1518
Anal. Chem. 1985, 57, 1518-1522
In Vitro Measurement of Dopamine Concentration with Carbon Fiber Electrode Akitane Akiyama*
Department of Electronic Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227, Japan Takeshi Kat0 a n d Kayoko Ishii Department of Life Chemistry, Graduate School at Nagatsuta, Tokyo Institute of Technology, Yokohama 227, Japan Eiichi Yasuda Research Laboratory of Engineering Materials, Tokyo Institute of Technology, Yokohama 227, Japan
Microcomputer-controlled potentiostatlc pulse polarizatlon techniques have been applied to the analysls of dopamlne wlth carbon fiber electrodes. By the Introduction of the electrochemical treatment before every measurlng pulse, the sensitivity and reproduciblllty were remarkably Improved. Dlfferenl carbon flbers gave different stability and sensitlvlty, and HTA-7 was preferred for anaiytlcal purposes. The dlameter of the electrode is 7 pm. The detection llmlt is less than 2 nM. The separation of dopamine from other catechols and Indoles Is satisfactory. The adsorptlon of dopamlne at the carbon fiber surface has been assumed, and the Isotherm has been dlscussed.
The measurement of dopamine concentration in the brain is now an important subject in brain research. Dopamine (DA) is easily oxidized at an anodically polarized electrode, so that electrochemical techniques are applicable. In consideration of the dimension of electrodes, carbon fibers often were selected as electrode materials. Initial attempts have been with normal pulse polarography (1-4); however, the detection limit is far higher than the expected value for extracellular DA. In order to enhance the sensitivity, differential pulse voltammetry has been widely used (5-9). Recent development has proceeded in two ways; one is the use of multifiber electrode (10) and another is the use of differential normal pulse voltammetry (11). The electrochemical techniques reported above are all potentiostatic pulse polarization techniques. It was thought that the precise control of pulses would give better results; therefore, a microcomputer-controlled system has been applied to this investigation. It is widely known that the surface pretreatment of solid electrodes determines the success of an electrochemical experiment; therefore, investigators have been examining the use of the electrochemical treatment of carbon fiber electrodes (12, 13). Rigid control of the preelectrolysis (activation) in this investigation provides excellent sensitivity, selectivity, and reproducibility. EXPERIMENTAL SECTION Microcomputer System. The microcomputer system designed and constructed in our laboratory for various electrochemical experiments was employed. A computer-controlled micropotentiostat was newly designed for this experiment and connected to the system. The main operational amplifiers used are AD515 and CA3130. The precision is 25 pA/digit, and the response time is adjusted to 0.5 ms. The microprocessor is MC6800, and the system contains 32K RAM, 4K EPROM, 16 channels of differentialAID, 4 channels of D/A, 3 programmable timers (MC6840),an arithmetic processor (9511A),2 asynchronous
communications interface adapters (MC6850),8 peripheral interface adapters (MC6820), and a general purpose interface adapter (MC68488). A programmable potentiostat, a graphic plotter, an automatic buret, and other 1/0 devices are connected to the system. The software used is essentially GAME which is widely used among Japanese amateurs, and most of the experimental routines are written in machine language. Working Electrode. The working electrodes were carbon fibers and fabricated in the method reported by Ponchon et al. (1). Epoxy resin was used for the shield and silver paste for the electrical contact. After the fabrication, the electrical resistance was checked and electrodeswhich had abnormallyhigh resistance were thrown away. The surface of carbon fibers is covered by other materials; therefore, an electrochemical treatment was necessary to remove them, and anodic and cathodic triangular polarizations were applied in situ for the newly prepared electrodes before experiment. Fibers tested were HTA-7 (Toho Beslon Co. LTD., Japan), MH-45 (Toho Beslon), heat treated HM-45 at 1000 "C under argon, and highly crystalized fibers which were synthesized by a vapor-phase growth method (14). The data shown in this paper are mainly those obtained by use of HTA-7. The length of the electrode was 500 pm, and the diameter was 7 pm. The length of 50 pm was also tested, and it was found that it had enough sensitivity for DA if the current sensitivity was increased by a factor of 10. Electrochemical Cell. An electrochemical cell was specifically designed for the in vitro study. The cell was a 100-mL separable flask with reference and auxiliary electrode compartments, all of which were connected through ball joints, so that the experiment can be performed under deaerated conditions. The reference electrode is a Ag/AgCl electrode and potentials in this paper are reported vs. Ag/AgCl. Experiments were performed in a constant temperature room at 25 "C. Chemicals and Electrolyte. The supporting electrolyte was mainly phosphate-buffered saline solution (PBS), although Krebs-Ringer bicarbonate buffer (pH 7.2) was also used for the reference. DA.HC1, 3,4-dihydroxyphenylaceticacid (DOPAC), 3-methoxytyramineHC1(Me-DA),5-hydroxytryptamine.creatinine sulfate (5HT),and 5-hydroxyindoleacetic acid (5HIAA)were dissolved in high concentration and stored in a freezer. The stocked solutions were thawed and diluted before addition to the cell. Ascorbic acid (AA) was immediatel? dissolved in the buffer before addition to the cell. RESULTS AND DISCUSSION Basic Electrochemical Procedure. The electrochemical technique employed in this investigation is essentially potentiostatic pulse polarization; however, an activation signal which is an anodic-cathodic triangular wave (the slope is 10 V/s) is introduced immediately before every measuring pulse. The potential profile is sketched in Figure 1. The parameters for measuring pulses are decided as follows. The length of first pulse ( T I )is 460 ms because it took about 250 ms to obtain a steady current in the current-time tran-
0003-2700/85/0357-1518$01.50/00 1985 American Chemical Society
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985 P
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Flgure 1. Potential profile of one measurement. P,: rest potential (-200 mV). ACT activation wave and Pa should be positive. T,: rest
time between activation and measurement. MES: measuring pulses. The first pulse is P I (mV) for T , (ms) and the second P2 (mV) for T 2 (ms). sient. At the end of first pulse, the current is measured every 625 ps for 160 ms, turns out 256 points, and added. The value is shown as a steady-state intensity (Il)in this paper. The length of second pulse is 22.5 ms. The polarization potential changes from PIto Pz and, after 2.5 ms, the current is measured every 625 ps for 20 ms, which is one cycle of the electric line (50 cycles/s). The current after 1ms instead of 2.5 ms gave the same value. The 32 measured points are added and divided by 8, which is some sort of average. The sensitivity was enhanced by this procedure without losing the reproducibility. This value is presented as an intensity (Iz) in this paper. After the second pulse, the potential is returned to the original value of -200 mV (P,) and kept at this value until next measurement. The data must be taken in a given duration of time (T,) in order to obtain reproducible results. The most important parameter in this method is the positive limit of the activation wave, Pa. The value is determined by observing the change of the electrode state during measurements, in a “feed-back’’ manner as follows. The computer program measures the initial intensity (12)of the second pulse at a given value of Pa. After T,, the intensity (12)is measured. The change in the intensity (Iz2- Izl)is divided by four and added to the value of Pa. The value is the positive limit (Pa) in the next measurement. The data are taken consecutively, and, after a certain period of time, I2 as well as Pa become constant. The value of Pais fixed at the constant value, and chemicals are added for the analytical experiments. The range of values for Pawas from 1950 to 2150 mV for HTA-7 and from 1450 to 1700 mV for HM-45. It was found that only a few millivolts change in Pacaused the pulse current drastically; however, if Pawas properly chosen, repeated measurements give the same value. The time durations (T,) were tested between 1 s and 20 min, and the reproducibility was excellent. The time duration (T,) chosen in the present study was mostly 1 min. Response of Dopamine. The intensities (Iz)of the second pulse are blotted in Figure 2 as a function of PI with (marked by 0,A,and 0)and without DA (marked by 0 and H). The concentration of DA is 1 X M, and Pzis equal to PI + 100 mV. The most sensitive value of PI for DA determination is 150 mV. This gives a value of P2 (250 mV) that is the same as the peak point in the voltammogram of DA (1X M) at the carbon fiber electrode. The effect of the rest time (TI in Figure 1) between the activation wave and the measuring pulses upon the intensities is shown in Figure 3 in the presence of DA (0) and the absence of DA (0).It is seen that the activity of the electrode surface in PBS decreases with T,. It is observed that the longer T , causes the further decrease of intensity; therefore, it was
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electrodes. concluded that the rest time (TI)should be as short as possible. The experiment with 1 X M DA, however, shows that there is a maximum in the intensity-rest time curve. The reason will be discussed later. In the following experiments, 3 s is chosen for TI. The concentration dependence and the reproducibility of the intensity are shown in Figure 4. A 100-pL quantity of DA solution (lo4 M) was added to the 100 mL of electrolyte. The electrolyte was stirred with air bubbles, and the measurement was performed after l min. The results in Figure 4 were obtained with three different electrodes. It was seen that the reproducibility is excellent. The detection limit is improved if the current sensitivity was properly chosen. The micropotentiostathas been designed
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Flgure 8. DA intensity curve in phosphate-buffered saline with ascorbic acid (AA), 5-hydroxyindoleacetic acid (BHIAA), and DOPAC. AA: I O 4 M. 5HIAA: lo-' M, DOPAC: lo-' M.
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Flgure 9. Intensity for DA and intensity for Bhydroxytryptamine (5HT) as a function of DA Concentration. P, for DA: 250 mV. P, for 5HT: 450 mV. 5HT of 1.5 X M was added initlally. (0): DA, (0): 5 Ht.
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again with use of two operational amplifiers (OPA 104). The intensity for DA in a low concentration range was tested with this potentiostat and plotted in Figure 5. The results in Figure 5 were obtained with one electrode, and the duration of time (Tm) was 30 s. The data show that the detection limit of DA concentration is less than 2 nM. Note that the activation wave should be from anodic to cathodic, in other words, a cathodic end. When the activation wave was reversed (that is an anodic end), the intensity for DA remarkably decreased as shown in Figure 6 (plotted as 0 ) . The electrochemicallyactive surface is only obtained by an anodic-cathodic treatment. The deaeration of the electrolyte by nitrogen gas was performed, and the effect of dissolved oxygen upon the sen-
8
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Figure 10. Intensity for 5HT and intensity for DA as a function of 5HT concentration. DA of 3 X M was added initially. Other condffions
were the same as those in Figure 9.
sitivity of DA was tested. The result is shown in Figure 7 (e in the figure represents a deaerated experiment and 0 an aerated experiment). It is seen that the presence of oxygen enhances the sensitivity toward DA. The response of DA is not inhibited by the presence of AA M), and 5HIAA (lo4 M). The inM), DOPAC tensity in the electrolyte with the three organics is plotted against DA concentration in Figure 8. In the Figures 8-11, negative points on the horizontal lines show the reproducibility without DA or 5HT. These results show the greatest possibilities for the electrode to be used in vivo.
ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Figure 11. Intensity for DOPAC and intensity for DA as a function of DOPAC concentration. P, for DOPAC: 350 mV. P, for DA: 250 mV. DA of 3 X IO-' M was added initially. (0): DOPAC. (0): DA.
Figure 12. Intensity vs. DA concentration curve for the estimation of surface excesses. (- -): For the straight line from points 0 with the least squares.
Separation of Dopamine from Other Chemicals. DOpamine a n d 5-Hydroxytryptamine. The parameters selected for 5HT are P1= 350 mV and P2 = 450 mV, and other parameters are the same as those of DA. Firstly DA solutions were added to the electrolyte which contained 5HT (1.5 X M), and the intensities both for DA ( 0 )and 5HT (0) are plotted against DA concentration in Figure 9. The intensity of DA increases with the DA concentration; however, the intensity of 5HT does not increase at all. Secondly, 5HT solutions were added to the electrolyte which contained DA (3 X lo-' M), and the intensities for 5HT (0) and DA (0)are plotted in Figure 10. The sensitivity for 5HT is higher t h m that for DA, so that the increment of 5HT concentration was 5X M. It should be noted here that the intensity of DA decreases slightly with the 5HT concentration. The result suggests the coadsorption of both 5HT and DA at the electrode surface. A part of the adsorbable sites for DA might be occupied by 5HT, so that the intensity of DA decreased. This will be discussed in detail later. The same kind of behavior was obtained when MeDA was added to the DA containing electrolyte; that is, the adsorption of MeDA is also suggested. Dopamine and 3,4-DihydroxyphenylaceticAcid. In the voltammogram of DA (1X loF5M), the anodic oxidation peak was 250 mV and the cathodic reduction peak was 200 mV; however, in the voltammogram of DOPAC (1 X low5M), there was no clear peak and the oxidation wave of DOPAC gradually increased from about 300 mV. This suggests that the electrochemical reaction of DA is more reversible than that of DOPAC. Although various values of P1and P2 were tested, the sensitivity for DOPAC was not improved. Gonon et al. (11) reported that DA and DOPAC cannot be separated though their electrodes were 100 times more sensitive for DA than for DOPAC. With our electrodes, DA and DOPAC can be separated from each other. The reason for the discrepancy is not clear but one of the possible explanations is that the electrochemical activation of the carbon fibers was different from each other. In the present study, a steady-state value (Il)of a polarization potential was 450 mV for the analysis of DOPAC. The steady-state values must be controlled by the diffusion of DOPAC, so that the reproducibility is very poor because the movement of electrolyte around the electrode greatly affects the anodic current. The intensities for DOPAC (11at 450 mV) (0) and the intensities for DA (I2)( 0 )are plotted in Figure 11as a function of DOPAC concentration. The experiment was performed by the addition of DOPAC solution into the electrolyte which contained DA (3 X loF7M). As shown in Figure 11,increase in DOPAC concentration did not cause the intensity increase for DA. All these data show the possibility of the measurement of DA concentration
without any interference by the presence of other chemicals. Electrode Materials. For analytical purposes, the lifetime of the electrode should be as long as possible and the measured values must be highly reproducible. As was seen above, HTA-7 is very reproducible as a solid electrode. One of the reasons why the electrode is so reproducible is the renewal of the electrode surface by the electrochemical treatment; indeed, the electrode becomes thinner after a long period of anodic polarization. Some layers of carbon atoms must be removed from the surface of the electrode by the triangular activation polarization used before each measurement, so that the carbon fiber for this purpose should be homogeneous in the radial direction. From electron-microscopic observations, it has been reported that HTA-7 was homogeneow but HM-45 was not (15). In the case of HTA-7, more than a thousand data points could be taken from one electrode; however, the number of replications was a factor of 10 less with HM-45. Both HM-45 and heat-treated HM-45 gave the same kind of results except in the initial period of experimentation. The sensitivity toward DA and other chemicals was about 10 times higher than those obtained with HTA-7. Unfortunately, the reproducibility was poor as was described. The high sensitive electrode loses the activity whether the electrode was stored in air or in PBS. A vigorous electrochemical treatment was necessary for the reactivation, and, sometimes, the nature did not return to the original one. This procedure shortened the life of the electrode. HM-45 may be preferable for a differential experiment of short time. Highly crystallized fibers synthesized by a vapor-phase growth method (14), especially the heat-treated fibers, have extremely low electrical resistance. The residual currents of the electrodes were about 10 times higher than those of HTA-7 and HM-45 electrodes. The electrode also had the sensitivity for DA; however, there seemed to be no special advantage for analytical uses. Mechanism and Isotherm. The DA concentration vs. intensity curves shows that there are two parts: one is a linear increase at higher concentrations and another is a logarithmic increase at low concentrations, which is shown in Figure 12. It was assumed that the anodic current was the sum of the oxidation of adsorbed DA and the oxidation of DA around the electrode. At higher concentrations, the adsorbed DA should be saturated and, therefore, the intensity increases linearly according to the linear increase of DA concentration around the electrode. As shown in Figure 3, the electrochemical activity monotonously decreases with the rest time between the activation and the measuring of the pulses; however, in the presence of DA M), the intensity increases initially. The initial increase may be caused by the increase of the adsorption of
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ANALYTICAL CHEMISTRY, VOL. 57, NO. 8, JULY 1985
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Flgure 13. Adsorption isotherm of DA. The surface coverage was approximately between 0.2 and 0.7.
DA at the electrode surface. There is a maximum around 2 s, and the intensity decreases after that period. It was assumed that the amount of adsorbed DA becomes constant at 2 s and then the adsorption of DA at the electrode comes to equilibr ium The adsorption isotherm was obtained as follows. First, a straight line was obtained with the least squares of the experimental points ( 0 )in Figure 12. The linear portion was extended to the lower concentration range, and the increase in the oxidation current except by adsorbed species was predicted. The predicted value was subtracted from the experimentally obtained intensity increment. The resulting value was considered as the equilibrium surface excess at a given DA concentration. The surface excess is plotted against the natural logarithm of DA concentration (nanomolar) in Figure 13. It was assumed that the adsorption of DA is saturated at high DA concentration (the linear portion in Figure 12), and the saturated surface excess was predicted. The surface coverage of the experimental points in Figure 13 was between 0.2 and 0.7. The linear relation in Figure 13 shows that the adsorption of DA at the carbon fiber electrode obeys a Tempkin isotherm in this region. The reduction in the intensity of DA was observed by the presence of 5HT and MeDA, which may suggest the competition adsorption of organics; indeed, the adsorption isotherm
.
of 5HT was similar to that of DA. Amino groups in organics may play an important role in the adsorbability. The desorption of anodized adsorbed DA might be suggested by the facts that the intensity of 5HT did not change by the presence of DA as seen in Figure 8 and that, in the voltammogram,the reduction peak height was about one-third of the oxidation height. It was shown that the adsorption of DA did not seem to occur at the anodically treated electrode surface. It was thought that the adsorbed oxygen would have inhibited the adsorption of DA. However, the adsorption of DA was helped by the presence of dissolved oxygen. The reason is not known, but the complex of DA with oxygen may play an important role in the adsorption. ACKNOWLEDGMENT We thank E. Endo and Toho Beslon Co. for kindly supplying highly crystallized fibers and HM-45 fibers, respectivdly. We also thank H. Miyazaki for useful discussion. Registry No. Dopamine, 51-61-6.
LITERATURE CITED Ponchon, J.-L.; Cespuglio, R.; Gonon, F.; Jouvet, M.; Pujol, J.-F. Anal. Chem. 1979, 51, 1483-1486. Ewing, A. G.; Dayton, M. A.; Wightman, R. M. Anal. Chem. 1981, 53, 1042-1 047. Plotsky, P. M.; De Greet, W. J.; Neill, J. D. Brain Res. 1982, 250, 251-262. Ewlng, A. G.; Alloway, K.; Curtis, S. D.; Dayton, M. A.; Wightman, R. M.; Rebec, G. V. Brain Res. 1983, 261, 101-108. Buda, M.: Gonon, F.; Cesuuglio, R.; Jouvet, M.; PuJoi, J. F. Eur. J. fharmacol. 1981, 73, 61168. Gonon, F.; Buda, M.; Cespuglio, R.; Jouvet, M.; Pujol, J. F. Brain Res. 1981, 223, 69-80. Brazell, M. P.; Marsden, C. A. Br. J . fharmacol. 1982, 75, 539-547. Plotsky, P. M. Braln Res. 1982, 235, 179-184. Buda, M.; Simoni, G. D.; Gonon, F.; Pujol, J. F. Brain Res. 1983, 273, 197-206. Forni, C.; Nieoullon, A. Brain Res. 1984, 297, 11-20. Gonon, F. G.; Navarre, F.; Buda, M. J. Anal. Chem. 1984, 58, 573-575. Gonon, F. G.; Fombarlet, C. M.; Buda, M. J.; PuJol,J. F. Anal. Chem. 1981, 53, 1386-1389. Ikeda, M.; Miyazaki, H.;Mugitanl, N.; Matsushita, A. Neurosci. Res. 1984, 1 , 171-184. Koyama, T.; E. Oyo Butsuri 1973, 42, 690. Dietendorf, R. J.; Tokarsky, E. folym. Eng. Sci. 1975, 15, 150-159.
RECEIVED for review November 19,1984. Accepted February 11, 1985.
