1042
Anal. Chem. 1981, 5 3 , 1842-1847
lifetime of 4 days (4). Both the response slope and the lifetime are known to be independent upon the amount of biocatalytic activity present at the electrode surface ( 2 , 3 ) . The use of rabbit muscle tissue slices in the construction of an AMP membrane electrode increases the amount of biocatalytic activity approximately 50 times over the enzyme-based system. This increase in the activity results in a sensor with excellent electrode characteristics including a response slope of 57 mV/decade and a lifetime of at least 28 days. This shows the effectiveness of using tissue slices over isolated enzymes in situations where the latter have insufficient biocatalytic activity.
rsoC
LITERATURE CITED (1) (2) (3) (4) (5)
L - _ - l - . - L 4- -
(8) (7) (8) (9)
4-----4
-Log AMP, (MI
(IO)
Flgure 7. Response of the tissue-based AMP electrode to AMP at various electrode ages: (0)day 2; (A)day 5; (0) and day 28.
different days. It can be seen from these results that the response of this electrode system is quite reproducible and reliable from electrode to electrode. Typical electrode characteristics for the previously reported enzyme-based AMP membrane electrode include a slope of 46 mV/decade change in AMP concentration and a useful
(1 1) (12) (13)
Frlcke, G. H. Anal. Chem. 1980, 52, 259R-275% Masclni, M.; Liberti, A. Anal. Chlm. Acta 1974, 68, 177-184. Gullbault, G. G.; Tarp, M. Anal. Chlm. Acta 1974, 73, 355-365. Papastathopoulos, D. S.; Rechnitz, G. A. Anal. Chem. 1978, 48, 862-864. Rechnitz; G. A,; Arnold, M. A.; Meyerhoff, M. E. Nature (London) 1979, 278, 466-467. Arnold, M. A,; Rechnltz, G. A. Ana/. Chim. Acta 1980, 113, 351-354. Arnlod, M. A.; Rechnltz, G. A. Anal. Chem. 1980, 52, 1170-1174. Arnold, M. A.; Rechnitz, G. A, Anal. Chem. 1981, 52, 515-518. D'Orazio,P.; Meyerhoff, M. E.: Rechnitz, G. A. Anal. Chem. 1978, 50, 1531- 1534. Wheeler, T. J.; Lowensteln, J. M. Biochemistry 1980, 19, 4564-4567. Vadegama, P. In "Ion-Selective Electrode Methodology"; Covlngton, A, K., Ed., CRC Press: Boca Raton, FL, 1979; Vol. 2, Chapter 2. Noda, L. In "The Enzymes": Boyer, P. D.,Ed.: Academic Press: New York, 1973 Vol. 8, p 279. Webster, H. L. Nature (London) 1935, 172, 453-454.
RECEIVED for review February 2, 1981. Resubmitted May 4, 1981. Accepted July 20,1981. We are grateful to the National Science Foundation (Grant No. CHE-7728158) for supporting this research.
Pulse Voltammetry with Microvoltammetric Electrodes A. G. Ewing, M. A. Dayton, and R. M. Wightman" Depattmenl of Chemistry, Indiana University, Bloomington, Indiana 47405
Microvoltammetric electrodes can be used to provide reproduclble results In an extremely complex chemlcal medluml the mammalian brain. Because of the small size of the microelectrodes, current on the back step of a chronoamperometric experlment is essentlally nonfaradalc and can be used for residual current correction. The use of normal pulse voltammetry mlnlmlres electrode response deterloratlon caused by fllmlng of the electrode surface by electrogeneratedproducts.
The response Of these electrodes In is to that of other electrodes; it Is not altered by the repetition rate of the pu's@s9and dopaminel a Of prime neurochemlcal Interest, can be partially resolved from 8scorblc add and dihYdroxYPhenYlacetlc acid by in VIVO VOltammetry.
Continuous monitoring of electroactive species in complex media with solid voltammetric electrodes is a very difficult problem because the electrochemical response tends to deteriorate with time. The goal of in vivo voltammetry and of 0003-2700/81/0353-1842$01.25/0
most electroanalysis schemes is to measure the concentration of electroactive species by the observed current and to identify the species via half-wave potential. This goal requires that the electrode retain constant Properties throughout the experiment* When the electrode surface cannot be renewed during the experiment, alternate procedural strategies to this In this paper, we demonstrate problem must be that voltammograms can be Obtained with microvoltammetric electrodes using a pulse potential wave form. The microvo~~etric are fabricakd from carbon fibers and have previously been shown to exhibit several unique properties that facilitate electroanalysis (1,2). The utility of these electrodes is demonstrated here by voltammograms in an extremely complex chemical environment, the mammalian brain. In the early 1970s, €3. N. Adanis recognized that several neurotransmitters (small molecules which relay information between neurons) are easily oxidized and, thus, should be determined with carbon electrodes. This observation led to the development of a number of analytical techniques for the determination of these compounds (3) including the use of 0 1961 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
voltammetric electrodes iaurgically implanted in the brain of living animals (4). Several investigators have shown that distinct changes in oxidation current occur at these electrodes during pharmacological, physiological, or behavioral manipulations of the animal (5-14). Many of these experimenh have been conducted in a brain region known to have high concentrations of the easily oxidized neurotransmitter dopamine. However, the actual identity of the substances giving rise to the electrochemical response is still unclear (5,7, 10, 13). Most of the experiments directed at identifying the substances oxidized have used phairmacological rather than electrochemical evidence because of the possibility of changes in the electrochemical propertiles of the electrode. These changes, arising from surface alterations, appear to have two major sources. One surface change arises from electrode filming by protein adsorption. This phenomenon has been studied at platinum (15) and carbon electrodes (16) and results in distortion of the shape of cyclic voltammograms in brain tissue for injected (17)or endo,genous compounds (6) by a shift in the peak potential. This type of filming is relatively difficult to circumvent although protein adsorption does appear to be potential dependent (16). The second, and perhaps more severe problem, i s the filming by electrogenerated products from compounds known to be present in the brain (see below). As will be shown, stablle reproducible voltammograins can be obtained with microellectrodes in brain tissue using a poltential wave form similar to that employed in normal pulse voltammetry. This approach was originally suggested for solid electrodes by G. C. Barker in 1962 (18)since it prevents large concentrations of electrogenerated species from accumulating at the electrode surface. The utility of this approach has been demonstrated by Lane and Hubbard (19) and by Anderson and Bond (20). However, it has not been widely employed because of the considerable residual currents a t solid electrodes. With microelectrodes, this current can be minimized, permitting sensitive measurements. This allows the use of voltammograms for meawring the time course of changes in chemical concentrations in the brain and also for identifying the half-wave potential of these compounds. EXPERIMENTAL SECTION Chemicals. Dopamine-HCl, homovanillic acid (HVA), dihydroxyphenylacetic acid (DOPAC),and d-amphetaminesulfate (Sigma Chemical Co., St. Louis, MO), a-methyldopamine (a gift from Merck, Sharpe and Dohme, Ftahway, NJ), and ascorbic acid, chloral hydrate, and ferricyanide (Mallinckrodt, St. Louis, MO) were used as received. Solutions were prepared daily by using water distilled in glass from alkaline permanganate. Ascorbic acid, DOPAC, a-methyldopamine, and d-amphetamine sulfate were dissolved in saline for in vivo injections. All solutionswere purged thoroughly with nitrogen. Electrodes. Carbon fiber electrodes were prepared as previously described (1) using Thornell P-55 fibers (Union Carbide Corp., New York, NY). The assembled electrode consists of a glass pipet which is closedl down around a carbon fiber. Epoxy between the fiber and glasci minimizes leakage into the pipet, and the active surface area of 'the electrode is best represented by a disk whose radius is defined by that of the fiber. Electrode response was optimized by pretreatment of the electrode with repetitive cyclic voltammograms from 0 to 1.8 at 250 V s-ll in pH 7.4 citrate-phosphate buffer. Apparatus. In this work, a three-electrode potentiostat of conventional design was employed with special precautions for low current measurementca. The power supply and the BC lines were well isolated from the amplifier section of the potentiostat. The circuit was assembled on a locally constructed printed circuit board. The amplifier driving the auxiliary electrode (356N, Signetics) had a time coristant of 100 ps. Connection to the electrodes was with coaxial shielded cables. The key to the low noise measurements is the current transducer. In this work a low noise, field effect transistor input operational amplifier in the current follower mode was used (AD515K, Analog Devices). The
18413
features of this amplifier which make it ideal for these types of measurements are the low input bias current (less than 150 f.A max), high temperature stability (drift rated at 15 pV/"C), and moderately fast slew rate (at unity gain 0.3 V/ps). The feedback loop for the current transducer contained a lo* R metal-oxide film resistor (MOX 400, Victoreen). Subsequent gain of thie transduced current wa3 made with an inverting amplifier (356N, Signetics) with a 10 ms time constant. For experiments with a series of repetitive pulses (single potential measurementa--Figure l), 92 ms potential steps from 0.0 V to 0.4 V vs. SCE were employed at a frequency of 0.25 Hz. ThLe amplified output signal was integrated during the last 34 ms of the potential step with a V/F converter, converted to the analog domain, and displayed as a single point on a strip chart recorder. Filtering on the output of the analog converter circuit was adjusted so that 90% response was obtained after 15 pulses. The circuit diagrams for the pulse generator and output processor have been published elsewhere (21). This instrument also generates a ramp for linear voltammetryor a series of pulses of increasing magnitudle (and of adjustable duration) for normal pulse (previouslyreferred to as scanning chronoamperometry (21))or differential normal pulse voltammetry. In the scanning pulse modes, the current is also sampled over two 60-Hz periods at the end of each potentiid step and is again sampled at an equal time after the potentid step. This second current from each step can be added to that acquired during the pulse to obtain residual current correction (see below). The current on the previous pulse can alternatively be subtracted from that on the present pulse to provide a differential normal pulse output. The output filtering is decreased for the scanning experiments to obtain a voltammogram free from distortion. The time between scans is controlled by the instrument so that continuous repetitions of the applied wave form can be readily obtained. Single-step chronoamperometry data were acquired and analyzed with a previously described, computercontrolled system (1). Surgical Procedure. Male Sprague-Dawleyrats were anesthetized with chloral hydrate (400 mg/kg) and placed in a stereotaxic apparatus. Working electrodes were implanted at a rate of 0.3 mm/min into the caudate nucleus using the coordinates (AP,+3.0; L, h2.3; H, -4.0) obtained from the atlas of Pelligrirno and Cushman (22). During implantation, the electrode potential is maintained at -0.2 17 vs. SCE, and the electrode is maintained at virtual ground throughout the entire experiment. The auxiliay electrode was a screw attached to the skull. A saturated calomlel electrode (SCE) was placed in contact with the cortex using a normal saline salt bridge fabricated from a disposable pipet tilp. The temperature of the rat was monitored with a rectal probe and a temperature of 31 O C was maintained with a heating pad and temperature contrlol unit (EKEGElectronics Co., Vancouver, Canada). Small amounts of chloral hydrate were administered at regular intervals to maintain a relatively constant level of anesthesia throughout the experiment. RESULTS AND DISCUSSION Single Potential Pulse Measurements. We have prleviously shown that a linear relationship between current arid concentration is obtained for the reduction of ferricyanide from to M. However, as can be seen in ref 1, our previous measurements with commercial instrumentation ,st M concentrations gave relatively poor values which had a low signal-to-noise ratio. With our locally designed instrumentation,we examined the two-electron oxidation of DA from to lo-' M. A logarithmic plot of the current vs. concentration was lintm with a slope of 0.957 and a correlation coefficient of 0.9997. The data obtained a t lo-' M DA are shown in Figure 1. The residual current in the buffer was determined from 0 to 5 min. At 5.5 min, sufficient IM DA solution was added to the buffer to give a solution that was 1.1X lo-' M in DA. After approximately 16 pulses the average current increased approximately 0.6 PA. Since the current was sampled for 34 ms, this corresponds to 20 fC or the oxidation of approximately 60 OOO molecules. Considering the very small numbeir of molecules oxidized per potential step, we consider the data in Figure 1 to have a very good signal-
1844
ANALYTICAL CHEMISTRY, VOL. 53, NO.
12,OCTOBER 1981
-7
1x10
M
DA
a
.o1
I
.I J ( S )
10
T i m e (rnin)
I
Flgure 1. Change in average current at a microvokammetric electrode for a change in DA concentration from 0 to 1.1 X lo-' M. Insert: Single current-time curve for a potential step in buffer solution. Arrows indicate beginning and end of sampling period.
Flgure 3. Ratio of backstep current (ib) to frontstep current (if) as a function of step time (7) for reduction of ferricyanide in 1 M K,SO,, pH 3.0: circled dots, experimental; line, from eq 3.
compound at a conventionally sized electrode (r > O , l ) , the current when the potential is returned to the base line following a potential step of duration 7 to the diffusion-controlled portion of a voltammetric wave has a significant faradaic component. Kambara (24)has shown that the faradaic current (ib) when the potential is returned to the initial potential is
In this equation, which was derived for a planar electrode, A is the area of the electrode, D is the diffusion coefficient, Cb is the bulk concentration of the electrolyzed species, and t is the total time measured from the beginning of the potential pulse. Evaluation of this equation at t = 27 gives a ratio of -&/if = 0.293 (%), where if is given by the Cottrell equation. At a carbon fiber electrode, we have shown that the current follows an equation of the form 0
.2
.4
.6
E (V) Figure 2. Normal pulse voltammograms of 20 pM DA in pH 7.4 citrate-phosphate buffer, 5 mV s-' scan rate, 1 s per step: (A) 57 ms pulse time. (B) 92 ms pulse time. (C) 92 ms pulse time, backstep corrected.
to-noise ratio. During a single 92 ms step, approximately 250000 molecules are oxidized, a minute fraction of the total molecules in the cell. Charging Current Correction with Pulse Potential Scans. When the potential is scanned to obtain a currentvoltage curve, the detection limits decrease because less electronic filtering can be applied and residual currents tend to obscure the response. Ideally, charging currents should be drastically reduced relative to faradaic currents at microvoltammetric electrodes because the faradaic current is linearly dependent on the electrode area while the double layer charging current is exponentially dependent on the area. In fact, carbon fiber electrodes exhibit significant charging currents which have been attributed to surface pits and cracks (23). These charging currents dictate that potential pulses of long duration be employed to maximize the faradaic to charging current ratio. For example, as shown in Figure 2, normal pulse voltammetry at a carbon fiber with 57 ms pulse time results in a current-voltage curve which is sharply ramped. Even with potential step times of 92 ms, the limiting current appears to be a function of potential. The charging current's contribution to the current-voltage curve can be decreased by adding the current of the step back to the base or initial potential to that measured during the potential pulse at microelectrodes. If the charging current is primarily of a capacitive nature, the current should be equal in magnitude and opposite in sign at equal times during and after the potential pulse. During electrolysis of a reversible
where r is the electrode radius. Division of eq 1 by eq 2 and evaluation at 27 gives
-ib/if = 0.293/(1
+ 5.5D1/2~1/2/n1/2r)
(3)
Although eq 3 is an approximation (an equation for the current of the reverse step for neither a sphere nor a disk has been reported), it accurately predicts that faradaic current at 27 is negligible for sufficiently long values of 7. Experimental verification of the trends indicated by eq 3 is given in Figure 3. This treatment shows that the primary component of the current at 27 is the residual current even in the presence of a reversible faradaic species. Therefore, correction of the voltammograms can be obtained by adding the reverse current to the forward current during data processing. As is apparent in Figure 2, the current-voltage curves are much less distorted by nonfaradaic contributions. Advantages of Pulse Voltammetry. Pulse voltammetry greatly enhances the reliability and stability of the electrode response in comparison to other scanning techniques. This is demonstrated in Figure 4 where normal pulse voltammograms are compared to conventional cyclic voltammograms of HVA at carbon fiber electrodes. Homovanillic acid is a major metabolite of dopamine in mammalian brain. It is well documented that the oxidation products of this compound form a partially insulating film on the electrode (6). With the pulse wave form, less of these products are formed and the electrode maintains a constant signal. This is in contrast to cyclic voltammetry or any technique where the excitation wave form is at potentials where electrolysis occurs for an extended period of time (semidifferential analysis, differential pulse
p;I
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
18415
B
I
2
:.
.... . .
.::::.
. . I /
..I.(
0
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.5
. . . . . .. . ..
1.0
c . .
a
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Background corrected voltammograms of 100 pM homovanilllc acki In pH 7.4 citrate-phosphate buffer: (A) cyclic voltammetry; (B) normal pulse voltammetry. Scan number indicated by numbers, 20 mV s-’ scan rate 92 ms pulse time, and 2 s per step. Figure 4.
A
/ *
. i ’
I
100pA .
6,l
. , -* :
.... ,. I i
0
.4
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Figure 5. Voltammograms from an electrode implanted in the caudate nucleus of an anesthetized rat: (A) cyclic voltammetry, (B) backstep corrected normal pulse voltammetry. Numbers refer to scan after Implantation. Conditions are as in Figure 4.
voltammetry, etc.). Although the concentration of HVA in mammalian brain is considerably lower than that employed in this illustrative example, the deterioration of electrochemical response with voltammetry is also observed when the measurements are made in the brain of anesthetized rats (Figure 5). The pulsed voltammogram retains its initial form throughout the measurement interval. ‘The advantages of pulsed wave forms in in vivo electrochemistry have been recognized by others, and thus, chronoamperometry has been the most popular electrochemical scheme used in in vivo electroanalysis (6, 7,lO-12,14).However, with the backstep corrected method, the advantage of pulsed wave forms can be maintained with the additional feature that reproducible current-voltage curves can be obtained. Voltammograms in Eirain Tissue. As shown in Figure 5 , voltammograms obtained in brain tissue with microvoltammetric electrodes have no distinct features and differential normal pulse voltammograms are similarly nondescrilpt. In a previous report, we demonstrated that while dopamine in pH 7.4 buffer exhibits a rapid charge transfer rate at microvoltammetric electrodes, ascorbic acid and dihyclroxy-
-.2
0
.2
.4
.6
E (V)
Figure 6. Backstep corrected normal pulse voltammograms of substances Injected into brain tissue. (A) a-methyldopamine, (B) ascorblic acid, (C) dihydroxyphenylacetic acid. Conditions are as in Figure 4.
phenylacetic acid, both anions at this pH, exhibited slow electron transfer rates)resulting in drawn out current-voltage curves (2). Identical behavior is observed in vivo, as shown in Figure 6. In this experiment, 100 nL of each of the tested substances (0.1 M) was injected into the brain. a-Methyldopamine rather than dopamine was injected to prevent enzymatic degradation by monoamine oxidase. The electrochemistry of this compound is identical with dopamine at carbon fibers. The syringe needle for injection was placed at a distance from the electrode tip that was approximately 5 times the radius of the injected droplet. Thus, these normal pulse voltammogramsrepresent the response of the electrodle to these substances diffusing through brain tissue. The maximum concentration of each of these substances was approximately 500 pM following dilution and diffusion to the electrode surface. The voltammograms in Figure 6 were obtained when the apparent concentrationhad decreased to --BO pM by diffusion awa:y from the electrode. They directly illustrate the attenuated response of carbon fiber electrodes to ascorbic acid and IIOPAC and demonstrate that low concentrations of dopamine can be observed in the brain. In addition, the similarity of shape of the voltammograms obtained in brain tissue (Figure 5B) and that for injected AA (Figure 6B) support the contention that ascorbic acid is a major contributor to the in vivo voltammogram obtained from the caudate nucleus of the rat (7, 9,13). Perturbation of Brain Tissue. Investigators using carbon electrodes of 50-100 ,um radius in vivo with repetitive chronoamperometry have! observed that constant values of the current are measured when the animal is under constant controlled conditions, unless the time interval between pulses is varied. If the time interval is increased, the value for the chronoamperometric current increases, and, conversely, the current decreases with a decreasing time interval. This has been interpreted by assuming a small “pool” of extracellular fluid is at the tip of the electrode (26,27). The electroactive contents of this pooll are depleted by electrolysis and are refiied by transport into this “pool”. According to this model, a longer time between pulses results in more complete equilibration of the cloncentrationsin the pool with those of the surrounding brain tissue. This phenomenon distorts both the magnitude and the time scale of the observed concentration changes.
1646
ANALYTICAL CHEMISTRY, VOL. 53, NO. 12, OCTOBER 1981
0
2
4 t(min)
6
Flgure 7. Average current from repettthre chronoamperometry, 92 ms pulse time, -0.2 to +0.35 V pulse ampliude. Output flltering adjusted to 90% response with a single step. At the left arrow, the time between steps was changed from 4 to 10 s. The time per step was Increased again to 30 s at the other arrow.
This type of perturbation of the measured response should be minimized at microelectrodes since their small size ensures less tissue damage than is caused by larger electrodes. The perturbation can also be minimized with short pulse times which decrease the depletion caused by electrolysis. To confirm these hypotheses, we have systematically investigated the effect of the time interval between constant potential pulses of 92 ms duration at carbon fiber electrodes implanted in vivo. In contrast to carbon paste electrodes (26),backstep corrected chronoamperometric results show theoretical faradaic behavior at times as short as 70 ms. As shown in Figure 7, only very small changes in the background signal are observed with changes in pulse interval (-2%). However, these changes are insignificant compared to those reported in the above references with larger electrodes. (A 7.4-fold increase in background currents is predicted with a change in time between steps from 4 to 30 8.) This result suggests that direct concentration measurements can be made with carbon fiber electrodes because they are considerably less perturbational to brain tissue. Furthermore, the time course of changes in concentration detected by amperometry with microelectrodes should directly correspond to the concentration fluctuations of electroactive substances in extracellular fluid. Post-in-Vivo Response. Despite the constancy of the electrode response in vivo, microvoltammetric electrodes are changed by in vivo experiments. When voltammograms are obtained in pH 7.4 solutions following in vivo use, the response is attenuated (-50%) and all of the compounds are less reversible. We have arbitrarily chosen the post-in-vivo responses to calibrate the electrode response. Since the response of the electrode does not alter during an experiment, we believe that the deterioration occurs during electrode implantation. Mass transfer is greatly accelerated at microelectrodes which would lead to this immediate deterioration. Pulse voltammograms for a mixture of 30 pM DA and 200 pM AA and for AA alone are shown in Figure 8. Point by point subtraction of these two voltammograms leads to a corrected voltammogram that is identical in shape with that for DA alone (comparison of Figure 2 and Figure 8 illustrates typical pre- and post-in-vivo voltammetric response). Subtraction to obtain the response of DA alone is possible with the electrodes employed here but not at electrodes of larger radius, since the multiplicative effect of the catalytic reaction is minimized (2). In Vivo Response to Amphetamine. A pulse voltammogram obtained in vivo is shown in Figure 9B. If the entire voltammogram is assumed to be ascorbic acid, the calculated concentration (based on post-in-vivo calibration) is -500 pM. This is considerably higher than that reported by other investigators, but this difference presumably arises because our results are not distorted by an isolated “pool”. A normal pulse voltammogram obtained after an intraperitoneal injection of amphetamine (1.85 mg kg-l) is shown in Figure 9A. Amphetamine is a drug that is well documented to release dopamine from neuronal tissue into extracellular fluid (28)
-.2
.2
0
.4
.6
E (V)
Flgure 6. Backstep corrected voltammograms in pH 7.4 buffer following 8 h of in VIVO amperometrlc measurements: 5 mV s-’ scan rate, 175 ms pulse time, 7 s per step; (A) 200 pM ascorbic acid plus 30 pM dopamine, (B) 200 pM ascorbic acid, (C) the difference of A and B. I
I
I
,
I
A .
I
100 pA
’0
# . . . . . I
C.
72
0
.2 E (V)
.4
,6
Figure 9. Backstep corrected voltammograms from an electrode implanted in the caudate nucieus of an anesthetized rat: 5 mV s-’scan rate, 175 ms pulse time, 7 s per step; (A) 45 min after intraperitoneal injection of amphetamlne (1.85 mg kg-I), (B) 1 min before amphetamine administration, (C) difference of A and B.
and has been used by numerous investigators to demonstrate the utility of in vivo electrochemistry. With electrodes which cannot distinguish DA and AA, a rise in the composite signal has always been observed and has been assumed to result from increased DA release from nerve tissue. Therefore, the expected result from subtracting the two voltammograms if only dopamine increases in concentration is a curve similar to the dopamine voltammogram in Figure 8C, the response of this same electrode following the in vivo measurements. In fact, this is not the case (Figure 9C)and this experiment has led us to believe that amphetamine has additional effects on the chemical composition of extracellular fluid in anesthetizedrata. In preliminary experiments we have found that amphetamine causes a large increase in easily oxidized substances in animals having one of two caudate nuclei totally depleted of dopamine by chemical lesioning. This unexpected result, which is in
Anal. Chem. 1981, 53, 1847-1851
direct contrast to other reports (5, 7 , 1 3 ) ,is currently being investigated. CONCLUSION This paper demonstrates that unmodified electrodes f a bricated from carbon fibers have several ideal properties for electroanalysis in a complex environment such as mammalian brain of an anesthetized animal. At constant potential, dilute solutions of DA can be detected. Backstep correction provides residual current correction for scanning pulsed wave forms. The pulsed wave form prolongs the usable lifetime of the electrode. Their small size, coupled with their rapid response, virtually eliminates artifacts caused by the oxidation of in vivo substances. Since chemical reactions following heterogeneous charge transfer are not apparent with these electrodes, digital subtraction or deconvolution techniques can be employed. While the electrode response certainly deteriorates in vivo, it deteriorates rapidly and then remains constant. Whiile the results presented here do not clearly indicate what compounds are changing in concentration with pharmacological manipulations, they do suggest that the interpretation of in vivo amperometric results is extremely complex. Experiments to clarify these results are in progress. ACKNOWLEDGMENT Helpful discussions with R. Ensman, El. Williamson, and R. Withnell are gratefully acknowledged. LITERATURE CITE11 Dayton, M. A.; Brown, J. C.; Stutts, K. J.; \Nightman, R. M. Anal. Chem. 1980, 52, 946-9!50. Dayton, M. A.; Ewing, A. (2.; Wightman, R. M. Anal. Chern. 1980, 52, 2392-2396. Adams. R. N. Anal. Chern. 1978, 48, 1126A-1138A. Klssinger, P. T. Anal. Chem. 1977, 49, 447A-456A. Lane, R. F.; Hubbard, A. T.; Fukunaga, K.; Blanchard, R. J. Braln Res. 1976, 114, 346-352. Wightman, R. M.; Strope, E.; Plotsky, P.; Adams, R. N. Braln Res. 1978, 159, 55-68.
1847
Conti, J. C.; Strope, E.; Adarns, R. N.; Marsden, C. A. Llfe Sci. 1978, 23, 2705-2716. Lane, R. F.; Hubbard, A. T.; Blaha, C. D. J. Nectroanal. Chem. 1971), 95. 117-122. Lane, R. F.; Hubbarcl, A. T.; Blaha, C. D. Bioelectrochem. Bioenerg. 1978, 5, 504-525. Huff, R.; Adarns, R. N.; Rutledge, C. 0. Braln Res. 1979, 1721, 369-372. Marsden, C. A.; Conti, J.; Strope, E.; Curzon, G.; Adams, R. N. Brah Res. 1979, 171, 85-99. Lindsay, W. S.; Kiuort, 8. L.; Justice, J. 8.; Salamone, J. D.; Neill, C). 8.J. Neuroscl. Meth. 1980, 2 , 373-388. Gonon, F.; Buda, M.; Cespugiio, R.; Jouvet, M.; Pujol, J.-F. Nature (London) 1980, 286, 902-904. Huff, R. M.; Adams, R. N. Neuropharmacology 1980, 19, 587-590. Ossendorfavl, N.; Prad%, J.; Pradleova, J.; Koryta, J. J. Electroanal. Chem. 1975, 58, 285-261. Mattson, J. S.; Jones, T. T. Anal. Chem. 1976, 48, 2164-2167. McCreery, R, L.; Drelilng. R.; Adarns, R. N. Brain Res. 1974, 721, 23-33. Barker, G. C. I n "Prlogress in Polarography"; Zuman, P., Koithoff, I., Eds.; Interscience: lUew York, 1962; Vol. 2, pp 41 1-427. Lane, R. F.; Hubbard, A. T. Anal. Chem. 1978, 48, 1287-1292. Anderson, J. E.; Bond, A. M. Anal. Chem. 1981, 53, 504-508. Ewing, A. G.; Withneil, R.; Wightman, R. M. Rev. Sci. Instrum. 198.1, 52, 454-458. Pellegrino, L. J.; Cushrnan, A. J. "A Stereotaxic Atlas of the Rat Brain"; Appleton Century Crofts: New York, 1967. Dietz, R.; Peover, M. E. J. Mater. Sci. 1971, 6 , 1441-1446. Kambara, T. Bull. Chem. SOC.Jpn. 1954, 27, 523-526. Bard, A. J.; Faulkner, L. R. "Electrochemical Methods: Fundament& and Applications"; Wiley: New York, 1980. Cheng, H. Y.; Schenk, J.; Huff, R.; Adams, R. N. J . Nectroanal. Chem. 1979, 100, 23-31, Lindsay, W. S.; Justice, J. B. Comput. Chem. 1980, 4 , 19-26. Von Voightlander, P. F.; Moore, K. E. J. Pharmacol. Exp. Ther. 1978, 184, 542-552.
RECEIVED for review ,4pril8, 1981. Accepted July 20, 1981. This research was supported by NSF Grant BNS 81-000441. M.A.D. is a combined Medical-Ph.D. candidate, Indiana University. R.M.W. is the recipient of a Research Career Development Award :from the National Institutes of Health (Grant No. 1 KO4 NS 00356).
Analog Instrument for Oxidative and Reductive Potentiometric Stripping Analysis Joan K. Christensen, Kristlan Keiding, Lars Kryger," Jean Rasmussen, and Hans J. Skov Department of Chemistry, Aarhus University, Langelandsgado 140, DK-8000 Aarhus C, Denmark
An Instrument for potenllometric strlpplng analysis is described. With a three-state potentlostat module, both loxldative and reductive potentlometric stripping analysis can be carrled out. I n thls manner, elements such as selenium, sulfur, the halldes, and manganese are added to the llst of substances whlch can be determined by potentiometric: strlpplng analysls. The Instrument can be extended by 8 dlfferentlator module. This module transforms the basic POtentlal vs. time curve to a dtfferential potentlogram, where the analytlcal Information Is represented as peaks. Examples of stripping potentlograms obtalned with the device are given, and those aspects of the analytical performance of the PO.. tentlometrlc stripping technique which depend on instrurnental factors are discussed.
Potentiometric stripping analysis is a relatively new technique for the determinatimon of trace elements in solution (I,
2). In general, the elements which can be determined b y potentiometric stripping analysis are those which can also be determined by voltammetric stripping analysis, and the results obtained by the two types of stripping techniques compare well. However, there me differences which may appear when, for example, samples contain organic traces: with voltammetric techniques, and in particular with pulsed voltammetric techniques, dissolved reversible couples produce interfering signals. This is not the case with potentiometric stripping analysis ( 3 , 4 ) . Potentiometric stripping analysis for elements which can be reduced at a mercury electrode to form dilute amalgams and which exhibit diffusion-controlledredissolution can be determined with very simple instrumentation comprising (apart from the electrochemical cell) a potentiostat, a high-impedance amplifier, a timer switch, and an x-t recorder (I). Such instrumentation is now commercially available (5). This paper describes an instrument for the potentiometriic stripping determination of a larger selection of elements than those determined with oxidative potentiometric stripping
0003-2700/81/0353-1847$01.25/00 1981 American Chemical Society
