1752
Anal. Chem. 1987, 59, 1752-1757
Temporal Characterization of Perfluorinated Ion Exchange Coated Microvoltammetric Electrodes for in Vivo Use Eric W. Kristensen, Werner G. Kuhr, and R. Mark Wightman*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
The temporal response of perfluorhated ion exchange (PFIE) coated, microvoitammetric electrodes to rectangular concentration changes has been evaluated. These devices, with mkrometer t i i dlmendons, have been designed for use as in vivo probes of eiectroactlve neurotransmitters such as dopamine. The penneath of dopamlne at electrodes wlth PFIE fibns thinner than 200 nm Is sufficiently fast that distortion of the concentration pulse is not apparent. The penneation rate of Ru(bpy)t+ Into these flbns varies wtth the appHed potential waveform. At constant applied potential the measured permeation rate Increases with repetitive exposures. However, wlth fast-gcan cycllc voltammetry (200 V s-’) repeated at 1-s Intervals the permeation time for Ru(bpy);+ remalns reiatively constant wlth repetitive exposures. Physical diffuslon through these films predominates for this complex when sampled In this manner. The thickness of each lndhridual Him can be determined from the permeation raies. The diffusion coefficient for dopamine in these thin PFIE films is found to decrease with a decrease in sodium ion concentratlon in solution. Voltammograms of dopamlne In PFIE lkns are slmiiar to those in solution except at high loading of the film where the buffer capacity can be exceeded. The electrodes are found to respond rapldly to the stlmuiated secretion of dop amine in the brain of an anesthetized rat.
In recent years there has been considerable interest in developing methods to measure the secretion of small molecules, known as neurotransmitters, from neurons deep inside the brain ( I ) . The interaction of neurotransmitters with specific receptors is one of the major modes of communication between neurons (2). The ability to detect neurotransmitters directly inside the brain, but exterior to neurons, would provide a direct method to understand this mode of chemical communication. Most neurotransmitters are small molecules, and some are easily oxidized. This enables voltammetric detection. Voltammetric probes of micrometer dimensions have been developed that cause minimal damage to tissue and are sufficiently large that extracellular measurements are assured (3). However, the specificity of the response of these probes has been questioned ( 4 ) ,and the signal tends to deteriorate within minutes after implantation ( 5 , 6 ) . Recently, these two problems have been minimized with the use of films made of Nafion brand perfluorinated in exchange membrane coated on the electrode as developed in Adams’ laboratory (7, 8). Nafion, a cation exchange polymer, has been extensively investigated as a membrane on the surface of voltammetric electrodes (9-19). This membrane can exclude anions from the electrode surface and thus impair their detection. The partition coefficients for cations into the membrane have been shown to have large values, and their accumulation inside the membrane facilitates electrochemical detection (19-22). Thus, Nafion provides a simple method to enhance the selectivity of voltammetric measurements. The polymer is available in a commercial, dissolved form, and the electrode surface can
be covered by dip-coating the electrode in the polymer solution and evaporating the solvent. This facilitates electrode modification at microvoltammetric electrodes. The utility of Nafion-coated microelectrodes has been demonstrated in the mammalian brain by detection of dopamine, an easily oxidized neurotransmitter, which contains a protonated amine group a t physiological p H (7, 8, 19). Although Nafion provides increased selectivity, it may distort the temporal response of the sensor. Diffusion coefficients of cations in the polymer tend to be greatly reduced compared to solution values. In fact, the rates of diffusive mass transfer for hydrophobic substances such as Ru(bpy)? are sufficiently low that transport of charge through the membrane is more efficient by a mechanism that involves intermolecular electron exchange (11-18). The time for diffusion-limited permeation through a membrane following instantaneous exposure to a chemical substance can be described mathematically by (23)
C,(t)/C, = m
1 - (4/7r)
C [ ( - l ) n / ( 2 n + l ) ]exp (-Dm(2n + 1)27r2t/412)
n=O
(1) In this equation C,(t) is the concentration of the substance in the membrane adjacent to the electrode, C1is the concentration of material in the membrane a t the membranesolution interface, D , is the physical diffusion coefficient within the membrane, t is the time since the chemical substance was exposed to the membrane-solution interface, and 1 is the thickness of the membrane. The concentration Cl is related to the solution concentration by the partition equilibrium coefficient, k D (20-22). The flux of material across the membrane-solution interface is usually assumed not to be rate limiting (9, 10, 24, 25). From eq 1 the initial measurable flux on the electrode side of the membrane will occur a t D,t/12 > 0.1 (23). Thus, to achieve the rapid temporal response required for detection of neurotransmitters in vivo, a membrane with minimal thickness is required. As will be shown, films of Nafion can be applied to carbon-fiber electrodes that are sufficiently thin that rapid permeation of dopamine is observed. The membrane contents have been sampled with fast-scan cyclic voltammetry, repeated at constant time intervals (26). In this way, the contents of the film are maintained in their original, reduced form for the majority of the time. To determine the approximate film thickness, the permeation of Ru(bpy)?+ has been examined under conditions where diffusional permeation as described by eq 1 is the predominant mode. In addition to demonstrating the physical and chemical properties of these ultrathin films, this paper describes the suitability of these sensors as in vivo probes of neurotransmitter release.
EXPERIMENTAL SECTION Reagents. All chemicals were reagent grade and used as re-
ceived from commercial sources. Solutions were prepared from doubly distilled water. Solutions of dopamine (3-hydroxytyramine
0003-2700/87/0359-1752$01.50/00 1987 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
hydrochloride, Sigma, St. Louis, MO) and ascorbic acid (Sigma) of the desired concentration were prepared by diluting a stock solution prepared in 0.1 N HC104into a pH 7.4 buffer (60 mM NazHPOl and 30 mM NaHzPOl (150 mM Na+)) or a buffer containing 4 mM Na2HP04and 2 mM NaH2P04(10 mM Na+). Solutions of Ru(bpy)32+(tris(2,2’-bipyridyl)ruthenium(II) chloride hexahydrate, Strem Chemicals, Inc., Newburyport, MA) were freshly prepared in 0.2 M Na$O1. All solutions were continuously dearated with N2 prior to use. Electrode Construction. Beveled microvoltammetric carbon-fiber electrodeswere prepared in glass capillariesas previously described (27). The result is an electrode with a smooth elliptical carbon surface, with a active sensing area of approximately 2 X lo4 cm2,surrounded by a thin wall of glass. Electrodes were not pretreated electrochemically (28-30) because this can cause adsorption of dopamine. Nafion Coating Procedure. A solution of Nafion (10% by weight of 1100 equivalent weight polymer) was prepared by placing a vigorously stirred commercial solution (5% by weight in 80% lower aliphatic alcohok and 20% water [Solution Technology Inc., PA]), into a boiling water bath until the solution volume was reduced by 50%. The alcohol-water solvent will evaporate at room temperature. Thus it is recommended that this solution be stored in an airtight vial and be replenished approximately every 3-6 months. Electrode tips were initially soaked in the 10% Ndion solution for 10 min and then allowed to dry for 10 min under a heat gun. The humidity at the electrode tip appears to affect the rate of solvent evaporation. The electrode tips were then rapidly (-0.1 s) dipped into the Nafion solution five times with approximately 2 s between dips, followed by 10 min under the heat gun. This procedure was repeated and the electrodes were allowed to dry at room temperature overnight. Thinner films can be made by increasing the time the electrode tip is dipped into the Nafion solution. It appears that the coating on the electrode tip disaolves when placed in the Nafion solution. Temporal Response Measurement. The temporal response of a microvoltammetric electrode to the rapid application of electroactive species was measured with the flow injection analysis (FIA) system previously described (31). Electrochemical measurements were made either at fixed applied potential or by fast-scan cyclic voltammetry. In both cases an IBM Personal Computer (Boca Raton, FL) with locally written software, a Labmaster interface card (Scientific Solutions, Solon, OH), and locally constructed potentiostat of conventional three-electrode design were employed. The time constant of the potentiostat’s potential driver and current transducer were 10 ps and 10 ms, respectively, for constant potential measurements and were 10 and 12 p s , respectively, for fast-scan cyclic voltammetric measurements. The voltammetric waveforms were obtained from a function generator (Wavetek Model 143, San Diego, CA) and were triggered by the computer. A saturated sodium calomel reference electrode was used in all experiments. To ensure that the observed temporal distortion was due to the Ndion membrane and not the dispersive elements within the FIA system the following protocol was adopted. Initially the microvoltammetric electrode was centered by sight in the stainless-steel tube (0.8-mm i.d.), which served as the exit of the FIA system. The electrode was lowered into the tube to a depth of 5.0 mm with a micromanipulator. The current response to a 10 p M dopamine pulse at this position was measured at a constant potential of 0.6 V vs. SSCE. The position of the microvoltammetric electrode was then adjusted in the plane perpendicular to the solution flow until the leading edge of the current response was nearly instantaneous and occurred 2.03 f 0.03 s (n = 13) after the injection of dopamine (31). The time delay was subtracted from the measured data to correct for the time in the transport tubing. The time required for the current to change to 50% of the limiting value was 0.08 & 0.03 s (n = 13) at uncoated electrodes. Voltammograms with Nafion-coated electrodes were obtained at various scan rates up to 300 V s-l. The voltammograms were repeated at intervals sufficient to allow diffusion-layer relaxation (32-34). Background currents were removed by subtracting the current before exposure to electroactive species (26). In Vivo Voltammetry. Male, Sprague-Dawley rats were anesthetized with chloral hydrate and placed in a stereotaxic
.
1753
.
I....,....I....,....I....,....I....,....I....~
0
4
(SI Figure 1. Amperometric response (0.6 V vs. SSCE) to a 10 MM pulse of dopamine before (A) and after (B) the Nafion coating. The response to 100 pM ascorbic acid pulse was 250 pA before the Nafion coating and (C) 1.3 pA after the Nafion coating. Amperometric response (1.2 V vs. SSCE) of a Nafion-coated electrode to the first 10-s 100 pM Ru(bpy):+ concentration pulse (D) followed 4 min later by the second exposure (E). Time
frame. The skin was retracted and holes were drilled in the animal‘s skull for insertion of the electrodes at the appropriate locations. Nafion-coated microvoltammetric electrodes were lowered into the anterior striatum, a region of the rat brain which has a large number of dopamine nerve terminals. The auxiliary and reference electrodes were placed in contact with the brain on the animal‘s skull. To induce dopamine release, the nerve fibers projecting into the anterior striatum were stimulated electrically. A stimulating electrode was lowered into the medial forebrain bundle and, at the appropriate time, a pulse train (60 Hz) of constant-current, biphasic pulses (200-3300 FA) was applied for 2 s. Voltammetric measurements were synchronized with the stimulus so that the data was acquired between the pulses. The electrode was then removed from the brain and characterized in the flow injection system. Further details of the in vivo experiment are given elsewhere (35). Curve Fitting. The values of the parameters in eq 1 were optimized with a nonlinear regression program to obtain the best fit to the transient shape of the measured permeation curve. The peak current is related to Co(t)by normalizing the data to the limiting peak current. The curve fitting program was based on the modified simplex algorithm of Aberg and Gustavsson (36).
RESULTS Response of Nafion-Coated Electrodes to Dopamine. The temporal response of a Ndion-coated electrode (constant potential of 0.60 V) to a concentration pulse of dopamine (10 pM) is compared to that of an uncoated electrode in Figure 1. In both cases the buffer contained 150 mM Na+. The distortion caused by the Nafion is sufficiently small that the existence of the membrane could be questioned. However, the Nafion membrane is clearly present as seen by the attenuated current measured in response to the electroactive anion, ascorbate (100 pM).The oxidation current is 0.5% of its precoated value for this electrode. No difference was observed in the time course between the first and subsequent exposures of the electrode to dopamine. Dopamine permeation was also examined by fast-scan cyclic voltammetry. This technique allows voltammetric characterization of the permeant species as well as the time for permeation. Triangular waveforms (200 V s-l) were repeated a t a fixed time interval, and the electrode potential was maintained at a nonoxidizing value for the majority of the time (Figure 2). Dopamine permeation rates can be examined by a plot of the current at the peak potential for dopamine from successive voltammograms. Although large residual currents are observed a t these rapid scan rates, digital subtraction of voltammograms obtained before and during exposure to dopamine results in well-shaped voltammograms even at rela-
1754
ANALYTICAL CHEMISTRY,
VOL. 59, NO. 14, JULY 15, 1987 -4 0
0
I
T l m r (s)
i,(nA)
0
0
Tlmr
I
( 8 )
0
Time
(0
40
Figure 3. Peak current response at 200 V s-' in response to a 50-s 100 pM concentration pulse of Ru(bpy):+ before Nafion (+) and after the Nafion coating. The time between the first (0)and second (0)
exposures after the Nafion coating was 20 min. The solid lines for the response of the Nafion-coated electrodes are calculated from eq 1. Both curves were corrected for residual current.
A
0
C
Figure 2. Voltammetric response of a Nafion-coated microvoltammetric electrode (radius = 4.32 pm) to a 10 pM dopamine pulse. Upper: The potential waveform of the 200 V s-' triangular wave; E,,, = -400 mV; E,,, = 900 mV; repeated every 100 ms. Mile: Peak current (i,) from the average current between 550 and 600 mV on the
positive sweep. The average current for the first five scans (residual current) was -13.3 nA. The subtracted voltammogram (C) was constructed by averaging the first five scans (voltammogram A) and subtracting this voltammogram from the average voltammogram of the hst five scans (voltammogram B). The peak current for the subtracted voltammogram (C) was 0.70 nA at 0.57 V vs. SSCE. tively low concentrations (26). Temporal Characteristics of Ru(bpy)3*+. Ru(bpy),*+ was selected to provide an electrochemical procedure to determine the thickness of these membranes. The electrochemical behavior of this compound in Nafion has been characterized (11-18), and the physical diffusion coefficient for this compound is estimated to be 1 X lo-" cm2 s-l (18). The response of a concentration pulse was examined at a fixed potential (1.2 V). A delay can be seen in the onset of the oxidation current on the first exposure to Ru(bpy)gP+(Figure l ) , as expected for a species with a reduced rate of mass transport through the membrane. However, the permeation rate increases with time in contradiction to that predicted for diffusion-electrolysis processes. Furthermore, when the membrane is exposed to Ru(bpy),*+ for a second time, the current-time curve is dramatically different from that of the first exposure. Fast-Scan Cyclic Voltammetry of R ~ ( b p y ) in ~ ~Nafion + Films. These amperometric results are likely to be a result of two competing mechanisms for charge transport through the membrane. Before electrolysis occurs on the first exposure, charge is transported simply by the physical diffusion of incoming Ru(bpy)?+. However, when the electrode is operated in an amperometric mode, Ru(bpy)?+ is generated throughout the film, which may alter the permeation process. If the potential of the electrode is kept at a value that ensures that the oxidation state of the species in the membrane is the same as in solution, then only one partner of the redox couple exists and complications in competing permeation pathways should not exist. To approximate this condition, the concentration in the membrane has been sampled with fast-scan voltam-
metry (200 V s-l) repeated a t relatively long intervals (15). Under theses conditions, generation of R ~ ( b p y ) ~occurs ,+ for only 4 ms, and its concentration in the film is kept at low levels by the electrode rest potential for the remainder of the time. In this way, permeation should be approximated by eq 1. The success of this approach is demonstrated in Figure 3. A finite delay time between exposure of the membrane to R ~ ( b p y ) and ~ ~ +the appearance of oxidation current at the electrode is clearly observed. The slowly rising signal, representing R ~ ( b p y ) , ~permeation, + fits well to the expected curve shape described by eq 1. To obtain the fit illustrated in Figure 3, a value of D, of 1 X lo-" cm2 s? was employed, and the thickness of the film was determined by nonlinear regression. The membrane thickness obtained from this simulation was 180 nm, which agrees remarkably well with the estimates from electron microscopy (vide infra). The thicknesses of the seven other electrodes tested in this manner were determined to be 70,110,120,140,170,450, and 530 nm. The peak current at steady state is larger at coated electrodes than a t uncoated electrodes because of the large partition coefficient for this compound (18, 22). When the concentration of R ~ ( b p y ) in ~ ~the + FIA system is reset to zero, the concentration of electroactive species in the membrane slowly diminishes. In thicker films loss of R ~ ( b p y ) , ~is+not observed (11-17). In the experiments reported here the film is constantly exposed to the carrier stream, which will facilitate outward transport. However, even 20 min after the initial exposure, 15% of the original concentration of R ~ ( b p y ) , ~still + exists in the film as determined by fast-scan cyclic voltammetry. Despite this, a second exposure to R ~ ( b p y ) , ~results + in a very similar permeation time to that observed initially. Values of the membrane thickness obtained during the second exposure were generally within 10% of that determined on the first exposure. Determination of Membrane Thickness by Electron Microscopy. Seven Nafion-coated microvoltammetric electrodes were investigated with a scanning electron microscope. A relatively smooth uniform membrane, without pinholes, was observed over the tip of the electrode. In one case the Nafion membrane peeled away from the glass capillary. This allowed the thickness of the membrane to be estimated as 200 nm. Characterization of Dopamine Permeation w i t h 150 m M Na+. Five of the electrodes whose thicknesses were determined with Ru(bpy)32+were also employed to characterize further the permeation of dopamine into Nafion. To maximize the temporal resolution of the experiment, voltam-
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
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X lmax
0
0
Figure 4. Left panel. Peak current response at 200 V s-' to a 10-s 10 pM dopamine pulse after the Nafion coating and simulated response: (0)response in 150 mM Na+ solution: (0)response in 10 mM Na' solution; (-) simulated response by eq 1 employing D, = 1.5 X lo4 om2 s-' (for curve A) and 5.9 X lo-" cm2s-' (for curve B) with I = 120 nm (as determined by Ru(bpyh*+ permeation, see text). Right panel. Voltammograms (50 V s-l)of 100 pM dopamine in 150 mM Na' (-) and in 10 mM Na' (-): S = 0.30 and 2.5 nA for A and B, respectively; S' = 2.5 and 19.4 nA for the solid and dotted line, respectively. mograms were obtained at 200 V s-l every 200 ms. A measurable temporal distortion to the dopamine pulse was observed only for those electrodeswith membrane thicknessesthat were greater than 400 nm. In these two cases, the plot of i, vs. time following exposure to 10 pM dopamine had the shape predicted by eq 1. Nonlinear regression, employing the membrane thickness obtained from the Ru(bpy)32+study, gave a value for D, of 1.5 X lo4 cm2 s-l, which is in agreement with previous estimateslg [(1-3) X cm2 s-l]. Dopamine disappeared from the film when the dopamine was removed from the buffer with a rate similar to that for permeation. To determine whether permeation times depend on concentration, dopamine concentrations from 1pM to 1 mM were examined at three electrodes with thicknesses of 80,110, and 120 nm. At all three electrodes, the shape of the permeation curves was not a function of concentration. The value of i, at steady state was linear with dopamine concentration (r = 0.9991, n = 3). Peak currents for dopamine oxidation were approximately 4 times greater than at a bare electrode.
Characterization of Dopamine Permeation from Buffer Containing 10 mM Na+. The partition equilibrium coefficient into Nafion films in these experiments should be a function of the NaC concentration in solution. To test for this, the three electrodes that were used to examine the concentration dependence for dopamine permeation were also examined in the presence of 10 mM Na+. At 200 V s-l the peak current a t coated electrodes for 10 pM dopamine was approximately 8.5 f 1.7 times larger for the low Na+ concentration than for 150 mM Na+. The peak current was linear from 1to 100 pM (r = 0.9990, n = 3) while nonlinear behavior was observed from 200 pM to 1 mM. In addition to the increase in peak amplitude, the permeation time is considerably altered at the lower Na+ concentration (Figure 4). The diffusion coefficient under these conditions was determined to be (7 f 3) X lo-" cm2 s-* by nonlinear regression of the data and by assuming that the film thickness was unchanged. Voltammograms at low concentrations of dopamine (10 p M ) were identical at both high and low concentrations of Na'. However at higher concentrations of dopamine (>lo0 pM)larger differences became apparent. The wave for the reduction of the electrolysis product split
Time (e)
6
Figure 5. Comparison of the normalized peak current for dopamine measured at 300 V s-' In the FIA system (+) in the presence of 150 mM Na+ and that obtained in vivo (0)during electrical stimulation of dopamine neurons. Peak concentration measured in vivo is 6 pM.
into two waves at scan rates over 20 V s-l in the low Na+ media (Figure 4).
Nafion-Coated Electrodes as in Vivo Probes of Dopamine. Dopamine can be observed in the anterior striatum either as a result of injection of this substance into the brain environment or as a result of electrical stimulation of dopamine fibers that project into the nerve terminal area of dopamine neurons (3,37,38). When a Nafion-coated working electrode is placed in the nerve terminal region, an increase in the oxidation current at the peak potential for dopamine can be detected during a stimulation. The peak currents from repeated voltammograms during and after the stimulus allow the temporal resolution of the stimulated release (Figure 5). Voltammetry during the stimulations shows that the released substance is identical with dopamine. More complete evidence for the identification has been previously reported (38). To ensure that the concentration transient measured in vivo is not distorted by the Nafion membrane, the response time of the electrode after implantation was measured with the FIA system with a Na+ concentration similar to that found in the extracellular fluid of the brain (Figure 5). It can be seen that the release and subsequent disappearance of dopamine in vivo are not significantly distorted by the Nafion-coated microvoltammetric electrode. Therefore, the disappearance rate of dopamine in vivo is a direct measure of the lifetime of dopamine in extracellular fluid. The stability of Ndion-coated microvoltammetric electrodes with respect to dopamine detection was examined before and after the in vivo experiment. These electrodes maintained a greater degree of their sensitivity to dopamine than did uncoated electrodes. DISCUSSION Nafion films on microvoltammetric electrodes greatly enhance the utility of these electrodes as chemical sensors. The films provide a considerable degree of protection to the electrode surface, which prevents diminution of the voltammetric signal in vivo. Of prime importance is the observation that the response time to changes in dopamine concentration is rapid. This enables the rapid changes in dopamine concentration to be measured in real time. The partial exclusion of anions, demonstrated here and elsewhere (19),is also important for a sensor that is to be used in a chemically complex mixture such as brain tissue. The advantageous properties of Nafion-coated electrodes are realized to a greater degree when the membrane contents are sampled by fast-scan cyclic votammetry. First, the digital subtraction technique on successive voltammograms enables
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ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
a clear "fingerprint" to be obtained for the substances in the film. Thus, identity of the detected substance is based on not only permeation of the film but also voltammetry. Second, rapid-scan voltammetry restricts the diffusion layer to the interior of the film. At a scan rate of 200 V s-l, electrolysis occurs for only 4 ms in each scan. For a substance such as dopamine (D, cm2 s-l) this means that the diffusion layer extends only 20 nm into the film. This results in an enhanced value of the peak current because of the high value of the partition equilibrium coefficient. In addition, voltammograms obtained in vivo and in vitro both are from the voltammetrically generated flux in the film. Thus, the validation of the concentrations determined in vivo is more likely correct, because the differences between brain and solution diffusion coefficients do not effect the current. The partition coefficient is affected by pH and ionic strength, but these are selected to be similar in the test solutions as in the brain. Furthermore, by restriction of the diffusion layer to the interior of the film, electrolysis products are less likely to be able to escape into the extracellular environment. This feature is important since dopamine o-quinone is likely to be a neurotoxin. The rapid response times for dopamine permeation arise because the Nafion films are very thin. As shown by eq 1, permeation times are critically dependent on this parameter when low values of diffusion coefficients occur. Indeed, equilibration times of several days are required for some inorganic cations into thicker (2 pm) Nafion membranes (22). Outward transport must occur over the same time interval as inward permeation and both of these processes must be rapid for a useful in situ sensor. We have used the permeation of Ru(bpy)? as a probe of the film thickness since traditional methods for thickness profiling are not useful for such films. This compound has a sufficiently low value for its diffusion coefficient in Nafion that temporal distortion is readily apparent. Permeation of R ~ ( b p y ) into ~ ~ +Nafion films on electrodes has previously been studied amperometrically. Under these conditions, the concentration gradient is affected by electrolysis as well as permeation (18,24,25). Although this can be accounted for mathematically (39-43), electrochemical generation of the redox partner, Ru(bpy)?+, alters the permeation time as observed in our amperometric experiments. In fact, a difference in the permeation time between the first and second exposure to Ru(bpy),3+is indication of charge transport via intermolecular electron exchange. When the film contents are sampled by fast-scan cyclic voltammetry (200 V s-l, electrolysis time 4 ms) repeated at 1-s intervals, permeation times become much more reproducible. Because the gradient induced by electrolysis is small, eq 1can be used to describe the relative concentration changes from the peak current of the voltammograms. Thus, with the use of the physical diffusion coefficient for R ~ ( b p y ) ~estimates ~+, of the film thickness can be obtained. These estimates are in agreement with those obtained by electron microscopy. The ability to discriminate against the electron-hopping pathway deserves further comment. Fast-scan cyclic voltammetry allows this discrimination in two ways. First, the scan is sufficiently fast that electron hopping is unlikely to occur during the interval when the oxidized form is generated. Anson has estimated the diffusion-controlled rate constant in the film to be 4 X lo3 M-' for a complex of similar size (14). Since the concentration of Ru(bpy)32+is low for the majority of the time of permeation, electron hopping does not have time to be efficient. Estimates of the diffusion coefficient obtained by using the peak current from a single scan and the known partition coefficient concur with this assertion. Second, the concentration of the oxidized form is kept low by the
-
electrode. During the reverse scan, 60% of the oxidized form generated initially is reduced. In addition, the rest potential ensures that reduction continues after the scan is terminated. However, a worst case concentration of Ru(bpy)?+ can be calculated from the experimental data. Subtraction of the integrated reverse current from the integrated forward current of a single scan provides the number of moles produced during each scan. If it is assumed that this amount does not change between scans, then summation of this amount gives, by Faraday's law, the amount of Ru(bpy),3+ that accumulates during the 50-s permeation experiment (-4 X mol). In contrast, the amount of the reduced form in the volume of the film over the electrode is 4 X lo-" mol, as calculated from literature values for the partition coefficient. Since the half-life for a second-order reaction depends on concentration, the electron-hopping mass-transport mechanism is discriminated against by a factor of lo3 compared to the analogous amperometric experiment. In practice, the degree of discrimination is even greater because the concentration of Ru(bpy),3+ is decreased even more by electrolysis between scans. With films that have been characterized in this manner, other properties of the permeation of dopamine have been examined. In solutions containing 150 mM Na+, a physiological concentration for Na+, the voltammetric current is proportional to dopamine concentration. At 10 mM Na+ the partition of dopamine into the film increases as expected (22). In fact, under these conditions calibration curves for dopamine become nonlinear at high concentrations presumably because the film becomes saturated. Even more striking is the change in permeation time that occurs for dopamine when the Na+ concentration is lowered. This apparent correlation of permeation rate with Donnan potential has not previously been observed. Because of the decrease in diffusion coefficient, only a modest increase in peak concentration was observed with a decrease in ion strength. The peak current from substances in the film is proportional to k&u1/2. Voltammograms of dopamine in Nafion films are similar to those for dopamine a t uncoated electrodes. In solution, the electrochemical oxidation of dopamine involves the loss of two protons and two electrons. Assuming that this mechanism holds in the film, it is surprising that the protons which are generated do not affect the voltammetric shape, because the Nafion film is an unbuffered region. However, it appears that the pH on the interior of these thin membranes is governed by that of solution. This is possible in thin films because protons, which should have a rapid diffusion rate in Nafion, can remain in communication with solution (44). Deviations of the shape of the voltammograms from that expected are only obtained with high dopamine concentrations in solutions of low buffer capacity. Under these conditions, it appears that the pH of the membrane is altered by the protons generated during electrolysis (45). Voltammograms obtained in vivo during stimulation of a dopamine nerve bundle provide evidence that dopamine appears in the extracellular fluid immediately upon initiation of stimulation. The voltammogram of the observed species is identical with that observed for dopamine in solution (35). With uncoated electrodes, this is frequently not the case because insertion of the electrode into the brain alters the kinetics of electron transfer and, thus, the wave position (5, 6). In addition, the partial exclusion of anions reduces the possibility that an anionic substance, with voltammetrically identical behavior, is responsible for the observed signal. The rapid time response to concentration changes enables their measurement in real time. Thus, the residence time of dopamine in extracellular fluid of the brain can be measured. As can be seen, concentrations greater than 1 pM exist in the extracellular fluid for only a few seconds. Since the affinity
ANALYTICAL CHEMISTRY, VOL. 59, NO. 14, JULY 15, 1987
of a major class of dopamine receptors (Dl) for dopamine is in the micromolar range (46),concentrations less than this are unlikely to be effective in chemical communication. From this result, we postulate that, in the brain region examined, the caudate nucleus, the role of freely diffusing dopamine in the extracellular fluid is of little importance under normal physilogical conditions. This postulate will have to be tested with a kinetic characterization of the regulatory processes and by comparison with dopamine extracellular concentration fluxes in other brain regions. The in vivo sensor characterized here should be very useful in these types of investigations.
ACKNOWLEDGMENT Discussions with W. Grot and R. Adams are gratefully acknowledged. Registry No. Na, 7440-23-5; dopamine, 51-61-6; Nafion, 39464-59-0.
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RECEIVEDfor review December 9,1986. Accepted March 30, 1987. This research was supported by a grant from NIH (PHS R01 NS-15841).
