Anal. Chem. 1994,66, 285-289
Voltammetric Measurements of Reversible and Quasi-Reversible Redox Species Using Carbon Film Based Interdigitated Array Microelectrodes Osamu Nlwa' and Hlsao Tabel NTT Basic Research Laboratories, Nippon Telegraph and Telephone Corporation, Tokai, Ibaraki 3 19- 1 1, Japan A carbon-based interdigitatedarray (IDA) microelectrodewas applied to the highly sensitive voltammetric detection of reversible and quasi-reversible redox species in both the anodic and cathodic potential regions. Carbon film was prepared on a surface-oxidized Si wafer by the pyrolysis of 3,4,9,10perylenetetracarboxylic dianhydride and was designated as amorphous by X-ray diffraction and Raman spectroscopy. The carbon IDA microelectrodewasfabricated by photolithography and dry etching. The IDA electrode had 50 microband pairs. The bandwidth of the IDAs was 3 pm, and the gap between two microbands was 2 pm. In the measurement of ferrocene derivatives, the iR drop was negligible when the ferrocene concentration was less than 0.1 mM at a moderate scan rate due to the low resistivity of carbon film. The cathodic potential window without redox species extended further in the negative direction than that of platinum and gold IDAs. The collector current of ruthenium hexaammine in a generation collection measurement using an IDA agreed well with the theoretical value even if the sample concentration was in the tens of nanomoles per liter range due to the flat baseline current at the collector in the cathodic region and high collection efficiency. The generation-collection voltammogram of dopamine was greatly improved after pretreating only the generator electrode. A low detection limit of 10 nM and a wide linear range from 10 n M to 1 mM were obtained at the collector electrode because the collector response of dopamine was improved by the generator pretreatment without increasing the background current at the collector electrode. Arrays of microelectrodes have been of particular interest because of their electrochemical advantages such as high current density and fast response while the same magnitude of current is maintaining as with conventional electrodes. Recent lithographic techniques are being widely applied to the fabrication of microarray electrodes' because they enable small electrodes of any required shape to be realized with excellent reproducibility. Various types of array electrodes including microdisks,24 microbands,7s8and interdigitated array (IDA)?-13 have been reported. (1) Kitt1esen.G. P.; White, H. S.; Wrighton, M. S . J . Am. Chem.Soc. 1985,107, 7373. (2) Reller, H.; Kirowa-Eisner, E.; Gileadi, E. J. Electroanal. Chem. 1984, 161, 247. (3) Cassidy, J.; Ghoroghchian, J.; Sarfarazi, F.; Smith, J. J.; Pons, S . Electrochim. Acra 1986, 31, (6), 629. (4) Hepcl, T.; Ostcryoung, J. G. J. Elecrrochem. Soc. 1986, 133, 752. (5) Pcnner, R. M.; Martin, C. R. Anal. Chem. 1987, 59, 2625. (6) Horiuchi, T.; Niwa, 0.;Morita, M.; Tabei, H. J . Elecrroanal. Chem. 1990, 295, 25.
0003-2700/94/03660285$04.50/0 0 1994 American Chemical Society
We have developed highly sensitive electrochemical detection using IDA microelectrodes, because IDA electrodes have interesting characteristics when redox species are mea~ured.1"~~ Since each of the two electrodes in the IDA can be potentiostated separately, an electroactive species generated at one electrode (generator) can diffuse to the second electrode (collector). The redox cycling of the species takes place between the twin microelectrodes, resulting in a steadystate current. As a result, a large limiting current can be obtained with a high S/N ratio, due to the large number of redox cycling reactions between the two electrodes. However, most of the microarray electrodes were made of metal film, and so their narrow potential window in thenegative region may limit their application to reductive electrochemical reactions. On the other hand, carbon electrodes are widely used in electroanalytical chemistry. This is because the carbon electrode has a wide potential window, surface functional groups for chemical modification, and controllable surface activity resulting from pretreatment. l8 Various carbon-based arrays of microelectrodes have been fabricated by use of conventional techniques such as the suspension of carbon particles or fibers in an insulating matrix, the impregnation of porous carbons with an insulator, or the impregnation of the pores of a host membrane with conducting carbon p a r t i c l e ~ . ' ~ -However ~~ it is difficult to fabricate the array electrodes reproducibly because the arrangement of each electrode in the array is not controllable using conventional techniques. It is also difficult to fabricate microarray electrodes from carbon film with the lithographic technique. Since the (7) Bard, A. J.; Crayston, J. A.; Kittlesen, G. P.; Shea, T. V.; Wrighton, M. S. Anal. Chem. 1986, 58, 2321. (8) Matsue, T.; Aoki, A.; Ando, E.; Uchida, I. Anal. Chem. 1990,62,407-409. (9) Sanderson, D. G.; Anderson, L. B. Anal. Chem. 1985,57, 2388. (10) Chidsey, C. E.; Feldman, B. J.; Lundgren, C.; Murray, R. W. Anal. Chem. 1986, 58, 601. (1 1) Aoki, K.; Morita, M.; Niwa, 0.;Tabei, H. J . Efecrroanal.Chem. 1988.256, 269. (12) Niwa, 0.; Morita, M.; Tabci, H. J . Electrounal. Chem. 1989, 267, 291. (13) Aoki, A.; Matsue, T.; Uchida, I. Anal. Chem. 1990, 62, 2206. (14) Niwa, 0.;Morita, M.; Tabci, H. Anal. Chem. 1990.62.447. (15) Niwa, 0.;Morita, M.; Tabei, H. Electroanalysis 1991, 3, 163. (16) Takahashi, M.; Motita, M.; Niwa. 0.;Tabei, H. J . Elecrroanal. Chem. 1992, 335, 253. (17) Horiuchi, T.; Niwa, 0.;Morita, M.; Tabei, H. Anal. Chem. 1992,64,3206. (18) McCrecry, R. L. Eleciroanal. Chem. 1990,17, 221. (19) Weisshaar, D. E.; Tallman, D. E. Anal. Chem. 1983, 55, 1146. (20) Sleszynski, N.; Osteryoung, J.; Carter, M. Anal. Chem. 1984, 56, 130. (21) Wang, J.; Freiha, 9. A. J. Chromarogr. 1984, 28, 79. (22) Wang, J.; Zadeil, J. M. J . Elecrroanal. Chem. 1988, 249, 339. (23) Sylwester, A. P.; Clough, R. L. Synrh. Mer. 1989, 29, F253. (24) Wang, J.; Brennsteiner, A.; Sylwester, A. P. Anal. Chem. 1990, 62, 1102.
Analytical Chemistry, Vol. 66,No. 2, January 15, 1994 205
adhesion of sputter- or vacuum-deposited carbon film to the substrate is poor, it is easily stripped off during the microfabrication process. The low conductivity of these carbon films is also a problem because it causes a significant iR drop after microfabrication. Kaplan et al. prepared a highly conductive carbon film by the pyrolytic deposition of a sublimed aromatic compound,25 and its wide potential window and good electrochemical features were reported by Anjo's group.26 Recently, we succeeded in fabricating an IDA microelectrode using a carbon film on the basis of the above reports with some modifications.27 Since a carbon IDA electrode provides not only current enhancement by redox cycling but also a wide cathodic potential window, we were able to achieve highly sensitive voltammetric measurements in the negative potential region using the IDAs. In this paper, we examine the basic electrochemical properties of carbon-based IDA electrodes using various redox species and apply them to highly sensitive detection in the negative potential region. The electrochemical detection of quasi-reversible species with a pretreated IDA is also studied using dopamine as a sample because the surface activity of the two microband arrays can be independently controlled by pretreatment. EXPERIMENTAL SECTION Reagents. The 3,4,9,1O-perylenetetracarboxylicdianhydride (PTCDA) was obtained from Aldrich. Hexaammineruthenium chloride ([Ru(NH&]C13, ruthenium hexaammine), (ferroceny1methyl)trimethylammoniumbromide ([ C ~ H S F ~ C ~ H ~ C H ~ N Br,( Caq-ferrocene), H~)~] and dopamine were obtained from Tokyo Kasei. Phosphate buffer solutions (0.1 mol/dm3, pH 6 or 7) were used as the electrolyte (Nacalai Tesque). Sulfuric acid was obtained from Wako. All reagents were used as received. Carbon Film Formation. The formation of poly(perinaphthalene) (PPN) carbon film by the pyrolysis of PTCDA was reported by Kaplan25and Anjo.26 We prepared the film on a surface-oxidized silicon wafer by modifying their methods. In order to obtain a large area film with a flat surface, PTCDA was evaporated at a lower rate. A pyrolysis temperature (1000 "C) was adopted which is higher than the above method (850 "C) in order to increase film conductivity. The carbon film, which covered the wafer, had a silver metallic mirror-like appearance? The structure of the film was characterized using X-ray diffraction and Raman spectroscopy. Microelectrode Fabrication. The substrates coated with conducting carbon film were fabricated into IDA microelectrodes. Thermally oxidized 3-in. silicon wafers (Sumitomo Sitix Co., Osaka, Japan) were used as substrates. The carbon film was formed on the wafer as described above. A siliconbased positive photoresist28was spin-coated on the wafer. The electrode pattern was exposed with a mask aligner PLF-600F (Canon Ltd, Tokyo, Japan), developed in NMD-W developer (25) Kaplan, M. L.; Schmidt, P. H.: Chen, C.-H.; Walsh, W. M., Jr. Appl. Phys. Lett. 1980, 36, 867. (26) Rojo, A.; Rosenstratten, A.; Anjo. D. Anal. Chem. 1986, 58, 2988. (27) Tabei, H.; Morita, M.; Niwa, 0.; Horiuchi, T. J . Electround. Chem. 1992, 334, 2 s . 33. (28) Tanaka. A,; Ban, H.; Imamura, S.;Onose, K. J . Vuc. Sci. Techno/. 1989,87 (3), 572.
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(Tokyo Ohka, Kawasaki, Japan), rinsed in deionized water, and then dried in a nitrogen gas flow. The wafer was placed in DEM-451 reactive ion etching equipment (Anelva, Tokyo, Japan), and the carbon film not covered with resist was etched with oxygen plasma until the substrate was exposed. The resist pattern on the carbon film was dissolved with an alkaline solution. The wafer was then again covered with siliconbased positive photoresist. The resist was photolithographically patterned to insulate the lead region of the IDA from the solution. This process is simpler than the previous process because we can form the insulation layer without dry etching.27 The lead region of the IDA was covered with photoresist, and the IDA and pad regions were exposed to the air. Since this resist film (insulating film) turns into silicon oxide-like film by heating it above 200 "C, we can form an insulating pattern which is insoluble in the solvent. Apparatus. Electrochemical properties were measured using a twin potentiostat HECS 990 (Huso, Japan), a potential sweep unit HECS 980 (Huso), and an X-Y recorder 3025 (Yokogawa). The two microband electrodes in the IDA were connected to a dual potentiostat. The Ag/AgCl reference and platinum auxiliary electrodes were immersed in the solution. The IDA electrode surface was observed with an SEM (JSM-840, JEOL) and a high magnification SEM (JSM-890, JEOL). Procedure. The electrode was characterized with cyclic voltammetry or generation collection voltammetry. The solutions were not deoxygenated before measurement. Phosphate buffers (pH 6 or 7) containing aq-ferrocene, ruthenium hexaammine, or dopamine were used as sample solutions. In the conventional cyclic voltammetry measurement, the potential of one electrode in the IDA was cycled and the other was disconnected from the potentiostat. In the generationcollection voltammetry measurement, one electrode in the IDA was fixed at a constant potential and the other potential was swept. In measuring dopamine, the electrode surface was pretreated by potentiostating the electrode at 1.8 V vs Ag/AgCl in 0.1 M sulfuric acid solution. The conductivity of the carbon film was measured with the conventional twoprobe dc technique at room temperature. RESULTS AND DISCUSSION Characterization of Carbon-Based IDA Electrode, Before the IDA was microfabricated, the structure of the carbon film was characterized by X-ray diffraction and Raman spectroscopy. Figure l a shows an X-ray diffraction chart of the carbon film fabricated on the silicon wafer. The peak observed at 25.4' corresponds to an interplanar spacing of 0.36 nm. However, this spacing is wider than that of single crystal graphite (0.335 nm).18 The peak shape in Figure l a is also broader than that of crystalline carbon. The Raman spectrum of the carbon film is shown in Figure 1b. Two large peaks were observed at 1590 and 1340 cm-I, showing that the graphite microcrystals are small. These results suggest that the carbon film has a structure similar to a glassy carbon.'* The conductivity of the carbon film measured with the two-terminal method is about 250 S/cm, which is similar to that of commercially available glassy carbon electrodes. With the SEM observation of carbon film based IDA, a well-defined micrometer-order grating can be seen.
Platinum IDA
Gold IDA
Carbon IDA
C 4-3
i
U
10
1.0
0.8
,1-
0
20
80
60
40
20 I degree L
1.6
1340
1500
lo00
500
250
Wavenumber / cm-' F@ro 1. X-ray diffraction (a) and Raman spectrum (b) of carbon film deposited on an oxldlzed slllcon wafer.
0
90
a
: Glassy carbon (BAS) : Carbon film (as deposited)
"1
0
: Carbon based IDA
70
60
0
1.0 -1.0
0
1.0
Flguro 3. Gemrationcollectionvoltammogramsat the platinum, gold, and carbon IDA electrodes in pH 7 phosphate buffer (0.1 M) without the sample: a, c, and e, potentialwas heldat 0 V (collectorelectrodes); b, d, and f, potentialwas cycled (generator electrodes). Each IDA has a 3-pm bandwidth and 2-pm gap.
1590
m
1.0 -1.0
Potential 1 V vs Ag/AgCl
Carbon film as deposit
I
.
0
0 ...... ..............................I ..................................... ..... *
A
\ %hetical
50
The resistance of the carbon microband region in the IDA
is higher than that of bulk carbon film because each band electrode is very narrow and thin. Therefore, the effect of microband resistance on iR drop was estimated using watersoluble ferrocene derivatives (aq-ferrocene) as a standard redox species. Figure 2 shows the peak separation of aq-ferrocene at carbon film and carbon IDA electrodes compared with that of a commercially available glassy carbon electrode. In order to obtain a conventional voltammogram, the potential of only one electrode in the IDA was swept at a scan rate of 50 mV/s.
The voltammogram of 1 mM aq-ferrocene at the IDA electrode shows larger peak separation than that at the other two. The resistance of one carbon band electrode in the IDA whose length, width, and thickness are 2 mm, 3 pm, and 0.1 pm, respectively, is 260 kQ, when the conductivity of the carbon film is 250 S/cm. Since the current flowing at each band electrode is 0.032 pA for 1 mM aq-ferrocene, the iR drop in the band electrode is calculated as 8.3 mV. This result indicates that the larger peak separation at the carbon IDA than at the other two electrodes is caused by the microband region resistance. The peak separation obtained at these three electrodes is similar to the ideal value when the aq-ferrocene concentration is less than 0.1 mM. This is because the iR drop at the IDA is calculated as less than 1 mV when 0.1 mM aq-ferrocene is measured. This indicates that the carbon IDA electrode has sufficient conductivity when it is applied to the detection of trace amounts of electroactive species. Highly Sensitive Voltametric Measurement in the Cathodic Potential Region. The potential window of the carbon electrode is wider than that of metal electrodes, particularly in the cathodic potential region. In a previous paper, we confirmed that carbon film has a wider potential window than platinum film.27 However, the smaller background current at the collector electrode when the generator electrode is scanned in the far-negative region is of greater importance in terms of achieving a lower detection limit. This is because measurement using the collector current is more sensitive than that using the generator current since the collector electrode is fixed at a constant potential and is free from the noisy charging current in the generation collection measurement. Figure 3 shows the results of generation-collection voltammetry at platinum-, gold-, and carbon-based IDA electrodes in pH 7 phosphate buffer without the sample. In this measurement, the collector potential was held at 0 V vs Ag/ AgCl and the generator potential was cycled between -1 and 1 V. The generator electrode of the carbon IDA is similar to commercially available glassy carbon because it has a wider potential window than that of metal electrodes, particularly in the cathodic potential region, resulting from the substantial overpotential of the hydrogen and oxygen reduction. Analytcal Chemistry, Vol. 66,No. 2,Janwty 15, 1994
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-
4 10-
\
c9 A
: Generator (cathodic)
0
: Collector (anodic)
0
10
-8
Q
0
10
-7
lo
-3
10
Concentration / M Flgure 4. Calibration curve of ruthenium hexaammine at a carbonbased IDA electrode. The IDA has a 3-pm bandwidth and 2-pm gap. Solid line in the inset is theoretical line calculatedwith Aoki's equation."
On the other hand, a large background current was observed at the collector electrode of the platinum IDA in the cathodic region. Since thedistance between thegenerator and collector electrodes is very small, some portion of the species such as oxygen or hydrogen reduced at the generator may reach the collector electrode and increase the collector background current. The collector current at the gold IDA is much lower than that of platinum, but it is still larger than that of the carbon IDA. Thevariation in thecarbon IDAcollector current is within 200 pA from -1 to 0 V. This flat collector baseline is the most suitable for achieving a low detection limit in generation-collection measurements. The generation-collection voltammogram of ruthenium hexaammine at the carbon IDA contains little current other than the ruthenium hexaammine limiting current compared to that measured at the metal IDA because both the generator and collector electrodes of the carbon IDA have a large potential window in the cathodic region. The collection efficiency of ruthenium hexaammine is more than 95% at the IDA with a 3-pm bandwidth and 2-pm gap. Figure 4 shows the calibration curves of ruthenium hexaammine at a carbon IDA electrode. A detection limit of the generator electrode was 1 pM, which is much higher than that of the collector. This is because the charging current and the Faradaic current of coexisting species such as oxygen reduction overlap the ruthenium hexaammine generator current. On theother hand, a linear relationship was obtained between the concentration and the limiting current at the collector from 0 to 1 mM. The collector current shown in the inset in Figure 4 agrees well with the theoretical current calculated using Aoki's equation'' because the collection efficiency is very little charging and Faradaic current other than ruthenium hexaammineoxidation current. This indicates that the measurement in the cathodic region using the collector current is accurate even in the nanomole region. The detection limit at the collector was 10 nM, which is equivalent to that of ferrocene derivatives using a metal IDA in the anodic potential region.I4 This low detection limit at the collector electrode was achieved because the current was enhanced by redoxcycling and the collector current contained less background current in the cathodic potential region than that of the metal IDA electrode. 288
Analytical Chemistry, Vol. 66, No. 2, January 15, 1994
Electrochemical Measurement of Dopamine Using the Electrochemically Pretreated Carbon IDA Electrode. It is difficult to estimate an analyte concentration quantitatively when the electron transfer between the analyte and electrode is slow. With a carbon electrode, theelectrochemical response of such an analyte can be improved by various types of pretreatment such as electrochemical,29~30 in situ laser,31or supersonic activation techniques.32 Electrochemical pretreatment is the most convenient method because it does not require extra equipment and is very easy to use with the electrode in situ. The electrochemical detection of catecholamines has attracted considerable interest for biological and clinical application^.^^,^^ Electrochemical pretreatment is also employed for the detection of catecholamines, because their response at an untreated carbon electrode is not electrochemically r e ~ e r s i b l e ~and ~ , is~ ~significantly -~~ improved by pretreatment. Catecholamine can also be detected selectively by using a pretreated carbon electrode in the presence of L-ascorbic acid, which is a well-known interferent in biological sample^.^^,^^ The cyclic voltammogram of dopamine using one electrode of a carbon IDA after electrochemical pretreatment at 1.8 V vs Ag/AgCl for 5 min shows a sharper response than that measured before pretreatment. The peak separation of dopamine was similar to the theoretical value after pretreatment. Optical microscope observation revealed that the band electrodes in the IDA became darker, suggesting that the surface reflectance decreases after pretreatment. Figure 5 shows high-magnification SEM photographs of carbon band electrode surfaces before and after pretreatment. The carbon electrode surface is flat before pretreatment (Figure Sa), but after pretreatment it becomes much rougher with many small pores of around 10 nm in diameter. This result clearly indicates that the surface area of the IDA electrode increases after electrochemical pretreatment. The problem with electrochemical pretreatment is that it increses the charging current and makes the potential window narrower. This is because it reduces the overpotential of coexisting electroactive species such as oxygen molecules or hydrogen ions. The increased surface area after pretreatment also increases the charging current. Figure 6 compares generation and collection voltammograms at a carbon IDA electrode under different pretreatment conditions. Unlike cyclic voltammetry using a conventional electrode, the surface activity of two microbands in the IDA can be controlled independently in the case of generationcollection voltammetry. The electrochemical response of dopamine is greatly improved by pretreating only the generator electrode. An interesting point is that the generator pretreatment only influences the electrochemical response. For example, curve b, which was obtained by pretreating the generator electrode, is the same as curve d obtained by (29) Ponchon, J. L.; Cespuglio, R.; Gonon, F.; Jouvet, M.; Pujol, J.-F. Anal. Chem. 1979, 51, 1483. (30) Gonon, F.; Buda, M.; Cespuglio, R.; Jouvet, M.; Pujol, J. Nature 1980, 286, 902. (31) Poon, M.; McCreery, R. L. Anal. Chem. 1986, 58, 2745. (32) Zhang, H.; Coury, L. A., Jr. Anal. Chem. 1993, 65, 1552. (33) Adams, R. N. Anal. Chem. 1976, 48, 1126A. (34) Wightman, R. M.; May, L. J.; Michael, A. C. AMI. Chem. 1988,60,769A. ( 3 5 ) Ewing,A. G.; Dayton, M. A.; Wightman, R. M. Anal. Chem. 1981,53,1842. ( 3 6 ) Saraceno, R . A . ; E w i n g , A . G . A n a l . C h e m . 1988, 6 0 , 2016.
collector
collector
r ?
! I
collector -0.2
0
0.2
0.4
0.6
0.8-0.2 0
collector 0.2
0.4
0.6
0.8
Potential / V vs AgIAgCI
r
(b) Figure 5. Hlghmagnetiflcatlon SEM photographs of lhe c a r b n IDA surface before (a) and aner (b) electrochemical pretreatment.
pretreatingbothelectrodes. It isalsoclear fromacomparison ofcurves a and c that collector pretreatment does not improve the response. Since the collector current flows by reducing almost all the dopamine molecules oxidized at the generator electrode, an improvement in the generator reaction also increases the collector response. And if the collector reaction ofdopamineis slowerthan thegeneratorreaction, theelectron transfer between the oxidized dopamine and the collector electrode can he increased easily by making the collector potential more negative. Therefore, generator pretreatment is moreeffective than collector pretreatment for thegeneration collection measurements. These results also indicate that a sharp response can be obtained from quasi-reversible electroactive species without increasing the background current at the collector, because their electrcthemical response can be improved without pretreating the collector electrode directly. Thelinear relationship between thedopamineconcentration and the limiting current is obtained from 10 nM to 1 mM using an IDA collector electrode whose generator electrode
Figure (1. Generatkm-wlbctbn voltammogramsof IOOpMdopamlne lnpH6phosphatebufferatcarbrrbasedIDAelectmdeswkhdlfferent pretreatment condiilons: (a)neitherelectrodepretreated;(b)generata electrde pretreated; (c) collector electrode pretreated; (d) botb electrodes pretreated. The collector potential was held at -0.2 V. and the generator potential was cycled at a scan rate of 50 mVls. Thtt IDA bandwidth and gap are 3 and 2 pm. respectively.
has been electrochemically pretreated. This high sensitivity and wide linear range are achieved because the dopamine response is improved as a result of generator pretreatment while a low background current at the collector is maintained.
CONCLUSION A carbon-hased interdigitated array microelectrode was applied to the highly sensitive voltammetric measurement of reversihleand quasi-reversible redox speciesin both theancdic and cathodic potential regions. The resistance of the IDA does not influence theiRdropwhen theanalyteconcentration is lower than 0.1 mM. Ruthenium hexaammine in the tens ofnanomoles per liter rangecan bemeasuredaccurately using the collector current by generation collection voltammetry due to the current enhancement by redox cycling and low collector background current in the cathodic region. In the dopamine measurement, a low detection limit of 10 nM and a wide linear range were obtained because the dopamineresponsewas improved by pretreating thegenerator while maintaining a low background current at the collector. Recelved fw 1993.’
revktw August 3. 1993. Accepted November
4,
*Abstract published in Adoonce ACS Absrrocrr. Dcscmbcr I, 1993.
AnaWcal Chemlslry. Vd. 66, No. 2, Januaty 75, 1994
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