Correspondence Anal. Chem. 1994,66,3500-3502
Subfemtomole Detection of Catecholamine with Interdigitated Array Carbon Microelectrodes in HPLC Hisao Tabel,'lt Masaya Takahashi,* Satoshl Hoshino,tl§ Osamu Niwa,t*§and Tsutomu Horiuchit9S NTT Basic Research Laboratories, Nippon Telegraph and Telephone Corporation, Tokai, Ibaraki 3 19- 1 1, Japan, and NTT Interdisciplinary Research Laboratories, Nippon Telegraph and Telephone Corporation, Musashino, Tokyo 180, Japan
An IDA carbon microelectrode for HPLC was fabricated by photolithographic techniques from the carbon film of pyrolyzed 3,4,9,10-perylenetetracarboxylic dianhydride on thermally oxidized silicon wafers. The IDA electrode consisted of 300 pairs, the finger widths and gaps were 5 pm, and each finger was 2 mm long. The electrode in a small-volumethin-layer cell incorporated in a microbore HPLC system achieved a low detection limit for dopamine due to current enhancement by redox cycling and low background noise at the carbon IDA microelectrode. The detection limit of 0.5 fmol of dopamine can be realized because of the low noise level and high current density. Microbore high-performance liquid chromatography (HPLC) has received much attention because a small volume sample can be analyzed and the low dispersion in the column increases the sensitivity.14 These characteristics make it suitable for the analysis of trace solutes in a sample which is usually determined by microdialysis in neurotransmitter research. In this situation, an electrochemical detector is expected to play an important role with regard to high sensitivity. A microelectrode array is a good candidate for the electrode in a flow-celldetector, and carbon microelectrodes are especially well suited for measurement because of their high signal-to-noise ratio and high current density. These characteristics have already been reported for random and linear arrays of carbon microelectrodes in flowing ~treams.53~ A metal film-based interdigitated array (IDA) microelectrode has been shown to be a highly sensitive electrochemical detector in HPLC due to a high mass-transfer flux resulting from redox cycling at the IDA.' With IDA electrodes, a "IT Basic Research Laboratories. Research Laboratories. 8 Present address: NTT Basic Research Laboratories, Morinosato Wakamiya, Atsugi, Kanagawa 243-01. Japan. (1) Goto, M.; Nakamura, T.; Ishii, D. J. Chromatogr. 1981, 226, 33. (2) Durkin, T. A.; Caliguri, E. J.; Mefford, I. N.; Lake, D. M.; Macdonald, I. A.; Sundstrom, E.; Jonsson, G. Life Science 1985, 37, 1803. (3) Kendrick, K. M. Curr. Sep. 1990, 9, 136. (4) Cheng, F. C.; Kuo, J. S.;Shin, Y.; Lai, J. S.;Ni, D. R.; Chia, L. G. J. Chromatogr., Biomed. Appl. 1993, 61.5, 225. (5) Magee, L. J., Jr.; Osteryoung, J. Anal. Chem. 1989, 61, 2124. (6) Wang, J.; Brennsteiner, A.; Sylwester, A. P. Anal. Chem. 1990, 62. (7) Takahashi, M.; Morita, M.; Niwa, 0.;Tabei, H. J . Elecfroanal.Chem. 1992, 33.5, 253. t
* N I T Interdisciplinary
3500
Analytical Chemistry, Vol. 66,No. 20, October 15, 1994
product generated at one band electrode collects at one of the adjacent band electrodes and reverts to its initial state when the potential of the adjacent band electrode is sufficient for the reverse reaction. Then, having reverted to its initial state, the species collects again at the first electrode. As this redox cycle repeats, the currents of both the anodic and cathodic electrodes are increased.*v9 However, current enhancement by redox cycling is less effective in a flow stream than in a stationary solution. This is because a rapid flow stream interferes with the redox cycling. Microbore columns are frequently used for biological analysis because very small samples must be analyzed. The combination of a microbore column and an IDA carbon microelectrode is expected to improve sensitivity because an HPLC system with a microbore column operates at a slower flow rate than one with an ordinary size column. In addition, an IDA carbon microelectrode is expected to have an excellent signal-to-noise ratio since it has a wider potential window and larger overpotentialgJO for oxygen reduction and hydrogen evolution than previously reported metal-based IDAS." In this paper, we demonstrate the high current response of catecholamine measured with an IDA carbon microelectrode incorporated in a microbore HPLC system and the subfemtomole detection of dopamine.
EXPER I MENTAL SECT1ON IDA Carbon Microelectrode Fabrication. The IDA carbon microelectrode was fabricated by photolithographic techniques from the carbon film of pyrolyzed 3,4,9,10-perylenetetracarboxylic dianhydride on thermally oxidized silicon wafers as reported p r e v i o ~ s l y . ~The J ~ IDA electrode consisted of 300 pairs, the finger widths and gaps were 5 pm and each finger was 2 mm long. A carbon IDA was used without pretreatment, since the peak separation of catecholamine is even smaller than that at a well-polished GC electrode. This is because the plasma reactive etching method applied at the (8) Aoki, K.; Morita, M.; Niwa, 0.;Tabei, H. J. Electroanal. Chem. 1988,256, 269. (9) Tabei, H.; Morita, M.; Niwa, 0.;Horiuchi, T. J. Electroanal. Chem. 1992, 334, 25. (10) Niwa, 0.;Tabei, H. Anal. Chem. 1994, 66, 285. (11) Niwa, 0.;Morita, M.; Tabei, H. Anal. Chem. 1990, 62,447.
0003-2700/94/0366-3500$04.50/0
0 1994 American Chemical Society
1 DHBA
T
-"
Anode
0
2 ?A
LJ
0
4
8
1
2
0
4
8
1
2
(a> (b) Retention Time / min Figure 1. Typical chromatograms of catecholamine measured with the IDA carbon microelectrode: (a) dual mode and (b) single mode. The cell thickness is 12 pm, volume flow rate Is 0.05mL/min, and injection volume is 5 pL.
end of the fabrication process may remove surface contamination. Reagents. A standard catecholamine solution was obtained from Katayama Chemical Industry (Osaka). It contained 10 mM each of epinephrine (E), norepinephrine (NE), dopamine (DA), and L-dopa. An internal standard solution was also obtained from Katayama Chemical Industry, which contained 10 mM of dihydroxybenzylamine (DHBA). These reagents were used as received. The mobile phase consisted of 0.85 M sodium acetate, 0.09 M tartaric acid, 0.5 mM ethylenediaminetetraacetic acid disodium salt, 0.8 mM l-octanesulfonic acid sodium salt, and 5.5% acetonitrile. Apparatus and Procedure. The liquid chromatography system used to characterize this electrode consisted of an 880PU pump (Japan Spectroscopic Co., Tokyo) and a CC-4 HPLC system incorporating an LC-4B amperometric detector from Bio Analytical Systems (BAS), West Lafayette, IN. The thickness of the cell channel was 12 pm, and a 100-mm X 1-mm (i.d.) SepStick reversed-phase column (BAS) was employed. The column was kept at 30 "C using a BAS LC22A system. To obtain the amperometric response for catecholamine, the potential of the anodic array of the IDA electrode was held at +0.6 V vs Ag/AgCl and that of the cathodic array was held at -0.005 V.
RESULTS AND DISCUSSION Figure 1 shows typical chromatograms of catecholamine measured with an IDA carbon microelectrode in which both
0 ' 4 '
8 ' i2
Retention Time / min Flgure 2. Anodic chromatograms of low-concentration samples in a dual mode: (a) 3.5 fmoi(700 pM), (b) 2.5 fmol(500 pM), (c) 1.5 fmol (300 pM), (d) 0.5 fmol (100 pM), and (e) background chromatogram for mobile phase.
the anodicandcathodicarraysare potentiostated (dual mode). The injected sample was a 5-pL solution which contained 10 nM of catecholamine and DHBA. The flow rate was 0.05 mL/min. A large front peak, generated by the sample solution used as the mobile phase, was observed at retention times of 2.5 and 3.5 min. In addition, gentle background noise peaks generated by the solvent were observed at about 7 and 14 min (shown in Figure 2). The L-dopa peak cannot be distinguished because it overlaps the front peak observed at about 2.5 min. The collection efficiency which is defined as the ratio of cathodic to anodic current at the IDA increases when the redox cycling number increases." As a result, a higher peak current is expected through the use of the IDA electrode. The collection efficiency was 70% for DA in HPLC. However, the collection efficiency for E was 60% which was lower than that of other analytes. Since E is not stable after electrochemical oxidation and changed to the indole form as a result of the cyclization reaction, some of the E molecules may be chemically changed before reaching the cathode array Analytical Chemistty, Vol. 66,No. 20, October 15, 1994
3501
electrode after electrochemical oxidation.'* The collection efficiency of aq-ferrocene is more than 90%" and that of dopamine is at least 80%13 in a stationary solution. These values are greater than that in a flow cell, suggesting that the flow stream reduces the collection efficiency. In our flow-cell experiment we used an IDA which had a 5-pm band width and gap. The above values are higher than those obtained in this work. This lower collection efficiency is due to the flowing stream and may also be due to the fact that part of the product at one band electrode is collected not only by the adjacent band electrodes but also by the auxiliary electrode which faces the IDA electrode in a cell with a narrow gap. Although the value of 70% is not as high as anticipated, it can be expected that redox cycling will increase the peak current. The peaks of chromatogram (b) were measured with the same IDA in which only the anodic array was potentiostated (single mode). As expected, the dual mode chromatogram exhibits a higher current peak than in the single mode. These high current values are due to the redox cycling of analytes at the anode and cathode electrode arrays in a flowing stream. However, the current peak in the dual mode was only 1.4 times higher than that in the single mode. It is considered that redox cycling occurred effectively between the anodic array and the auxiliary electrode even in the single mode because the thickness of the cell channel was 12 pm, which is not very different from the IDA gap of 5 pm. The current density of the single mode must be higher than that in an ordinary flow-cell detector with a thick channel because the current peaks in the single mode are already enhanced by redox cycling between the anode and the auxiliary electrode. Similar phenomena have also been reported for the microtip electrode of a scanning electrochemical microscope and in a voltammetric measurement using a narrow gap micro-macro twin electrode in a stationary s o l ~ t i o n . ' ~ Therefore, J~ the amplification factor which is the ratio of the peak current measured in the dual mode to that measured in the single mode is not very high. The peak current density corresponding to 100 nM DA, which was 12 nA/cm2, was measured with a commercially available glassy carbon (GC) disk electrode for a 12-pmthick cell in the single mode.I6 On the other hand, the current density of the DA at the carbon based IDA was 9 times higher than that at the GC electrode, and we could obtain a larger peak current than with the G C electrode. Figure 2 shows anodic chromatograms of catecholamine and DHBA obtained using an IDA electrode operating in a dual mode for low concentration samples. A chromatogram without a catecholamine sample is also illustrated in Figure 2. The injected moles are (a) 0.5 fmol(lO0 pM), (b) 1.5 fmol (12) Hawley, M.;Tatawawadi,S.;Adams,R.N. J . Am. Chem.Soc. 1967,89,447. (13) Niwa, 0.; Morita, M.; Tabei, H. Ekctroanalysis 1991, 3, 163. (14) Bard, A. J.; Fan, F.-R.F.; Kwak, J.; Lev, 0. Anal. Chem. 1989, 61, 132. (IS) Horiuchi, T.; Niwa, 0.; Morita, M.; Tabei, H. J. Electrochem. SOC.1991, 138, 3549. (16) Takahashi, M.;Morita, M.; Niwa, 0.;Tabei, H. Sensors Acfuarors E 1993, 13-14, 336.
3502
Analytical Chemistry, Vol. 66, No. 20, October 15, 1994
1x10.~ E 0 4
2 1x10'0
I
lxlo-"
I
o * A 0
OEA O A
A 1x10'2 1x101~
I
I
1~10-l5
,
I
I
,
, I ,I
,
,
1~10-l~
, ,
,
,~
1~10-'~
Moles injected / mol
Figure 3. Calibration curves for dopamine and dlhydoxybenzylamine measured with the IDA electrode: (0) dopamine; (A)dihydroxybenzyiamine.
(300pM), (c) 2.5 fmol(500 pM), (d) 3.5 fmol(700 pM), and (e) 0 fmol (injected only mobile phase solution). Although L-dopa, NE, and E are also included in the injected sample, their peaks overlap the front peaks in this low concentration range. In contrast, the current peaks of DA and DHBA can be distinguished from baseline noise in such a low concentration sample. Although the noise peak generated by the injected sample solution appears at 7 min between the DHBA and DA peaks, both peaks can be distinguished even in the 0.5 fmol (100 pM) sample, and they are proportional to the sample concentration. The noise level of the GC detector was 6 PA,' whereas the noise level observed using the IDA carbon microelectrode was less than 1 pA which is a little lower than that of an ordinary GC disk electrode. As the injected sample volume is 5 p L and the lowest concentration is 100 pM in this experiment, a dopamine detection limit of 0.5 fmol can be achieved because of the low noise level and high current density. Figure 3 shows the calibration curves for DA and DHBA. The plot shows good linearity from 0.5 to 50 fmol. In summary, an IDA carbon microelectrode for HPLC was fabricated by photolithographic techniques from the carbon film of pyrolyzed 3,4,9,1O-perylenetetracarboxylic dianhydride on thermally oxidized silicon wafers. The electrode in a small-volume thin-layer cell incorporated in a microbore HPLC system achieved a low dopamine detection limit of 0.5 fmol due to current enhancement by redox cycling and low background noise at the carbon IDA electrode. Other catecholamines such as E and NE may be detected with high sensitivity by shifting their retention time by changing the mobile phase. A lower detection limit can be expected if an IDA carbon microelectrode is used which has a smaller pattern size. Received for review November 5, 1993. Accepted June 15, 1994.* @Abstractpublished in Advance ACS Absfracfs,August 1, 1994.
