Anal. Chem. lB88, 60, 1268-1272
1268
Fast-Scan Voltammetry of Biogenic Amines John E. Baur, Eric W. Kristensen, Leslie J. May, Donna J. Wiedemann, and R. Mark Wightman*
Department of Chemistry, Indiana University, Bloomington, Indiana 47405
The use of fast-scan cycllc voltammetry Is explored as an analytical technlque for the detectlon of blogenlc amlnes. Cycllc voltammograms were recorded at a scan rate of 200 V 8-' at carbon-flber electrodes wlth and wlthout coatlng of a perfluorinated krrexchange materlal. Voltammograms were recorded In a flow Injectlon apparatus, and background subtraction was used to remove the resldual current. Voltammograms for the oxldatlon of 4-methylcatechol at uncoated electrodes had the peak amplltude expected for the prevlously publlshed oxldation mechanlm for catechols at Intermedlate pH. I n contrast, voltammograms for dopamlne, dlhydroxybenzylamlne,and norepln6phrkre showed much larger peak currents. SemRntegratiOn of these voltammograms ghres a peak-shaped curve lndlcatlve of adsorptlon. At coated electrodes, the voltammograms for anlonk spedes are greatly attenuated, whlle for the blogenlc amines the peak currents are larger than at uncoated electrodes. Fast-scan voltammetry Is shown to be particularly advantageous for the determlnatlon of 5-hydroxytryptamine. The short electrolysis tlme prevents the oxIdation products from forming an Insulating fllm on the electrode. Although adsorptlon of these compounds occurs In thelr reduced form, thls feature Is advantageous since It Increases the signal-to-nolse ratlo. Submicromolar detectlon Ilmlts can be achleved because of the preconcentratlon on the electrode surface.
Fast-scan cyclic voltammetry (>lo0 V s-l) is seeing increased use as an analytical method for the in vivo determination of the neurotransmitter dopamine (I, 2). This technique, originally developed by Millar (3),has been demonstrated to have many unique features when used with carbon-fiber microelectrodes. Most important is that it allows subsecond temporal resolution, which is necessary to observe the rapid concentration changes in the micromolar range that occur in the extracellular environment of the brain ( 4 , 5 ) . In addition, the use of rapid time scales discriminates against chemical events after the initial electron transfer that can alter the voltammetric characteristics of the species initially electrolyzed (6). The use of a perfluorinated ion-exchange coating over the carbon-fiber microelectrode has been shown to increase the selectivity of the technique (7). Despite the demonstrated experimental utility of this technique, little has been reported on the scope and limitations of this electroanalytical approach. Electroanalytical chemists have traditionally avoided cyclic voltammetry, especially a t fast scan rates, as an analytical technique. This is because of the significant background currents that occur. However, it has been shown that digital subtraction of the background scan, recorded in the absence of an electroactive species, can yield analytically useful data (8) without distortion of the faradaic signal (9). Furthermore, we have predicted that recognizable, subtracted voltammograms should be achieved at micromolar concentrations for a scan rate greater than 300 V s-' (9). In fact, submicromolar measurements have been * T o whom correspondence should be addressed.
achieved in previous neurochemical studies (I, 2). The objective of this study was to provide insights into the underlying reasons for the high sensitivity of fast-scan voltammetry for dopamine. Voltammetric results are shown for several other compounds of neurochemical importance. In addition, we evaluated the use of cyclic staircase voltammetry. From the results, it is possible to predict the types of compounds that are amenable to trace level detection by fast-scan voltammetry.
EXPERIMENTAL SECTION Electrodes. Microvoltammetric electrodes were prepared from 10 pm diameter carbon fibers sealed in glass capillaries and beveled on a polishing wheel with 1pm diamond paste (IO). The electrode surface is elliptical with a minor radius defined by that of the carbon fiber and a major radius that depends on the angle at which the surface is polished. The apparent radius was determined by steady-state amperometric measurements in solutions of known concentration. In some experiments, the area was verified by electron microscopy of the electrode dimensions. The electrodes were used without any other physical surface treatment or electrochemical activation. As noted in the text, some electrodes were coated with a thin film of a perfluorinated ion-exchange material, Nafion. The film was deposited on the electrode by a dip coating procedure (7) using commercially available solutions (Solution Technology, Rockland, DE). This procedure results in a thin film (-200 nm) that allows rapid, diffusion-controlled mass transport through the film. The reference electrode used was a sodium saturated calomel electrode (SSCE). Voltammetric Procedures. Except as noted, all experiments were conducted in a flow injection analysis system (11). Electrodes were lowered into the exit tube of a loop injector, and an aqueous buffer was pumped at a flow rate of 1 mL min-'. An IBM-XT computer (Boca Raton, FL) was used to control introduction of electroactive species to the electrode via a pneumatically controlled loop injector. The computer also triggered a function generator (Model 143, Wavetek, San Diego, CA) for cyclic voltammetry and acquired the data. For cyclic staircase voltammetry the waveform was computer-generated with a step potential, AE, of 24.4 mV. The effective scan rate was controlled by adjusting the step time (7). Current measurements were made 8 ps prior to the end of each step with a 12-bit analogldigital converter (Scientific Solutions, Solon, OH). The software employed limits the upper scan rate to 300 V s-'. Voltammograms were repeated at 200-ms intervals. Background subtraction of the voltammograms was accomplished with the use of data obtained prior to exposure of the analyte. The locally written software enables several voltammograms to be averaged together to improve signal-to-noise ratios. Alternatively, the current during a single voltammogram can be averaged over a desired potential range to provide a single value that is proportional to the concentration determined during the scan. Three-point moving averages were used as noted to smooth the data. Both two- and three-electrode potentiostats were employed without any measurable differences between the two. Signalto-noise ratios were determined with a commercially available potentiostat (E1400,Ensman Instrumentation, Bloomington, IN). The circuit diagram of the three-electrode potentiostat has been published previously (9). The instrument time constant (RC) was selected by using the following criterion: 24RC) < 1/(40nu) where u is the scan rate in volts per second and n is the number
0 1988 American Chemical Society 0003-2700/88/0360-1268$01.50/0
ANALYTICAL CHEMISTRY, VOL. 60, NO. 13, JULY 1, 1988
1269
Table I. Voltammetric Characteristics at Carbon-Fiber Electrodes at a Scan Rate of 200 V s-l Epa,
compd
i N a f b l ibarBe
iNiafliCdCdd
bare
0.72 f 0.44 5.9 f 2.2 10.9 f 1.7 16.0 f 1.7 67.3 f 5.7 0.15 f 0.04
585 f 80 580 f 150 360 f 51 420 f 20 430 f 20 370 f 20 440 f 10 475 f 30
AA
DOPAC 4-MC NE DHBA DA 5-HT 5-HIAA
0.65 f 0.21 1.3 f 0.03 1.1 f 0.24 1.5 f 0.54 1.4 f 0.3 0.14 f 0.01
m,, mVf
mve Nafion
440 f 530 f 580 f 510 f 470 f
22 50 40 55 20 410 f 20
bare
Nafion
660 f 90 740 f 140 490 f 56 460 f 40 430 f 20 400 f 40 240 f 40 290 f 35
610 f 69 570 f 70 610 f 70 610 f 110 225 f 60 150 f 40
aAveragesare the results from three different electrodes. In each case the voltammograms were obtained from the difference between 10 averaged scans in background solutions and 10 averaged from solutions containing depolarizer. Concentration of all species is 10 pM except those of AA, DOPAC, 5-HIAA (all 200 pM),4-MC (100 rM), and 5-HT (1pM). Errors are given as standard deviations. bMean anodic peak current measured at Nafion-coated electrodes. Mean anodic peak current measured at uncoated carbon-fiber electrodes. Calculated peak current from simulations as described in text. eMean anodic peak potential. 'Mean separation between anodic and cathodic waves. 4
D
1
n
F
E
1
"A
"A
m--J=50 pAs'/'
0
0 5
cc
35 E&
00 YE
05
0 0
-05
SSCE)
Figure 1. Subtracted cyclic voltammograms at a bare carbon-fiber microelectrode in pH 7.4 phosphate buffer recorded at 200 V s-': (A) 200 pM AA; (B) 200 pM DOPAC; (C) 100 pM 4-MC; (D) 10 pM NE; (E) 10 pM DHBA; (F) 10 pM DA. Each scan is the average of 10 subtracted voltammograms.
of electrons transferred (assumed to be 2 for all of the organic species). Theoretical voltammetric shapes were calculated by digital simulation (12) with the use of diffusion coefficients previously reported (13). The simulation assumes diffusion control, and this is found to be the case for nonadsorbed species even though the measurements are made in a flowing stream. This is because of the small dimensions of the diffusion layer at the scan rates employed. Semiintegration was as described previously (14). Reagents. All chemicals were reagent grade and were used as received from commercial sources. Solutions were prepared in doubly distilled water. A phosphate buffer was used throughout (pH 7.4), which contained 150 mM Na+ ions. RESULTS Fast-Scan Voltammetry of Ascorbate a n d Catechols. Voltammograms of several compounds were recorded a t 200 V s-l at uncoated carbon-fiber microelectrodes. The voltammetric characteristics are summarized in Table I. At this scan rate, all of the compounds examined are characterized by a large separation of the anodic and cathodic waves, indicative of the slow charge-transfer rates at carbon surfaces (14). The subtracted voltammograms of ascorbic acid (AA) and 3,4dihydroxyphenylacetic acid (DOPAC), both anions at pH 7.4, are shown in Figure lA, B. The anodic peak current for DOPAC is 70% of that expected for a quasi-reversible, diffusion-controlled, two-electron transfer with the rate-determining step being the first electron transfer and with a = 0.5. We have previously shown that this is the apparent mechanism of catechol oxidation at intermediate pH values (14). The cathodic wave for the voltammogram of DOPAC indicates a chemically reversible process as seen with the other catechols. The oxidation wave for AA is also significantly smaller than expected for a compound exhibiting a similar electrode mechanism. The cathodic wave in the voltammogram of AA
IC
05
O C E ( V v s S S C E I 0 5
00
-05
Figure 2. Semiintegrals of voltammogramsC-F of Figure 1: (A) 100 pM 4-MC; (B) 10 pM NE; (C) 10 pM DHBA; (D) 10 pM DA.
is small because of a subsequent follow-up reaction (15). The voltammogram of 4-methylcatechol (4-MC), a neutral compound at pH 7.4, is shown in Figure le. In this case the oxidation wave is identical in shape and magnitude with that expected for the established, diffusion-controlled electrode mechanism. The voltammetry of the compounds shown in 3,4-dihydroxybenzylamine Figure 1D-F, norepinephrine (NE), (DHBA), and dopamine (DA), respectively,gives peak currents that are larger than expected. All three of these compounds contain an amine side chain that is protonated at the pH used. Previously it has been shown that DA adsorbs to the surface of carbon electrodes that have been electrochemically pretreated, and this would explain why the current is larger than expected (16-19). However, for carbon paste (14) or polished carbon fibers (10) adsorption is not evident at scan rates less than 1 V s-l. Adsorption will be more apparent a t fast scan rates, because the diffusion layer dimensions are small and the fraction of the total current due to adsorbed species becomes greater. Semiintegral analysis was used to test the contribution of adsorption to the measured current (20). The semiintegral of 4-MC (Figure 2A) is sigmoidal, the shape expected for a voltammogram where the current is diffusion-limited. This result is in accord with the results of Michael and Justice (16). The semiintegrals of NE, DA, and DHBA (Figure 2B-D) all deviate from the ideal sigmoid and are peak-shaped. This indicates that there is a source of faradaic current other than from a diffusion-controlled process, most likely currrent due to the oxidation of adsorbed species. The semiintegrals of Figure 2 indicate that the contribution of adsorption to the peak current follows the order DA > DHBA > NE > 4-MC. Voltammetry at Nafion-Coated Electrodes. The carbon-fiber electrodes were coated with the perfluorinated ion-exchange material and the above experiments repeated. Voltammograms at 200 V s-l of the six compounds are shown
1270
ANALYTICAL CHEMISTRY, VOL. 60, NO. 13,JULY 1, 1988
- ,
cs
IO
sc
I
I ;5
E
II
3 r "5
co
0 5
05
SSCEl
Flgure 3. Subtracted cyclic voltammograms at a Nafion-coated carbon-fiber microelectrode. Ail condltlons are as in Figure 1. A
n
T
I
IC
1
I
I
35
I
- "
Time (s)
ground scans were averaged and subtracted from 30 averaged scans during exposure to DA. One three-point smooth was applied to the subtracted voltammogram. The horizontal line is zero current. (B) Current response versus tbne for a 10-s exposwe to 0.5 pM DA. Each point represents the current from one cyclic voltammogram (same conditions as in A) averaged over the entire oxidative wave, 0.3-0.8 V (vs SSCE). One three-point smooth has been applied to the current-time response. Voltammograms were repeated at 200-ms intervals.
in Figure 3. The anions AA and DOPAC are not incorporated into the film to a measurable degree, and no voltammetric information is present (Figure 3A,B). Table I compares with response of the coated electrodes to that of the bare electrodes. The neutral compound, 4-MC, shows less current at the coated electrode, while the catecholamines all show slightly greater current (Figure 3C-F). Semiintegral analysis indicates that adsorptive behavior a t the Nafion-coated electrodes occurs to a lesser degree than a t uncoated electrodes. The ratios of observed current in the Nafion film to calculated current at a bare electrode are also shown in Table I. Again, DA shows the greatest current enhancement. The Nafion coating also appears to cause each voltammogram to become more irreversible. Anodic peaks were shifted by 80-150 mV, while the peak separation increased by 80-210 mV after coating. Detection of Low Concentrations of Dopamine. The signal-to-noise ratios for DA a t perfluorinated-ion-exchange-coated electrodes were compared with the use of fast-scan cyclic and cyclic staircase voltammetry with subtraction in each case. The results were similar with each technique a t scan rates greater than 100 V s-l. As shown in Figure 4, submicromolar amounts of DA can be readily detected from the current averaged over the oxidative wave from repetitive voltammograms. Furthermore, the averaged voltammogram provides qualitative information about the detected substance. The drift apparent in the current-versustime curve is due to the alteration in background current that occurs during the 15-9 experiment. At scan rates less than
l 5
/ SSCEl
I
I
05
I
I
c5
0 0
1
/
-2c
E ( V v s . SSCE)
=
Figure 5. Background current-voltage curves resulting from application of cyclic (outer trace) and staircase (inner trace) waveforms at 200 V s-' to a carbon-fiber microelectrode in phosphate buffer (A) and in a dummy cell (B: R = 27 kQ; C = 33 pF). Inset shows scan rate dependence of peak background currrent: (0)cathodic peak current; (0)anodic peak current.
I
Figure 4. Detection of trace concentration of dopamine: (A) Background-subtracted, cy& voltammogram for 0.5 pM DA at a scan rate of 200 V s-' at a Nafion-coated carbon-fiber electrode. Ten back-
I
r log'
^ ^
" "
s
Figure 6. Delay time dependence of peak current for the oxidation of DA at Naflon-coated carbon-fiber microelectrode at v = 200 V s-' in 10 pM DA: (0)cathodic peak current; (0)anodic peak current.
5 V s-l, cyclic staircase voltammetry was the more sensitive technique. The failure of cyclic staircase voltammetry to provide significantly improved signal-to-noise ratios a t high scan rates was investigated further. The application of a staircase waveform to a purely capacitive system results in zero current when the current is sampled a t long intervals after each potential step. To test the response of our computer-potentiostat system, a series RC network was used as a dummy cell. The outer trace in Figure 5B is the charging current due to the application of a triangle potential waveform. This current is given by i, = uC,where u is the scan rate (200 V s-l) and C is the capacitance (33 pF). The inner trace is the current resulting from the application of a staircase waveform, and a large reduction in background signal is apparent. Shown in Figure 5A are the current-voltage curves resulting from the application of the same waveforms to a carbon-fiber microelectrode. The background cyclic and staircase voltammograms of Figure 5A exhibit waves that are uncharacteristic of a purely capacitive system, since they contain distinct peaks. A plot of the amplitude of the current peaks from background cyclic voltammograms, normalized by scan rate, versus the logarithm of the scan rate is shown in the insert of Figure 5. At scan rates greater than 5 V s-l, the slope of the plot is nearly zero for both the anodic and cathodic waves. A log-log plot for both the anodic and cathodic waves versus scan rate gives slopes of 1.01 and 0.99, respectively, with correlation coefficients of 0.999 in each case. Thus, the background current is due to surface phenomena, presumably from the oxidation and reduction of functional groups on the carbon surface. Cyclic staircase voltammetry does not completely discriminate against these waves, presumably because of slow kinetics. Figure 6 shows the effect of varying the delay time between scans on the peak current a t Nafion-coated electrodes with the use of cyclic voltammetry (u = 200 V s-l). The peak current is observed to decrease at delay times less than 50 ms,
ANALYTICAL CHEMISTRY, VOL. 60, NO. 13, JULY 1, 1988
1271
B I
I
LO
U
0.5
I
E (Vvs. SSCE)
0.5
0.0
J
Flgure 7. Cyclic voltammograms for 50 pM 5-HT at a carbon-fiber microvoltammetric electrode: (A) First (dashed iine) and 14th (solid line) background-subtracted scan obtained at a scan rate of 200 V s-’. (B) First (lower trace) and 14th scan (upper trace) obtained at a scan rate of 100 mV s-‘. The horizontal line is zero current.
whereas at delay times greater than 50 ms the peak current does not change. Maximum sensitivity, therefore, is obtained when sufficient time is allowed for the diffusion layer to relax following a scan. A t these scan rates the diffusion layer is almost entirely within the Nafion film, and thus diffusion in these films determines the time response of the system. Voltammetry of Hydroxyindoles. These techniques have also been used to investigate the voltammetry of 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleaceticacid (5-HIAA). 5-Hydroxytryptamine is a neurotransmitter while 5-HIAA is its major metabolite in the mammalian brain. Thus, in vivo investigations of 5-HT require a probe that distinguishes between it and its metabolite and that also discriminates against ascorbate, the distribution of which is ubiquitous throughout the brain. Previous voltammetric studies of 5-HT at physiological pH values have been hindered by the tendency of the oxidation products to form an insulating film on the electrode surface (21-23). This is shown for an uncoated carbon-fiber electrode in Figure 7 , where the first scan obtained in a solution of 50 p M 5-HT is compared to the 14th scan at a scan rate of 100 mV s-l. If the deterioration of the electrode response is caused by reactions of the oxidative products, this problem should be removed by fast-scan cyclic voltammetry, since this will decrease the time these products are present at the electrode surface. This appears to be the case because reproducible voltammograms can be obtained for repetitive scans at 200 V s-l (24). The voltammetric peak current for 5-HT oxidation is much higher than for DA, indicating a greater degree of adsorption (Table I). However, this is advantageous since it leads to a decrease in the detectable level (Figure 8). Voltammetry of 5-HT is little changed by the presence of Nafion on the electrode tip. In both cases, semiintegration indicates that the reactant is adsorbed. This adsorption is rapid, however, as indicated by the rapid response to a concentration pulse with or without Nafion at the tip. The amplitude of voltammograms for 5-HIAA (an anion at pH 7.4) is greatly attenuated with the Nafion coating.
DISCUSSION The data presented here clearly show that fast-scan cyclic voltammetry can be used as an analytical technique with microelectrodes. Compared to other electroanalyticalmethods for organic compounds, it offers some distinct advantages. First is the obvious advantage of time resolution. Second, when used in a repetitive mode as illustrated here, several scans can be averaged together to improve the signal-to-noise ratio. This feature is of importance for rapid changes in concentration that can be repetitively induced, such as during neuronal stimulation in vivo or in flow injection analysis. At 200 V s-l a single scan can be recorded in 10 ms. As long as the concentration does not change in that time interval, scans recorded a t different concentration values can be averaged together. The resulting voltammogram will then be that of the substance(s) detected during the overall averaged period.
L
0.8
*,
0.4
I
E ( V vs. SSCE)
.*
0.4
00
1
I
a8
E (V 04 vs SSCE) 00 0
Time IO (s)
0
Figure 8. Data from fast-scan voltammograms of hydroxyindoles: (A) Background-subtracted voltammograms for 1 pM 5-HT at a scan rate of 200 V s-’obtained at a carbon-fiber electrode (solid line) and at a Nafion-coated, carbon-flber electrode (dashed line). (B) Semiintegrations of the voltammograms in A; a solid line is at the carbon-fiber
electrode and dashed iine is at the Nafion-coated carbon-fiber electrode. (C) Background-subtractedvoltammograms for 200 pM 5-HIAA at a scan rate of 200 V s-’ obtained at a carbon-fiber electrode (solid line) and a Nafion-coated, carbon-fiber electrode (dashed iine). (D) Temporal response obtained during and after injection of a 10-s bolus of 1 pM 5-HT. The peak anodic current for successive vottammograms (same conditions as in A) is plotted as a function of time. The lower trace is the response observed at a carbon-fiber electrode. The upper trace is the response observed at a Nafion-coated, carbon-fiber electrode.
A voltammogram recorded at slow rates over the same time interval would contain information on the temporal concentration changes as well as the voltammetric information and would be difficult to interpret. The third advantage comes from the ability to provide qualitative analysis from the voltammogram. Voltammetry is usually a poor qualitative technique because compounds are distinguished by their E l j 2values alone. An additional identifier is provided at fast scan rates, the rate of heterogeneous electron transfer. For example, DOPAC can be distinguished from the other catechols because its voltammetry reflects its slower rate of electron transfer at carbon-fiber surfaces (Table I). The difference in voltammetric shape of the hydroxyindoles from that of the catechols permits clear distinction of these classes of compounds, even though their values are similar. Unfortunately, the ability to distinguish between NE and DA on the basis of kinetic effects does not seem sufficient under the conditions explored in this paper. A fourth distinct advantage is noted in the voltammetry of 5-HT. The oxidation products, which in this case form an insulating film on the electrode at conventional scan rates, are rapidly reduced at fast scan rates. Thus, their concentration is kept to a low value, and dimerization or polymerization reactions are discriminated against. In this way, the electrode is kept in a stable form and can be used continuously without the need for resurfacing. The chief disadvantage of fast-scan voltammetry is the large background current that must be removed to clearly observe the faradaic events. As has been discussed, the background signal remains fairly constant if the waveform is applied continuously at regularly spaced intervals and if the electrode is kept continuously immersed in solution (9). Furthermore, the Nafion film keeps the electrode surface in a uniform condition (7). The resolution of the digitizing device is a major limitation for low levels of detection. Cyclic staircase voltammetry should improve this situation if the background is purely capacitive (25). However, as shown by the data, the background signal contains additional components at car-
1272
Anal. Chern. 1988,60, 1272-1281
bon-fiber electrodes that are not removed by the staircase waveform a t fast scan rates. While the advantages listed above are general ones, the trace detection of DA and 5-HT with fast-scan cyclic voltammetry is a result of additional factors. As the data clearly show, biogenic amines adsorb to carbon electrodes in a reversible manner. Dopamine adsorption has been demonstrated at electrochemically treated electrodes as noted earlier, but this is the first time it has been reported a t unactivated surfaces. The adsorptionldesorption rates for DA are rapid, and thus, temporal information is still present while the faradaic signal is enhanced at bare carbon electrodes. At the Nafion-coated electrodes, the evidence for adsorption is less compelling for DA, but the large value of the partition coefficient (26)results in increased sensitivity. We have previously shown that linear calibration curves are obtained for DA with these electrodes (7). The combination of the noncapacitive nature of the background current and the surface preconcentration leads to the comparable sensitivity of cyclic voltammetry and staircase at fast scan rates. At more modest scan rates, surface processes are less predominant, and in the case of DA, the staircase technique is superior. Adsorption of 5-HT occurs to a greater degree, and thus this technique has even greater sensitivity for this compound. These data show that fast-scan cyclic voltammetry is useful as an analytical technique, but the sensitivity depends on the surface properties of the electrode and the nature of the analyte. Registry NO. AA,50-81-7;POPAC, 102-32-9; 4-MC, 452-86-8; NE, 51-41-2; DHBA, 37491-68-2; DA, 51-61-6; 5-HT, 50-67-9; 5-HIAA, 54-16-0; Nafion, 39464-59-0.
LITERATURE CITED (1) Stamford, J. A,; Kruk, 2 . L.; Miihr, J. BrainRes. 1988,387, 351-355. (2) Kuhr, W. G.; Wightman, R. M. Braln Res. 1986,381, 168-171. (3) Miliar, J.; Armstrong-James, M.; Kruk, 2. L. Brain Res. 1981,205, 419-424.
Ewing, A. G.; Bigelow, J. C.; Wightman, R. M. Science (Washington, D . C . ) 1983,221, 169-170. Church, W. H.; Justice, J. B. Anal. Chem. 1987,59,712-716. Stamford, J. A. Anal. Chem. 1986, 58, 1033-1036. Kristensen, E. W.; Kuhr, W. G.; Wightman, R. M. Anal. Chem. 1987, 59, 1752-1757. Millar, J.; Stamford, J. A.; Kruk, 2. L.; Wightman, R. M. Eur. J. Pharmacol. 1985, 709,341-348. Howell, J. 0.; Kuhr, W. G.; Ensman, R. E.;Wightman, R. M. J. Nectfoanal. Chem. Interfacial Electrochem. 1986,209, 77-90. Kelly. R.; Wightman, R. M. Anal. Chim. Acta 1988. 787, 79-67. Kristensen, E. W.; Wilson, R. L.; Wightman, R. M. Anal. Chem. 1986, 58, 986-988. Wipf, D. 0.; Deakin, M. R.; Kristensen, E. W.; Wightman, R. M. Anal. Chem. l98& 6 0 , 306-310. Gerhardt, G.; Adams, R. N. Anal. Chem. 1982,5 4 , 2616-2620. Deakin, M. R.; Kovach, P. M.; Stutts, K. J.; Wightman, R. M. Anal. Chem. 1988,58, 1474-1480. Wehmeyer, K. R.; Wightman, R. M. Anal. Chem. 1985. 57, 1969-1993. Michael, A. C.; Justice, J. B. Anal. Chem. 1987,59,405-410. Feng, J.-X.; Brazeii, M.; Renner, K.; Kasser, R.; Adams, R. N. Anal. Chem. 1987,59, 1863-1667. Kovach, P. M.; Deakin, M. R.; Wightman. R. M. J. Phys. Chem. 1986, 90,4612-4617. Sujaritvanichpong, S.; Aoki, K.; Tokuda, K.; Matsuda, H. J. Electroanal. Chem. Interfacial Nectrochem. 1986, 198, 195-203. Bowling, R.; McCreery, R. L. Anal. Chem. 1988,60,605-606. Poon, M.; McCreery, R. L. Anal. Chem. 1987,59, 1615-1620. Berniese-Genard, J. C.; Kauffman, J. M.; Hanocq, M.; Moiie, L. J. Nectfoanal. Chem. Interfacial Electrochem. 1984, 770,243-254. Wrona, M. 2.; Lemordant, D.; Lin, L.; Blank, C. L.: Dryhurst, G. J. Med. Chem. lD88,29, 499. Kruk, 2. L.; Armstrong-James, M.; Miiiar, J. Life Sci. 1980, 27, 2093-2098. Biiewicz, R.; Osteryoung, R. A.; Osteryoung, J. Anal. Chem. 1986, 58,2761-2765. Nagy, G.; Gerhardt, G. A.; Oke, A. F.; Rice, M. E.; Adams, R. N.; Moore, R. 8.; Szentirmay, M. N.; Martin, C. R. J. Electroanai. Chem. Interfacial Nectrochem. 1985, 188, 85-94.
RECEIVED for review October 7,1987. Accepted March 1,1988. This research was supported by NIH (Grant No. NS 15841). Leslie May is the recipient of a G*POP Fellowship and an Eli Lilly Fellowship.
Transient Response of the Two-Dimensional Glucose Sensor Joseph Y. Lucisano and David A. Gough* Department of Applied Mechanics and Engineering Sciences, Bioengineering Group, University of California, S a n Diego, La Jolla, California 92093
The two-dlmenslonal enzyme electrode has certaln advantages for the development of an Implantable glucose sensor, but detalls of the dynamic response of thls electrode have not been prevlously understood. A model and experimental observations are presented that demonstrate the effects of substrate concentratlon, mass-transfer parameters, lmmoblllzed enzyme actlvlty, geometry, and sensor slze. WRh approprlate deslgn, the translent response can be rapld and comparable to that of the one-dimenslonal sensor.
The enzyme electrode can serve as the basis of a sensor for glucose. In the sensor we are developing (I),the immobilized enzymes glucose oxidase and catalase are used in conjunction with electrochemical oxygen sensors. The enzymes catalyze the following overall reaction: glucose
-
+ 1/202gluconic acid
* A u t h o r to whom correspondence should b e addressed.
(1)
Glucose and oxygen diffuse into a gel containing the immobilized enzymes, where the reaction occurs. Excess oxygen not consumed by the reaction is detected by an oxygen sensor as a glucose-modulated, oxygen-dependent current, igmo A second oxygen sensor without the enzymes produces an oxygen-dependent current, io, which indicates the background oxygen concentration. The currents are subtracted by an appropriate method, and a glucose-dependent difference current, i,, results. The complete system is therefore composed of a glucose electrode containing the immobilized enzymes, a reference oxygen electrode, and a means of calculating the glucose-dependent difference current. One important application of a glucose sensor would be its use as an implant in the body to monitor blood or tissue fluid glucose concentration. Continuous, convenient glucose monitoring may make possible new approaches to the therapy of diabetes. There are, however, several difficult issues that must be satisfactorily resolved before such an application can be considered feasible. One problem is the “oxygen deficit” ( I ) , in which the relatively low concentration of oxygen in the body compared to that of glucose imposes a stoichiometric
0003-2700/88/0360-1272$01.50/00 1988 American Chemical Society
