, F.; Luque de Castro, M. D.; Valcircel, M. Anal. Chim. Acta 1986, 185, 87. Kiba, N.;Tomiyasu, T.; Furusawa, M. Talanta 1984, 31, 131. Bernt, E.; Gutrnann, I. I n Methods of Enzymatic Analysis. 2nd ed.; Bergmeyer, H. U., Ed.; Verlag Chemie: Weinheim, and Academic: New York, 1974; Vol. 3, pp 1499-1502. Lundquist, F. I n Methods of Enzymatic Analysis, 2nd ed.; Bergmeyer, H. U., Ed.; Veriag Chemie: Winheim, and Academic: New York, 1974; Vol. 3, pp 1509-1513. Official Methods of Analysis, 12th ed.; Association of Official Analytical Chemists: Washington, DC, 1975; p 603. Masoom, M.; Townshend, A. Anal. Chim. Acta 1984, 166, 111. Lizaro, F.; Rios, A,; Luque de Castro, M. D.; Vaicircel, M. Anal. Chim. Acta 1988, 179, 279.
RECEIVED for review December 3, 1986. Accepted March 9, 1987. The “Comisi6n Asesora de InvestigaciBn Cientifica y TBcnica” is thanked for financial support (Grant No. 2012/83).
Electrochemical Pretreatment of Carbon Fibers for in Vivo Electrochemistry: Effects on Sensitivity and Response Time Jian-Xing Feng,’ Michael Brazell, Kenneth Renner, Richard Kasser, and Ralph N. Adams* Department of Chemistry, University of Kansas, Lawrence, Kansas 66045
Oxidative electrochemical pretreatment of carbon flbers greatly improves thelr sensitivity for in vivo electrochemical detection of catecholamine species. I t is shown that the extent of the anodic potentlal excursion In the pretreatment is a major factor in both the sensltivlty and the response time of the resulting flber electrode. The high sensitlvlty for neurotransmitter specles such as dopamlne appears malniy due to adsorptlon on the oxldized carbon fiber surface states. Practlcal protocols for flber electrodes to be used In In vivo brain studies are evaluated. Permanent address: N a n k a i University,
Republic of China.
Two quite diverse applications have spurred studies of the electrochemical pretreatment of carbon fibers in recent years. The fibers are used extensively in resin composites to provide high strength-low weight structural materials, especially for jet aircraft. Oxidative treatments are known to improve the fiber-resin bonding and electrochemical oxidation is a versatile means of pretreatment. Sherwood and co-workers have utilized a variety of spectroscopic techniques to study the surface states of these pretreated fibers (1-5). Although of far less industrial importance, equally intense interest has developed in the use of carbon fiber microelectrodes as in vivo electrochemical detectors to detect biogenic amine neurotransmitters and their metabolites in the extracellular fluid
0003-2700/87/0359-1863$01.50/0 0 1987 American Chemical Society
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
(ECF) space of the brain (6-8). In this case, electrochemical pretreatment enhances resolution and sensitivity for brain measurements (9-12). The surface spectroscopy studies mentioned above provide ample evidence that various electrochemical pretreatments of carbon fibers give rise to surface oxide states (1-5). Indeed, an extensive literature illustrates that pretreatments of pyrolytic graphite, glassy carbon, and other carbonaceous materials used in electroanalytical practice produce some form of oxidized surface state, but their exact nature remains elusive. Kuwana and co-workers have reviewed the complexity and practical difficulties of analyzing the surface states of these and other chemically modified electrodes (13). The present study was undertaken to better understand the response characteristics of pretreated electrodes when used for in vivo electrochemistry. Electrochemically pretreated fibers have much greater sensitivity for catecholamines than do their untreated counterparts. However, they sometimes require considerable time to respond to changes in catecholamine concentrations, a disadvantage for rapid in situ measurements. These response characteristics have been studied as a function of pretreatment parameters, and several factors of primary concern for in vivo voltammetry applications have been elucidated. Another advantage provided by electrochemical pretreatment is the shifting of the oxidation potentials of catecholamines, ascorbate, etc., which results in improved voltammetric selectivity. While this is an important advantage, it was not part of this study. We are aware that in a study such as this one must be careful not to over-generalize about the effects of pretreatment. This investigation does not examine the nature of the surface states nor their possible involvement in electrocatalysis; it is limited to the effects of pretreatment on sensitivity to biogenic amines and electrode response time. While we do feel that the conclusions of these studies will apply qualitatively to most fibers used for in vivo electrochemistry, there will be quantitative differences. Fibers are prepared by various hightemperature graphitization procedures from different commercial polymer threads. In addition to size (diameter) differences, each brand has its own composition and thermal history, which may produce slightly different surfaces after a particular electrochemical pretreatment.
EXPERIMENTAL SECTION The carbon fibers used in this study were 3C-40 pm in diameter and were obtained from AVCO, Lowell, MA. The surface of these fibers has no external coating. A single fiber was placed inside a glass capillary (Kimble No. 34507) and the glass-fiber combination pulled on a vertical electrode puller (David Kopf, Tujunga, CA). With fine forceps, the glass was broken just beyond the point where it could be seen to join the fiber, and a suitable length of fiber was exposed. A good seal was achieved by backfilling the capillary with a low viscosity epoxy (Shell 815 Resin) mixed with graphite (UltraCarbon, Bay City, MI). The graphite resin mixture was loaded into the capillary with a syringe. Electrical contact was made by pushing a copper wire down the tapered neck of the capillary. After backfilling, the electrodes were placed vertically in an oven at 60 "C for 10 min to seal the fiber-glass junction. Following the curing, the exposed fiber was carefully cut to the desired length under a 40X microscope by using fine scissors. Normally, 2S300-pm lengths are employed for in vivo studies. In this work lengths (I) of 150, 300, 350, and 600 pm (cylindrical electrodes) were used, as well as ones cut flush with the capillary electrode. The latter, with 1 = 0, are designated disk electrodes. Their end surfaces were not polished and, depending on the cut, can be far from a true disk. It is evident that surface oxidation can be achieved by constant potential anodic electrolysis,potential cycling, controlled current electrolysis, or combinations of these approaches. However, those who use these electrodes for in vivo applications are not best served
by suggesting that they choose pretreatment techniques based on mechanistic details of electrochemical methodology. Thus we chose, as a starting point, the original pretreatment of Gonon et al. (6, 9) for 8-pm fibers. This essentially consists of a 0 to +3 V (vs. Ag/AgCl), 70-H~ triangular wave applied for a few seconds. This was modified by using lower frequencies and decreasing the potential excursion. The use of an anodic potential of +3 V in aqueous media is an "overkill" for surface oxidation-a considerable portion of the current goes to oxygen generation or other solution oxidation reactions. (It may be true that some of these processes are beneficial in the practical pretreatment.) It is also true that the real potential of the fiber is less than +3 V due to uncompensated tR loss (&). This loss can be estimated for microelectrodes as shown by Robinson et al. (14) and is ordinarily of little consequence when only picoamperes of current pass during in vivo applications. However, with the 0.2-0.4 mA currents that flow during pretreatments, if a given fiber resistance is several hundred ohms or more, the iRu loss can be appreciable. The pretreatment of fiber electrodes with high resistance often gives poor results, as indicated earlier (11).It suffices to say that Pretreatments that provide excellent sensitivity for catecholamines can be obtained with cyclic potential excursions only t o +1.3 or +1.5 V vs. Ag/ AgC1. The fiial pretreatments used herein are of two types, depending on the anodic potential excursion. The first, which produces fibers designated as high potential pretreated (HPP), cycled the potential from 0.0 to +2.6 V vs. Ag/AgCl for 30 s, using a 6-Hz triangular wave. This, in potential excursion, is close to the original pretreatment of Gonon et al. (6,9).The second style gives low potential pretreated (LPP) electrodes and employs the same 6-Hz triangular wave for 30 s with the smaller potential range of 0.0 to +1.3 V vs. Ag/AgCl. At the end of the pretreatment time, electrodes were physically disconnected from the potentiostat circuit and allowed to stand for 5 min in the buffer medium before further testing. The cycling frequency was lowered from the 70 Hz originally described (9) to 6 Hz (any convenient value in the range 1.5 to 6 Hz is satisfactory) because this rate is available on most commercial voltammetry instruments or laboratory-designed cyclic voltammetry equipment. Any equipment with current capability of 0.5 mA or more is satisfactory for pretreatments. The results and discussion will indicate how workers may need to modify the pretreatment protocols slightly to best suit their fibers and experimental requirements. The pretreatments themselves, as well as various cyclic and pulse voltammetry studies, were carried out with an EC 225-2A voltammetric analyzer (IBM Instruments) or a PAR-174 with a laboratory-built triangular wave imput. All pretreatments and electrochemical studies were carried out in phosphate-buffered saline solution, which is the background medium of choice for in vivo electrochemical studies. This solution, frequently abbreviated PBS, will be herein referred to simply as buffer. It contained 11.5 g of Na2HP04,2.47 g of NaH2P04.2H20,and 9.0 g of NaCl per liter with the pH adjusted to 7.5 by small additions of NaOH or HC1 as required. Dopamine hydrochloride (DA) and 5-hydroxytryptamine (5-HT) were obtained from Sigma Chemical Co. All other chemicals were reagent grade and all solutions were prepared from double-distilled water. The response times of electrodes were determined with a short-coupled type of gravity-induced flow injection apparatus. A small piece of ca. 50-pm-diameter polyethylene tubing was attached to the output of a Hamilton four-port valve (86729, Alltech, Deerfield, IL). The inlet ports were connected to elevated reservoirs of buffer only and buffer plus test solution (25 pM DA; 50 pM potassium ferrocyanide, or other electroactive system). Fast, uninterrupted flow switching of buffer or buffer-test solution was obtained by 90" rotation of the valve handle. The fiber electrodes were lowered a fixed depth into the outlet tube (reference and auxiliary were placed externally in the overflow) and the electrochemical response was monitored as the amperometric signal to +0.6 V vs. Ag/AgCl. A microswitch activated by a cam in the valve handle gave a timing pip on the recorder trace when the valve reached the full-open position. Two measurements were always recorded-an approximation of the earliest detectable amperometric signal, taken as 10% of the maximum response ( t 3 , and that for 90% of the maximum (tso).
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987 Ob
05
01
03
01
01
Table I. Capacity and Sensitivity of Carbon Fiber Electrodes after Electrochemical Pretreatment‘ geometric fiber length, pm 0 (disk) 150 300 450 600
Figure 1. Charging current measurements of untreated carbon fibers, charging current curves in buffer at indicated potential sweep rates.
The best estimate of the linear distance from the valve inner switching port to the electrode was ca. 2 cm. A volume flow rate of 1mL/min was used for all experiments. From the measured dimensions, the linear flow rate was approximated at 5 cm/s, which gives a theoretical value of 0.4 s for the time it took a switched pulse of electroactive species to move from the inner portion of the valve to the electrode tip. The oxidation of ferrocyanide gives rapid Nernstian behavior at platinum, so the response of a small platinum wire to 20 fiM ferrocyanide in buffer was used to test the apparatus. The value of tlo for ferrocyanide at platinum was 0.61 f 0.02 s (standard deviation) for eight determinations. Considering the geometric approximations as well as the hand-switching and pen recording system used, this agreement (within 100-200 ms of the calculated value) is quite satisfactory. A more sophisticated flow apparatus, which can examine pulsed changes in electroactive species with greater accuracy, was recently described by Kristensen et al. (15). As discussed later, our apparatus was used only to measure the relative changes in response time as a function of carbon fiber pretreatment. While quite precise measurements of tlo and t90 could be made, their absolute values are not to be taken as significant. The observed capacitance, C or Cow, of fiber electrodes before and after pretreatment was determined with i, = C dE/dt where i, is the charging current and dE/dt the potential sweep rate. Charging current curves in the buffer solution were determined from cyclic voltammograms obtained by using a potential sweep range from -0.2 to +0.6 V vs. Ag/AgCl. The filter of the IBM instrument was set at 0 for these measurements. By use of a wide range of sweep rates, charging current curves were generated and i, was evaluated from the anodic sweeps at a potential of +0.3 V. C at about +0.3 V is of interest because it is approximately the potential where catechols oxidize.
RESULTS AND DISCUSSION A. Capacitance and Sensitivity of Untreated Fiber Electrodes. Figure 1illustrates voltammograms obtained at a variety of sweep rates. The corresponding plots of i, (at +0.3 V) vs. sweep rate were all linear and C was evaluated from the slope of the latter plots. C was measured for cylindrical (length, 1 = 150, 300, 450, and 600 pm) and flush-cut disk electrodes ( I = 0) both before and after the electrochemical pretreatment protocol. There was a linear relationship between the observed capacitance and surface area for the untreated carbon fibers. For the disk electrode (1 = 0) the measured Cobsdwas 0.3 nF. With its very small area of -1.3 X cm2, its capacitance is about 23 pF/cm2. The value for the four cylinders of different lengths was calculated from the slope of the linear plot of C vs. area and is 6.8 F/cm2. The capacity characteristics of fiber electrodes appear not to be isotropic; Cobad of the end surface (disk, 1 = 0) is some 3.4 times greater than that of the cylindrical surface. Although the respective surface roughness could not be measured easily, i t is likely that the
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A,b,ccm2 1.3 X 2.0 x 3.9 x 5.8 x 7.7 x
10-4 10-4 10-4 10-4
CObsd/unit area, pF/cm2
sensitivity/ unit area, pA/cm2d
167 115 126 119 118
37 27 33 29 29
“High potential pretreatment, HPP, 0 to +2.6 V, 6 Hz, 30 s. bCalculatedarea from A = 1rr2(disk) + 21rrl (cylinder),where r = fiber radius and I = exposed fiber length, both in cm. e With the optical measuring equipment available, geometric areas are obtainable only to two significant figures. d1.6 X lom4M 5-HT in buffer solution used to determine Deak current in DPV mode. end surfaces have greater roughness and certainly more irregular geometry. It is also very likely that this difference gives a t least some of the apparent anisotropy. The sensitivity experiments were carried out by using differential pulse voltammetry (DPV) with hE = 50 mV, a potential sweep rate of 30 mV/s and sweep range from 0.0 to +0.6 V. The DPV peak current of a solution of 1.6 X M 5-HT in buffer solution was measured (EP= ca. +0.33 V vs. Ag/AgCl) a t each of the different length electrodes. The relationship between DPV current (ip) and electrode area was linear. If one calculates the sensitivity per unit area for this concentration of 5-HT, the value for the disk ( I = 0) is 11 pA/cm2. The value for the cylindrical surface, however, is only 2.4 pA/cm2. The former is about 4.5 times greater than the cylinder surface. Considering the uncertainty in estimating geometric areas for these fibers, this result is quite similar to the experimental capacitance differences observed for the two surfaces. B. Capacitance and Sensitivity of Carbon Fiber Electrodes after Electrochemical Pretreatment. CONand DPV sensitivities were carried out exactly as in the previous experiments using the same fiber electrodes, which now had been subjected to the H P P electrochemical protocol. The results are summarized in Table I. It can be seen that both the capacity and sensitivity of the electrode (for 5-HT oxidation) increase by almost an order of magnitude after pretreatment. The relative differences in capacity for the disk and cylinder surfaces are more nearly equal after pretreatment. It appears that with untreated carbon fibers the cylindrical or parallel surface is partially insulating in nature, resulting in low Cow and faradaic sensitivity, and this partial insulator effect is mostly removed by the rather drastic electrochemical pretreatment. It may be that not all fibers will show this anisotropy effect to the same extent, depending on their carbon structure or manufacturing differences such as external coatings, etc. Furthermore, the increase in sensitivity with pretreatment has some unusual characteristics, as shown in the next section. C. Sensitivity of Carbon Fibers to Ferrocyanide and Dopamine Oxidation as a Function of Pretreatment. A thorough examination was made of the voltammetric sensitivity of fibers for ferrocyanide vs. DA oxidation, before and after pretreatment. A total of eight electrodes, four each for ferrocyanide and DA, were cut as close as possible to 250 pm in length. This is a typical length used for in vivo work in our laboratory. The cyclic voltammograms of each compound were measured before and after the standard HPP (0 to +2.6 V, 30 s). A sweep rate of 100 mV/s was used, and all measured currents were corrected for background scans run with each experiment. Data from the four electrodes were averaged. They are plotted in Figure 2 in log-log form to accommodate
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
Current lnAl
2 5 pM DA
5pM DA
I
I I
25
UNTREATED
A
TREATED
Figure 3. Cyclic voltammograms of DA and ferrocyanide at untreated and pretreated fibers: potential sweep rate, 100 mV/s for all scans.
buffer solution, gives a memory effect, i.e., voltammetry shows surface-bound catecholamine response. Such behavior is an example of adsorption onto electrode surfaces. The surface response can be removed by a few cyclic scans. Qualitatively, the acid metabolites (like 3,4-dihydroxyphenylacetic acid, DOPAC) appear to be less adsorbed. This memory effect of DOPAC on pretreated fibers was first noted by Gonon et al. (9).
If then, as indicated above, the major part of the electrochemical signal for catecholamines at pretreated carbon fibers is due to adsorbed species, how do the electrodes correctly follow solution concentrations and function as in vivo electrodes to detect changes in the brain ECF? The answer is that only very low concentrations of catecholamines have been of interest for in vivo studies and this has fortuitously made the measurements fairly reliable. Calibration concentrations for in vivo work rarely exceed 100 pM and the ECF concentrations one wishes to detect vary from nanomolar only up to low micromolar levels. These concentrations presumably all lie on an approximately linearly rising portion of the adsorption isotherms for catecholamines, and so the adsorbed levels (at equilibrium) are representative of solution values. For DA at the pretreated fibers described herein, the linear concentration range extends to ca. 200-250 wM-at higher solution concentrations the response increment markedly decreases and a typical adsorption plateau is seen. At 6W700 KM,the pretreated fiber is only minimally sensitive to further increases of DA in solution. Fortunately for in vivo electrochemistry, even 200 pM is beyond physiological reality. As the literature attests, the electrodes have functioned quite well a t the low levels encountered. However, with such luck comes a corresponding price to pay. If one wishes to measure rapid in vivo responses, pretreated electrodes may take considerable time to reach their maximum response. The adsorption equilibrium, or some associated step, can be slow, and the slowness of response is directly related to the nature of the pretreatment-particularly the anodic potential to which the fiber is cycled. This factor and its manipulation are discussed next. D. The Slow Response of Pretreated Fibers to Biogenic Amines. There is nothing unusual or unexpected in the charging current behavior of carbon fiber electrodes. Despite the relatively high capacitance of 250-wm-length pretreated fibers (ca. 100 wF/cm2), their very small areas give values of actual Cobsdaround 30-50 pF. If one assumes as a worst case an uncompensated resistance of 1MQ, then the time constant (R,Cobd) of these electrodes is ca. 40 ms. Thus, the top limit for essentially all charging current decay can be considered as 5RC or ca. 200 ms. All carbon fibers in this study (pre-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
treated or not) gave buffer background currents, which decayed to almost zero in about 200 ms or less. Nor do pretreated fibers respond slowly to the oxidation of 20 pM ferrocyanide in buffer solution. Measured in the flow apparatus described earlier, five fibers subjected to the L P P (0 to +1.3 V, 6 Hz, 30 s) gave tlo and tw of 0.67 f 0.06 and 0.76 f 0.07 s, respectively. These values are not much different from those obtained with ferrocyanide at platinum wires. (At platinum, ferrocyanide oxidation gives essentially Nernstian behavior.) The H P P (0 to +2.6 V, 6 Hz, 30 s) rendered them a bit slower to ferrocyanide response, with tlo and tgOof 0.81 f 0.13 and 1.02 f 0.23 s, respectively. The response time to DA was also not increased much by the LPP-the tlo and tw of 9 fibers were 0.67 f 0.10 and 0.92 f 0.31 s. In contrast, these same electrodes subjected to HPP had t I oand tw values of 1.48 f 0.06 and 4.32 f 0.33 s. If one pretreats at even higher potentials, there is a pronounced increase in response time. Typically, a fiber treated at (2-3.2 V and 6 Hz for 30 s increased its tw for 10 pM DA to 8.0 s. When the anodic excursion of the pretreatment potential exceeds about +2.4-2.6 V vs. AgJAgCl, the surface oxidation states produced have much different properties than those obtained at lower potentials, resulting in a much prolonged response time. Since this delayed response is primarily observed with the biogenic amines and not with the nonadsorbed species ferrocyanide, one is tempted to relate it to some slow step in the adsorption. However, there may be other surface factors that contribute to the effect. It can be noted that other modes of electrochemical measurement (i.e., pulse voltammetry) may show even slower apparent response times for fibers pretreated at high anodic potentials. E. Significance of Pretreatment Styles. It is certainly true that the sensitivity of carbon fibers for biogenic amines increases markedly with increasing anodic potential pretreatments, and that this is a significant advantage. As has been shown, however, the highest sensitivity is gained at the expense of increased response times. The latter is of no particular consequence if one is studying, for example, systemic drug effects where the biogenic amine release into extracellular fluid gives rise to rather slow and large concentration variations. However, if one wishes to measure very transient pulses of biogenic amine overflow into extracellular fluid, which result from brief electrical stimulations or physiological stimuli, then the slow response electrodes are clearly unsatisfactory. For example, Kuhr and Wightman (20) have recently shown that very minimal electrical stimulation of the medial forebrain bundle produces dopamine release in the rat caudate, which can be detected by high-speed voltammetry. The maximum change in DA concentration persists for only 1-2 s. In such a case, electrodes that have response times of this magnitude or greater can at best “see” only some fraction of the available signal. Thus, sensitivity and response time are trade-offs which need to be tailored through pretreatment to the requirements of the in vivo experiment. The best compromise of properties for the carbon fibers used in this study appears to be the LPP at anodic potential excursions no greater than +1.3 V. Such electrodes have very good sensitivity for catecholamines, yet have response times of 1s or less. The exact protocol we have found most useful is pretreatment of the fibers at 0 to +1.3 V and 6 Hz for 30 s in buffer, followed by disconnecting all leads to the electrode and allowing the electrode to “age” for 5 min in the buffer. This aging process seems to give the fastest response times available. A similar aging after the H P P is of little or no help in reducing the response time resulting from this pretreatment. The surface state produced by the H P P appears to be quite different and inherently slow in its catecholamine response.
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The increases in sensitivity and delays in response times are believed to be due to some slow stage in the adsorptive interaction of the biogenic amines with the surface oxidation state on the carbon fiber, which is produced by the electrical pretreatment. While this paper was in preparation, a preliminary publication from Matauda’s laboratory (21) appeared on the behavior of dopamine at carbon fiber electrodes. These workers, using similar pretreatment procedures, concluded that multilayer dopamine adsorption occurs on strongly pretreated carbon fibers. Although they did not investigate response times, all elements of our studies are in good agreement with their findings and recent work by Kovach et al. (16). We believe that the present work can provide a sound rationale for choosing pretreatment protocols for carbon fibers used for in vivo electrochemistry applications. Different fiber types and the nature of the in vivo experiments will dictate slightly different pretreatment needs. It is recommended that future applications of pretreated fibers include experimental checks of the electrode response times. Once again, it must be emphasized that while we believe that the conclusions of this work will apply qualitatively to most pretreated carbon fiber electrodes, quantitative differences will surely exist because of different interactions between various pretreatment techniques and the composition and manufacturing mode of various commercially available carbon fibers.
ACKNOWLEDGMENT We thank R. J. Suplinskas for advice and for generously supplying the carbon fibers used. LITERATURE CITED Proctor, A.; Sherwood, P. M. A. J . Electron Spectrosc. Relat. Phenom. 1982,27,39-56. Proctor, A.; Sherwood, P. M. A. Carbon 1983,21,53-59. Proctor, A.; Sherwood, P. M. A. S I A , Surf. Interface Anal. 1982,4 ,
212-2 19. Kozlowski, C.; Sherwood, P. M. A. J . Chem. SOC.,Faraday Trans. 1
1984,80,2099-2107. Kozlowski, C.; Sherwood, P. M. A. J . Chem. SOC., Faraday Trans. 1 1985, 81,2745-2856. Ponchon, J.-L.; Cespuglio, R.; Gonon, F.; Jouvet, M.; Pujol, J.-F. Anal. Chem. 1979,51,1483-1486. Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1980,52. 946-950. ArmstrongJames, M.; Miilar, J.; Kruk, 2 . L. Nature (London) 1980,
288, 181-183. Gonon. F. G.; Fombarlet, C. J.; Buda, M. J.; Pujol, J-F. Anal. Chem.
1981,53, 1386-1389. Piotsky, P. M. Brain Res. 1982,235, 179-184. Gonon, F.; Buda, M.; Pujol, J.-F. I n Measurement of Neurotransmitter Release I n Vivo; Marsden. C. A., Ed.; IBRO Handbook Series: Methods in the Neurosclences; Wlley-Interscience: Chichester, U.K., 1984: Vol. 6,Chapter 7. Cespuglio, R.; Faradji, H.; Hahn, 2 . ; Jouvet, M. In Measurement of Neurotransmitter Release I n Vivo, Marsden, C. A,, Ed.; IBRO Handbook Series: Methods in the Neurosciences; Wlley-lnterscience: Chichester, U.K., 1984; Voi. 6,Chapter 8. Karweik, D. H.; Miiier, C. W.; Porter, M. D.; Kuwana, T. IndustrialApplications of Surface Analysis; ACS Symposium Series, No. 199;Casper, L. A., Powell, C. J., Eds.; American Chemical Society: Washington, DC, 1982;Chapter 6. Robinson, R. S.;McCurdy, C. W.; McCreery, R. L. Anal. Chem. 1982.
54 2356-2361. ~
Kristensen, E. W.; Wilson, R. L.; Wightman, R. M. Anal. Chem. 1986,
58,986-988. Kovach, P. M.; Deakin. M. R.; Wightman, R. M. J . Phys. Chem. 1986,
90,4612-46 17. Kovach, P. M.; Ewing, A. G.; Wilson, R . L.; Wightman, R. M. J . Neurosci. Methods 1984, 1 0 , 215-227. Galus. Z.;Schenk, J. 0.; Adams, R. N. J . Electroanal. Chem. Interfacial Electrochem. 1982, 135, 1-1 1. Aoki, K.; Honda. K.; Tokuda, K.; Matsuda, H. J . Electroanal. Chem. Interfacial Electrochem 1985, 182,267-279. Kuhr, W. G.;Wightman, R. M. Brain Res. lS86,381, 168-171. Sujaritvanichpong, S.;Aoki, K.; Tokuda, K.; Matsuda, H. J . Electroa nal. Chem. Interfacial Electrochem. 1986, 198, 195-203.
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RECEIVED for review October 16, 1986. Accepted March 26, 1987. The support of this work by NIH Grant NS08740 and a grant from the Advanced Technology Commission and Oread Laboratories, Inc., is gratefully acknowledged.
