Anal. Chem. 1989, 61 2323-2324
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CORRESPONDENCE Selectivity of Stearate-Modified Carbon Paste Electrodes for Dopamine and Ascorbic Acid Sir: A number of distinct voltammetric methods have been developed recently to detect the overflow of monoamines in brain extracellular fluid (1-5). For techniques that are designed to measure dopamine, a major limitation has been the inability to dinstinguish between currents arising from the oxidation of catecholamine and those due to the oxidation of ascorbic acid (6-9). A stearic acid modification of graphite paste electrodes has been reported to overcome this restriction (10, l l ) , and they have frequently been used with linear sweep voltammetry and chronoamperometry in attempts to monitor base line and drug-induced changes in dopamine levels in vivo (10, 12-15). However, there are some anomalies in the reported findings (2, g),in particular the estimation of the basal concentration of extracellular dopamine a t approximately 2 orders of magnitude greater than the value of approximately 50 nmol/L measured with other electrochemical methods (16, 17) and microdialysis probes (18). We now investigate this problem, and find that the response of stearate-modified electrodes is altered by brain tissue to such an extent that they no longer discriminate between the oxidation currents of dopamine and ascorbate and consequently, in their present form, cannot be used to measure levels of dopamine unambiguously in vivo. EXPERIMENTAL SECTION The stearate-modified electrodes were prepared as described by Blaha and Lane (IO) from 1.5 g of UCP 1-M graphite powder (Ultra Carbon Corp.) and 100 mg of stearic acid (Sigma Chemical Co.) dissolved in 1mL of Nujol oil (Aldrich Chemical Co.), packed into a Teflon-coated silver wire. The working diameter of the disk electrode was 250 pm. The unmodified carbon paste electrodes were prepared in the same way, but without stearic acid. Cyclic voltammetric experiments were performed at 25 "C, using a microcomputer based three-electrode system similar to that described previously (19), at 50 mV/s between 0 and 1000 mV vs Ag/Ag+ reference electrode (-15 mV vs SCE). For clarity, however, and because the systems used were essentially chemically irreversible under these conditions, only the oxidation phase of the waves is shown. The background current, measured in the absence of substrate, was subtracted from each voltammogram before analysis. The substrates, dopamine (Sigma) and ascorbic acid (BDH), were used as supplied. The experiments were carried out in phosphate buffered saline (PBS), pH 7.4; NaCl (0.15 mol/L), NaH2P04(0.04 mol/L), and NaOH (0.04 mol/L). The effect of brain tissue on the response of stearate-modified electrode to oxidation of dopamine and ascorbate was determined in separate experiments by measuring their response in vitro before and after a 24-h contact period with brain tissue.
RESULTS AND DISCUSSION The oxidation waves for dopamine and ascorbate at unmodified carbon paste electrodes are shown in Figure 1. As can be seen, there is no well-defined peak for either substrate and so that potential of maximum slope (20), Vs-, is used to compare the position of the two waves on the voltage axis and as an index of changes in electron transfer kinetics for ascorbate and dopamine. The foot potential (the potential a t which the current first reached 1 nA above the base line), V , is also used to compare the positions of the two waves on the potential axis. 0003-2700/89/0361-2323$01.50/0
Table I. Means f Standard Error of Mean (n = 3 ) O dopamine CPE SME TMSME CPE SME TMSME
VS-, mV 140 f 3 272 f 15 77 f 9
Vf, mV 68 f 12 140 f 10 40 f 5
ascorbic acid 565 f 45
880 f 55 275 f 10
300 f 28 487 f 22 27 f 27
"V?- for each substrate was significantly different for every combination of electrode pairs (unpaired two-tailed t test); P < 0.01. CPE, unmodified carbon paste electrode; SME, stearatemodified carbon paste electrode; TMSME, stearate-modified electrode after 24-h tissue treatment. V , potential maximum slope for the waves; V,, potential at wh% the oxidation wave has reached a current of 1 nA.
The introduction of stearic acid into carbon paste electrodes reduced the rate of charge transfer for both dopamine and ascorbate (Figure 1 and Table I), but, as expected (10, 111, the effect on the ascorbate anion was greater than that for the cationic dopamine species. Thus the modification increases the separation of the waves ( Vs-) for dopamine and ascorbate from 425 f 45 to 608 f 57 mV. However, after a 24-h period of contact with brain tissue, this separating power of stearate-modified electrodes in vitro was not only reversed but reduced to a value of 188 f 14 mV, which is less than that of unmodified carbon paste electrodes. Furthermore, the positions of the foot potential for the two waves virtually coincide after the electrode had been modified by contact with the tissue, indicating that a t any potential along the range 0-800 mV the electrode can no longer detect dopamine without interference from ascorbate (Figure 1 and Table I). The limit of detection for dopamine a t the stearate-modified electrode before and after tissue treatment is of the order of 5 and 10 pmol/L, respectively. These values indicate that the electrode would not be able to detect dopamine in the concentration range observed in vivo with other techniques (16-18). A further problem for the measurement of dopamine is posed by possible amplification of the signal in the presence of ascorbic acid due to electrocatalysis. This problem does not arise when ascorbate oxidizes at a less anodic potential than dopamine. However, shifting the ascorbate oxidation to higher anodic potentials, as was done with the stearatemodified electrode, enables the electrocatalytic process to take place (11). In the presence of ascorbate the oxidized form of dopamine, dopamine-0-quinone, oxidizes ascorbate present in the vicinity of the electrode, with the dopamine-o-quinone being reduced back to dopamine which can then be reoxidized a t the electrode (21-23). We did not address this problem for the stearate-modified electrode in the present study, since after treatment with brain tissue electrochemical discrimination of the substrates is lost; this renders studies on electrocatalytic interference of the dopamine signal superfluous. 0 1989 American Chemical Society
Anal. Chem. 1909.61,2324-2327
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estimate of the concentration of dopamine needed to produce a current of this size. Considering the ratio of 1OooO:1 for ascorbate to dopamine concentrations in the striatum, it is likely that the current recorded in vivo is due almost entirely to ascorbate. In conclusion, the results indicate that while stearatemodified electrodes have the desired properties for electrochemical discrimination of ascorbate and dopamine before they are implanted in brain tissue, these properties are lost after implantation. Taken together, the literature data and the present findings also suggest that these electrodes are neither selective nor sensitive enough to detect dopamine levels in vivo.
’A
LITERATURE CITED
c
250
mc
40s
boo
Figwe 1. Sections of the voltammograms (seetext) for dopamine (DA) and ascorbate (AA) in PBS, pH 7.4: (top) DA, 100 pmol/L; AA, 200 pmol/L; at an unmodlfied carbon paste electrode; (middle) DA, 20 pmol/L; AA, 200 pmol/L; at a stearate-modlfied carbon paste electrode; (bottom)DA, 100 WmollL; AA, 500 Imol/L; at a tissue-treated stearate-modified carbon paste electrode.
The effect of brain tissue on the stearate-modified electrode is consistent with recent studies of similar action by brain tissue on unmodified carbon paste electrodes (24) and with reports for surfactant action on carbon paste electrodes (25). It has been proposed that in such cases the surfactant solubilizes the oil and other hydrophobic elements of the paste, leaving behind a “clean” graphite surface. We propose that a similar mechanism occurs in vivo. The stearate-modified electrode implanted in brain tissue is introduced to the hydrophobic environment of lipids and proteins. These take the role played by the surfactants mentioned above and remove the oil and other hydrophobic /lipophilic moieties of the electrode surface, the result being a modification of the electrode surface and an increase in the rate of electron transfer as shown (Figure 1 and Table I). A reduction in sensitivity is found at the tissue-treated electrodes compared to the electrodes before treatment, and is most likely a result of partial blockage of the electrode surface due to the adsorption of lipids and proteins (26). A linear sweep voltammetric wave, attributed to dopamine oxidation at the stearate-modified electrode in vivo, has been reported by Lane et al. (14). The wave, centered at +lo0 mV vs Ag/AgCl, has a peak height of the order of 1nA. This peak occurs at a potential (vs SCE) corresponding to large ascorbate oxidation at the tissue-treated electrode. A current of 1 nA for dopamine in vitro would correspond to a concentration of approximately 5 wmol/L (10). Taking into account the restricted compartment environment of the electrode in the brain (27, 28), this concentration represents a gross under-
Marcenac, F.; Gonon, F. Anal. Chem. 1985,57, 1778-1779. Crespi, F.; Martin, K. F.; Marsden, C. A. Neurosclence 1988, 27, 885-896. Stamford, J. A. Anal. Chem. 1988,58, 1033-1036. Kasser, R. J.; Renner, K. J.; Feng, J. X.; Brazell, M. P.; Adams, R. N. Brain Res. l98& 475, 333-344. Ewing, A. 0.; Wightman, R. M. J. Neurochem. 1984. 43. 570-577. Adams, R. N.; Marsden, C. A. I n Hendbook of Psychopharmacdogy; Plenum Press: New York, 1982; Vol. 15, pp 1-74. Voltammefry in the Neurosclences; Justice, J. B., Jr., Ed.; Humana Press: Cllfton, NJ, 1987. Measurement of Neurotransmitter Release In Vivo; Marsden, C. A,, Ed.; IBRO Handbook Series; J. Wiley and Sons: Chichester, 1984; Vol. 6. Marsden, C. A.; Joseph, M. H.; Kurk, 2. L.; MaMment, N. T.; 0”eill. R. D.;Schenk, J. 0.;Stamford, J. A. Neuroscience 1988,25, 389-400. Blaha, C. D.; Lane, R. F. Brain Res. Bull. 1983, 10, 881-864. Gelbert, M. 8.;Curran, D. J. Anal. Chem. 1986,58, 1028-1032. Lane, R. F.; Biaha, C. D.; Hari, S. P. Braln Res. Bull. 1987, 19, 19-27. Broderick, P. A. Life Sci. 1985, 36, 2269-2275. Lane, R. F.; Blaha, C. D.; Phillips, A. G. Brain Res. 1988. 397, 200-204. Glynn, G. E.;Yamamoto, B. K. Brain Res. 1989, 481, 235-241. Gonon. F. G.; Navarre, F.; Buda, M. J. Anal. Chem. 1984, 56. 573-575. Kelly, R. S.;Wightman, R. M. Brain Res. 1987,423, 79-87. Church, W. H.; Justice, J. 6.. Jr. Anal. Chem. 1987,59, 712-716. ONeill, R. D.; Fillenz, M.; Albery, W. J.; Goddard, N. J. Neurosclence 1983,9 ,87-93. OMham, K. 8. J. Electroanel. Chem. 1985, 184, 257-287. Sternson, A. W.; McCreery, R.; Feinberg, 6.; Adams, R. N. J . Nectroanal. Chem. 1973,46, 313-321. Dayton, M. A.; Ewing, A. G.; Wlghtman, R. M. Anal. Chsm. 1980,52, 2392-2396. Kovach, P. M.; Ewing, A. 0.; Wilson, R. L.; Wightman, R. M. J. Neurosci. Mettwds 1984, 10. 215-227. Ormonde, D. E.; O‘Neill, R. D. J. Electroanal. Chem. 1989. 261, 463-469. Albahadily, F. N.; Mottob, H. A. Anal. Chem. 1987,59, 958-962. Nelson, A.; Auffret, N. J. Electroanel. chem. 1988,248, 167-180. Albery, W. J.; Goddard, N. J.; Beck, T. W.; Flllenz, M.; O’Neill. R. D.J. Electroanel. Chem. 1984, 161, 221-233. Cheng, H.-Y. J. Nectroanal. Chem. 1982, 135, 145-151.
Paul D. Lyne Robert D. O’Neill* Chemistry Department University College Dublin Belfield, Dublin 4 Ireland
RECEIVED for review April 28, 1989. Accepted July 20,1989. We thank EOLAS for a grant to P.D.L. under the Basic Research Awards scheme.
Estimating Error Limits in Parametric Curve Fitting Sir: The recent article by Phillips and Eyring in this journal (I) presented an interesting solution, based on the sequential simplex method, to the problem of the estimation of errors in nonlinear parametric fitting. The authors did not mention
two other simple, powerful, and reliable methods, the jackknife and the bootstrap (2-6). The need for simple and robust procedures to assess confidence limits in estimated parameters is widely perceived in
0003-2700/89/0361-2324$01.50/00 1989 American Chemical Society
