Anal. Chem. 1981. 53. 1386-1389
1380
and for typioal values (rn = 1 mg d,t d = 6 s) f = 52 Hz. It should always be possible, therefore, to eliminate the peak by increasing the square-wave frequency. For instruments which operate at fixed frequency, the peak position should move as the flow rate, delay time, initial potential, or step height are changed. As none of these parameters influence the peak position for a faradaic response, their systematic variation should identify unambiguously peaks due to the resonance phenomenon. Consider the instrument which operates a t 25 Hz. From eq 2 at 25 Hz
E,
- Ei
1.31 = AE- td
m
should ensure that in most practical applications it can be avoided.
LITERATURE CITED Ramaley, C.; Krause, M. S., Jr. Anal. Chem. 1889, 41, 1362-1365. Krause, M. S., Jr.; Ramaley, L. Anal. Chem. 1989, 41, 1365-1369. Christie, J. H.; Twner, J. A.; Osteryoung, R. A. Anal. Chem. 1977, 49, 1899- 1903. Turner, J. A.; Christie, J. H.; Vucovic, M.; Osteryoung, R. A. Anal. Chem. 1077, 49, 1904-1908. O'Dea, John J.; Osteryoung, R. A.; Osteryoung, Janet Anal. Chem. 1981, 53, 695-701. Yarnitzky, Chaim; Osteryoung, R. A,; Osteryoung, Janet Anal. Chem. 1980, 52, 1174-1178. Stojek, 2.; Osteryoung, Janet Anal. Chem. 1981, 53, 847-851. Brumleve, Tlmothy R.; O'Dea, John J.; Osteryoung, R . A.; Osteryoung, Janet Anal. Chem. 1981, 53, 702-706. French, A. P. "Vibrations and Waves"; W. W. Norton and Co.: New Yo*, 1971; p 121. Forham, S. Proc. R . SOC. London, Ser. A 1948, A194, 1
(4)
For reasonable values of m and t d (rn = 1.31 mg s-l, t d = 6s), E, - Ei = -5AE. Thus the peak position is not influenced strongly by changes in step height for normal operating conditions. To the extent the electrochemical system permits, the best way to move the resonant peak from the potential region of interest is to change the initial potential. The ability to influence the phenomenon through choice of parameters
RECEIVED for review March 23,1981. Accepted May 15,1981. This work was supported in part by Grant CHE7917543 from the National Science Foundation.
Electrochemical Treatment of Pyrolytic Carbon Fiber Electrodes F. G. Gonon,' C. M. Fombarlet,' M. J. Buda, and J. F. Pujol INSERM U 17 1, D6partement de M6decine Exp6rimentale, Universit6 Claude Bernard, 8, A venue Rockefeller, 69008 Lyon, France
Pyrolytlc carbon fiber electrodes Improved by electrochemical treatment appeared very useful for in vivo studies of dopamine metabolism in brain tissue. These treated electrodes used in comblnation wlth differentlal pulse voltammetry were capable of separating ascorbic acid from catechois and exhibited a very high sensitivity for some catechols. Parameters and experimental conditlons of this electrochemical treatment were explored.
In order to investigate the functional significance of brain catechols, it is of great interest to monitor their endogenous level from the brain tissue of live, freely moving animals and thus to avoid tissue sampling postmortem or tissue perfusion. Several in vivo voltammetric techniques have been developed for detection of catechol compounds which are synthesized by dopaminergic neurons (1-7). However, the major problem was the presence in brain tissue of high levels of ascorbic acid (AA)which oxidizes on untreated carbon electrodes a t the same potential as several catechols. Improvements of pyrolitic carbon electrodes by various treatments have been already reported. Kuwana and coworkers have treated pyrolytic graphite electrodes by radio frequency plasma in oxygen (8) and subsequent attachment of benzidine (9) or various quinones (10). Improvements of oxidation reaction of AA and of several other compounds have been demonstrated (9, 10). Blaedel and Mabbott (11) described electrochemical and chemical treatments of a pyrolytic carbon film electrode and thus obtained well-defined, reproducible current voltage curves. Various pyrolytic carbon fiber electrodes for in vivo application in brain tissue have been 'Present address: Laboratoire de Chimie Organique et Cingtique, Institut de Chimie et Physique Industrielles, 69002 Lyon, France. 0003-270018 1/0353-1386$01.25/0
recently developed (2, 7, 12, 13). The active surface of our electrode is a pyrolytic carbon fiber 8 pm thick and 0.5 mm long. In recent reports (14-17) we have shown that it is possible to resolve AA from catechols by means of this electrochemically treated carbon fiber electrode used in combination with differential pulse voltammetry. Then the catechol signal obtained in vivo from dopaminergically innervated tissue had been identified as the direct metabolite of dopamine (DA), Le., 3,4-dihydroxyphenylaceticacid (DOPAC) (24-17). Thus it appeared that this technique might be a valuable tool to study in vivo DA function. However, these in vivo studies give rise to several questions from the point of view of the electrochemistry. Actually, the separation of AA from catechol resulted from the electrochemical treatment which moved the AA signal to a more negative potential than that of the catechol signal. Moreover, the sensitivity of the treated carbon fiber electrode appeared 10 times higher for DOPAC than for AA and almost 100 times higher for DA than for DOPAC. Such improvement of sensitivity and selectivity of carbon fiber electrodes required very precise treatment conditions. This led us to explore in this paper several parameters of this treatment.
MATERIeLS AND METHODS Recording of the Voltammograms. A conventional threeelectrode system was connected to a pulse voltammetric system (PRGB Tacussel, Villeurbanne, France). The auxiliary electrode was a platinum wire. The Ag/AgCl reference electrode was prepared by stripping a 10 mm length of Teflon-coated silver wire (60.25 mm, Medwire Corp., Mt. Vernon, NY)and anodizing (+0.6 V for 1 min) in 1M hydrochloric acid. During experiments this AgCl-coated silver wire was directly immersed in the solutions. A commercially available Ag/AgCl reference electrode (C6 Tacussel, Lyon) has been occasionally used. Both electrodesobtained identical results. The potential of such reference electrodes slightly (+20 mV) differed from this of a calomel saturated electrode. The carbon fiber electrodes were prepared as previously described (7). 0 1981 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981
Figure 1. Voltammograms obtained with an untreated carbon fiber electrode. AA (200 pM) and DOPAC (20 p M ) were added to a PBS solution.
In order to obtain a complete polymerization of the resin, we kept these electrodes at 60 “C for 24 h. The parameters of the differentialpulse voltammetry used here were scan rate 10 mV/s and pulse modulation +50 mV in amplitude, 48 ms in duration, and 0.2 s in period. When phosphate buffered saline (PBS) solutions (KCl, 0.2 g/L; KH2P04,0.2 g/L; MgClz, 0.047 g/L; NaCl, 8 g/L; Na2HP04, 1.15 g/L; pH 7.4) contained AA and/or DOPAC, the concentrations of these compounds was always 200 pM (AA) and 20 fiM (DOPAC). The solutions were generally not deaerated since identical results were obtained with or without deaeration of solutions. AA, DA, and DOPAC solutions were tested at 25 “C within an hour following their preparations in order to avoid the influence of spontaneous oxidation. Electrochemical Treatments. Carbon fiber electrodes were immersed in the PBS solution. The first treatment was achieved by applying an alternating potential of a triangular wave form to the working electrode. In the standard conditions (as used in ref 14-17 and Figures 2 and 5A), the parameters of this treatment were the following: frequency of the alternating potential, 70 Hz; lower potential limit, 0 V, upper potential limit, +3 V, duration, 20 s. Several of these parameters have been independently investigated. The effect on the AA + DOPAC signal of various upper potential limits is shown in Figure 3 and the duration effect is in Figure 4. In these studies other parameters values were those of the standard conditions. In all these treatments the triangular wave form potential was applied via the PRG5 to the threeelectrode system. For this purpose the “pilot tension input” of the PRG5 was supplied by a conventional function generator (FG 600,Feedback Instruments Ltd., England; output impedance, 600 0). During the treatment the resulting current occurring through the working electrode was measured by means of an oscilloscope, the first channel of which was connected to the working electrode output in the back panel of the PRG5. The potential measured at this output is directly proportional to the current measured by the current-voltage amplifier of the PRG5 without filtering or damping. The second channel was connected to the pilot potential output to control the potential applied to the working electrode. The second treatment was always applied after the first one described above (standard conditions). It was achieved by applying to the working electrode a continuous potential of +1.5 V va. the reference electrode for 20 s. This was performed by means of the PRG5 alone, the “initial potential” of which was set at +1.5 V.
RESULTS Untreated carbon fiber electrodes gave poorly defined peaks a t +550 mV for DOPAC and +600 mV for AA (Figure 1). Moreover, these peak potentials varied from one electrode to another. The sensitivity of such electrodes was almost equal for AA and DOPAC (Figure 1). Mixed solutions of AA and DOPAC exhibited only one peak, which rapidly decreased in successive scans. When the standard treatment (0 to +3 V, 20 s) was applied to a carbon fiber electrode, voltammograms obtained from AA and DOPAC solutions exhibited two well-resolved peaks (Figure 2). AA peaked at -80 mV and DOPAC a t +70 mV. Both peaks increased during successive scans until an
1387
Figure 2. Equilibration of a carbon fiber electrode just after a standard treatment. Numbers indicate the order of the successive voltamme grams recorded every 1 min (AA 200 uM and DOPAC 20 pM excepted when “PBS” is mentioned).
Potential
of
treatment : from 0 V 2.4
2.8
3
3.4 v
3.2
A 16 11 2nA
u
0 3
7 0 - e 0 . 3 0 . 3
1_
0
.3
Flgure 3. Effect of various upper potential limits on the AA
1_
0
.3v
+ DOPAC
signal. For each treated electrode, voltammograms are recorded after equilibration (see Figure 2) first in AA DOPAC solution (higher traces) and second in PBS solution (lower traces).
+
equilibration of the electrochemical response was reached a t the 20th scan. Subsequent AA and DOPAC signals were stable. Then, when the electrode was immersed in PBS solution without AA or DOPAC, the voltammogram exhibited a remaining peak at +70 mV. This remaining peak did not change when several voltammograms were recorded from PBS solution. If the electrode was tested again in AA + DOPAC solution, the previous AA and DOPAC peaks were immediately obtained without the need for equilibration. Finally, as regards peak heights, Figure 2 shows that this treatment slightly decreased the sensitivity of the electrode for AA, while that for DOPAC increased by 1 order of magnitude. For each electrode the current induced by the standard treatment was measured. It reached a maximum value which was 296 f 28 pA (mean f standard deviation, 10 experiments). The standard treatment was also performed by using various solutions: deaerated PBS solution (by means of N2),PBS solution at pH 2.5 and at pH 8. These treated electrodes were then tested in the same conditions as in Figure 2. The voltammograms thus obtained from the test solution (AA DOPAC in PBS solution at pH 7.4) did not differ significantly from standard experiments. Figure 3 shows the effect of increasing upper potential limit of the treatment on the AA + DOPAC signal. The effect of the lowest treatment (0 to +2 V) was, first, to move the DOPAC and AA signals to +70 mV and, second, to improve the AA DOPAC peak definition. However, these effects progressively disappeared following successive scans. It appeared that a potential equal to or higher than +2.6 V was required to obtain a valuable separation of AA and DOPAC peaks. This separation arose from the moving of the AA peak from
+
+
1388
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9, AUGUST 1981 AA
Treatment from 0 to 3 V
0
0
t
DA
PES
0
0
0
0
0
0+.2v
Flgure 6. DA measurement with a double treated electrode. Numbers indicate the order of successive voltammograms recorded every 1 min (DA 0.1 pM and AA 200 pM in PBS solution). 0 .3
0
.3
0
.3
0 .3
0 .3v
Flgure 4. Effect of various treatment durations on the AA
signal (test conditions as in Figure 3).
+ DOPAC
Figure 5. Effect of the second treatment. Voltammograms were obtained: (A) after the standard treatment, (B) after the second one (test conditions as in Figure 3).
+70 to -80 mV. When the upper limit of the potential applied during the treatment was increased, the AA signal decreased and the DOPAC peak increased. But if the remaining blank peak appearing a t +70 mV in P B S solution was subtracted, then the signal due to DOPAC had the same amplitude when tested after every treatment (from +2.6 to +3.4). When the upper potential limit was extended to +3.6 V, the electrode was broken by this treatment. During these various treatments, the current occurring through each carbon fiber electrode was measured. The values of the maximum current were found to be 50,80,130,210, 260, 310, 350, and 410 pA when the upper limit of the treatment increased from +2 to +3.4 V, respectively. The influence of the frequency of the triangular wave potential has been explored from 10 to 1000 Hz. In all cases a 0 to +3 V potential was applied to the working electrode for 20 s. This parameter slightly affected the resulting test voltammograms. When a 10-Hz frequency treatment was applied, the subsequent test voltammogram was very similar to that recorded after a 7 0 - H ~treatment, the upper limit of which was equal to +3.2 V (Figure 3). The test voltammogram obtained after a 1000-Hz frequency treatment was almost identical with that obtained after a 0 to +2.8 V treatment applied at 70 Hz (Figure 3). Various durations of the standard treatment (0 to +3 V, 70 Hz) were tested (Figure 4). In all cases the separation between AA and DOPAC occurred. This parameter mainly acted on the sensitivity of the carbon fiber electrode to AA. When the treatment was extended up to 60 s, the electrode was broken. In order to decrease the amplitude in the remaining blank peak at +70 mV, we performed a second treatment after the standard one (Figure 5). Two durations of this treatment were tested (5 s and 20 s), but the resulting voltammograms were very similar. Moreover, this second treatment slightly improved the sensitivity of the electrode to AA. Such double
treated electrodes were used in vivo and then calibrated in vitro for DOPAC and AA (17). The DOPAC peak height linearly increased with DOPAC concentration (5-50 pM) and did not depend on the AA concentration in the solution (from 0 to 400 pM). The AA peak height linearly increased with AA concentration (50-400 pM) but was slightly depressed when the DOPAC concentration exceeded 20 pM. Chemical treatments of the carbon fiber electrode were also tried. The working electrode was immersed for 20 min in "03, H2S04, HzOz,and concentrated acid dichromate solution. In all these cases the resulting test voltammograms (AA + DOPAC in PBS) showed only one poorly defined peak. However, this peak moved to a less positive potential and its height was increased. The best results were obtained with concentrated acid dichromate solution. In this case the resulting voltammograms were very similar to those obtained after a 0 to +2 V electrochemical treatment (Figure 3). Finally, electrochemically treated carbon fiber electrodes were used for in vitro detection of submicromolar DA concentrations in the presence of 200 pM AA (Figure 6). As previously reported (14-1 7) DA peaked at the same potential as DOPAC and other unmethylated or unconjugated catechols such as norepinephrine, ~-3,4-dihydroxyphenylalanine, and 3,4-dihydroxyphenylethyleneglycol. But the sensitivity for DA was about 100 times higher than for DOPAC. Figure 6 shows that the maximum peak height for DA was not immediately obtained. In fact this DA peak took 10-20 min (with or without voltammetric recordings) to reach a maximum. Consequently, and in contrast to DOPAC or AA measurement, every time the DA concentration was modified, about 15 scans (recorded every minute) were necessary to obtain a steadystate signal. However, the electrode used in Figure 6 was tested as described in Figure 6 with increasing DA concentrations (from 50 nM to 1 pM) and a linear response of the steady-state signal was observed from 50 nM to 0.5 pM. For higher concentrations the maximum peak height measured at the steady state still increased but not as much as predicted with a linear response.
DISCUSSION The test voltammograms obtained from electrodes which were treated in the standard manner were not identical (compare Figures 2,3,4, and 5A). Moreover, the current which occurred through the carbon fiber electrode had a standard deviation of 10%. This latter variation can, however, be correlated to the resulting aspects of the test voltammograms. In fact the lowest maximum currents observed during standard treatments (in the range of 260-270 pA) correspond to treated electrodes which then exhibited tested voltammograms similar to those obtained with a 0 to +2.8 V treatment (Figure 3). This suggests that the relative lack of reproducibility could be due to some variation in the impedance of the carbon fiber electrodes. This suggests that the maximal density current, which reached about 2.5 A/cm2 during standard treatment, could be a parameter as important as the applied potential
ANALYTICAL CHEMISTRY, VOL. 53, NO. 9,AUGUST 1981
(Figure 3). However, diince the duration of the treatment greatly influenced the resulting signals (Figure 4), it might be suggested that the total amount of current occurring through the electrode during the treatment could also be a significant factor. The effect of chemical and electrochemical treatments on the pyrolytic carbon film electrode has already been reported (11). It has been shown that dichromate solution as well as electrochemical treatment (-4 V to +4 V applied using a square-wave generator far 4 min) improved the sensitivity and the reversibility in ferrirymide-ferrocyanide systems. However, electrochemical measurements were reported to be unreproducible except when the electrode was pretreated again just before each scan. These results are very close to ours with regard to chemical treatment as well as with the lowest electrochemical treatmen,t (0 to +2 V, Figure 3). The fact that their -4 to +4 V electrochemical treatment gave results similar to our 0 to f 2 V treatment could be explained by the fact that their electrode surface was more than 2000 times larger than ours. Thus, the current density applied to their electrode was presumably smaller thain that which occurred during our standard treatment. The most interesting feature of our electrochemically treated electrode is its ability to resolve catechols from AA which is achieved by moving AA redox potential to -80 mV. This potential correspond's very closely to the reversible formal potential given by Milnzzo (18). As regards peak heights (compare Figures 1 and 5A) the increase in sensitivity following electrochemical treatment was more than 1 order of magnitude for BOPAC. This sensitivity improvement is probably related to the DOPAC peak shifting and could be due to the improvement of the reversibility and consequently of the efficiency of the DOPAC oxidation reaction. With regard to DA, the increase in sensitivity was higher (more than 2 orders of magnitude). But in this case an adsorption mechaniwm might be suspected since a very progressive equilibratiorr phenomenon occurred every time the DA concentration was modified. Finally the AA peak height was increased by the less aggressive treatments (left part of Figures 3 and 4) but was depressed by the most aggressive ones (right part of the Figures 3 and 4). In light of these results, much more work is needed to support a valuable hypothesis about the mechanism by which such treatments act. However, it might be suggested that
0
1389
some unknown surface functionalities could be attached to the electrode surface during the treatment. The resulting improvement of sensitivity, reversibility, and selectivity could then be explained by a catalytic mechanism similar to that described by Kuwana and co-workers (9, 10): the oxidation of the compound present in the solution could be initiated by the previous oxidation of the surface functionalities. However, this study underlines the importance of the current which occurred through the carbon fiber electrode during the treatment. Moreover, these data might be very useful for obtaining desirable performance from pyrolytic carbon electrodes used for in vivo neurochemistry or for other purposes.
ACKNOWLEDGMENT We thank R. N.Adams for his helpful discussion and criticism of this work.
LITERATURE CITED Adams, R. N. Anal. Chem. 1976, 48, 1126A-1137A. a n o n , F.; Cespuglio, R.; Ponchon, J. L.; Buda, M,; Jouvet, M.; Adams, R. N.; Pujol, J. F. C. R . Hebd. Seances Acad. Sd. 1978. 286. 1203-1206. Lane, R. F.; Hubbard, A. T.; Blaha, C. D. Biodectrochem. Biosnerget. 1978, 5 , 506-527. Conti, J. '2%; Strope, E.; Adams, R. N.; Marsden, C. A. Life Sci. 1978, 23, 2705-2716. Lane, R. F.; Hubbard, A. T.; Blaha, C. D. J . Electroanal. Chem. 1979, 95, 117-122. Lane, R. F.; Hubbard, A. T.; Fukunaga, K.; Blanchard, R. J. Brain Res. 1976, 114, 346-352. Ponchon, J. L.; Cespuglio, R.;Gonon, F.; Jouvet, M.: Pujol, J. F. An&!/. Chem. 1979, 51, 1483-1486. Evans, J. F.; Kuwana, T . Anal. Chem. 1977, 49, 1632-1635. Evans, J. F.; Kuwana, T. J . Electroanal. Chsm. 11977, 80, 409-416. Chi-Sing, D.;Kuwana, T. Anal. Chem. 1978, 50, 1315-1318. Blaedel, N. J.; Mabbatt, G. A. Anal. Chem. 1978, 50, 933-936. Dayton, M. A.; Brown, J. C.; Stutts, K. J.; Wightman, R. M. Anal, Chem. 1980, 52,946-950. Armstrongdamas, M.; Millar, J.; Kruk, 2. L. Nature (London) 1980, 288, 181-189. Bud& M.: Gonon, F.; Cespuglio, R.; Jouvet, M.; Pujol, J. F. C. R . Hebd. Seances Acad Sci. 1980, 290,431-434. Gonon, F.; Buda, M.; Cespuglio, 8.;Jouvet, M.; Pujol, J. F. Nature (London) 1980, 286,902-904. Buda, M.; Gonon, F.; Cespuglio, R.; Jouvet, M.; Pujol, J. F. €or. J . PhWn7aCO/.,in press. Gonon, F.; Buda, M.; Cespuglio, n.;Jouvet, M.; Pujol, J. F. Brain Res., in press. Milazzo, G. In "Electrochimie"; Dunod Ed.: Paris, 1969.
R E C Efor~ review February 3,1981. Accepted May 11,1981. This research was supported by DGRST (Grants No. 7870156 and 7872788), INSERM IJ 171 and CNRS La 162.
