Anal. Chem. 2002, 74, 1349-1354
Determination of Thiols by Capillary Electrophoresis with Amperometric Detection at a Coenzyme Pyrroloquinoline Quinone Modified Electrode Takayo Inoue and Jon R. Kirchhoff*
Department of Chemistry, University of Toledo, Toledo, Ohio 43606
A chemically modified electrode has been developed as a detector for the sensitive and selective determination of thiol-containing compounds following capillary electrophoresis separation. Electrodes were constructed by entrapment of the coenzyme pyrroloquinoline quinone (PQQ) into a polypyrrole (PPy) matrix on a 245-µm graphite electrode during electropolymerization of pyrrole in the presence of PQQ. PQQ serves as an efficient biocatalyst to mediate the oxidation of thiols at a substantially reduced overpotential relative to an unmodified electrode. Furthermore, this design takes advantage of the pH-dependent reversible electrochemical properties of PQQ, which facilitates optimization of separation and detection conditions. The PQQ/PPy-modified electrode was incorporated as an end-column detector, and a separation of homocysteine, cysteine, N-acetylcysteine, and glutathione was developed. Detection limits for these four thiols were determined to be 11, 23, 104, and 134 nM, respectively, with mass detection limits ranging from 0.29 to 3.48 fmol. The PQQ/PPy electrode was also found to be very reproducible in run-to-run, day-to-day, and electrode-to-electrode comparisons. The utility of this electrode was demonstrated for the detection of cysteine in dietary supplements and human urine, resulting in excellent agreement with reported values. Capillary electrophoresis (CE) has been recognized as an attractive technique for the separation of mixtures, especially complex biological samples, due to the high separation efficiencies and very small amounts of sample required for analysis.1,2 UV/ vis spectroscopy is the most commonly used detection method to date for CE. However, the small path length across the capillary poses a limitation on sensitivity. In contrast, electrochemical (EC) detection has attracted significant attention as an alternative method for the detection of electroactive species because of the * Corresponding author: 2801 W. Bancroft St., Toledo, OH 43606. Phone: (419) 530-1515. Fax: (419) 530-4033, E-mail:
[email protected]. (1) Kuhn, R.; Hoffstetter-Kuhn, S. Capillary Electrophoresis: Principles and Practice; Springer-Verlag: Berlin, 1993. (2) Foret, F.; Krivankova, L.; Bocek, P. Capillary Zone Electrophoresis; VCH: Weinheim, 1993. 10.1021/ac0108515 CCC: $22.00 Published on Web 02/14/2002
© 2002 American Chemical Society
inherent advantages of simplicity, ease of miniaturization, high sensitivity, and relatively low cost.3,4 Electrochemical detection for CE is not without experimental difficulties. Electrode alignment and isolation of the detector from the separation voltage are two challenges, which have been addressed by devices for straightforward electrode alignment and decoupling strategies, respectively.5,6 An additional challenge for the analysis of many important analytes at solid electrodes, such as noble metals and carbon, is that direct detection is often complicated by slow electron-transfer kinetics and the need to apply extremely high overpotentials to initiate the oxidation or reduction reaction. This can be a serious disadvantage, especially in complex samples, because selectivity is inversely related to the magnitude of the applied potential. Thus, coupling a highly selective and sensitive EC detection system with the high separation efficiency of CE is very desirable. Detection of free thiols by EC approaches is an example of this latter case. Given the widespread involvement of thiols and the corresponding disulfides in many essential biological functions,7,8 much effort has been made to develop sensitive and selective methods for their detection; however, direct oxidation of thiols at solid electrodes is slow and requires a potential of at least 1.0 V to initiate.9,10 To address this problem, Rabenstein and Saetre11 introduced an indirect detection scheme based on the interaction of a thiol with a mercury electrode to form a mercury thiolate complex, which can be detected at a reduced potential of +0.1 V versus Ag/AgCl. Subsequently, mercury-based electrodes have been used as detectors for separation methods,12-15 including (3) Voegel, P. D.; Baldwin, R. P. Electrophoresis 1997, 18, 2267-2278. (4) Baldwin, R. P. Electrophoresis 2000, 21, 4017-4028. (5) Matysik, F.-M. Electroanalysis 2000, 12, 1349-1355. (6) Holland, L. A.; Lunte, S. M. Anal. Commun. 1998, 35, 1H-4H. (7) Friedman, M. The Chemistry and Biochemistry of the Sulfhydryl Group in Amino Acids, Peptides, and Proteins; Pergamon Press: Oxford, 1973. (8) Jocelyn, P. C. Biochemistry of the SH Group; Academic Press: London, 1972. (9) Mefford, I.; Adams, R. N. Life Sci. 1978, 23, 1167-1174. (10) Kreuzig, F.; Frank, J. J. Chromatography 1981, 218, 615-620. (11) Rabenstein, D.; Saetre, R. Anal. Chem. 1977, 49, 1036-1039. (12) Saetre, R.; Rabenstein, D. Anal. Biochem. 1978, 90, 684-692. (13) Saetre, R.; Rabenstein, D. Anal. Chem. 1978, 50, 276-280. (14) Bergstrom, R. F.; Kay, D. R.; Wagner, J. G. Anal. Chem. 1978, 50, 21082112. (15) Allison, L. A.; Shoup, R. E. Anal. Chem. 1983, 55, 8-12.
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electrode for CEEC and the separation and detection of several biologically interesting thiols, including the determination of cysteine in dietary supplements and human urine samples, are described. Figure 1. PQQ redox reaction.
CE, with both single-electrode detection for thiols16 and dualelectrode detection for simultaneous determination of thiols and disulfides.17,18 A different approach for thiol analysis has focused on the use of chemically modified electrodes (CMEs) with redox mediators to enhance detection at much lower potentials following CE separation.19 O’Shea and Lunte20 were the first to utilize a modified microelectrode detector of this type for the CE analysis of thiols. In this example, the electrocatalyst cobalt phthalocyanine21 was incorporated into a carbon paste matrix and found to detect cysteine down to 31 nM. A similar method has been used to measure free and total cysteine in urine.22 On the basis of the macroelectrode developed by Cox and Gray,23 a mixed-valence ruthenium cyanide modified microelectrode array was also fabricated for the simultaneous detection of thiols and disulfides by CEEC.24,25 In addition, Wang et al.26 recently used a bare carbon disk electrode at +1.1 V versus Ag/AgCl for the CEEC analysis of thiols. In previous work, we designed and characterized an amperometric sensor for the selective detection of thiols.27 This sensor incorporates the redox cofactor pyrroloquinoline quinone (PQQ) as a biocatalyst into a conducting polypyrrole (PPy) matrix on a glassy carbon electrode substrate. Thiol detection is mediated by their reaction with the entrapped biocatalyst and takes advantage of the pH-dependent reversible electrochemical reaction of PQQ (Figure 1) to enhance detection selectivity and sensitivity. In the presence of a thiol, PQQ is reduced to PQQH2, and the thiol is oxidized. Thiols are then quantitatively detected by monitoring the amperometric response from the reversible oxidation of PQQH2 to PQQ at the electrode. This cycle effectively regenerates the catalyst for further detection and permits detection of thiols at a reduced overpotential. Since the PQQ/PPy-modified electrode is prepared by electropolymerization, film thickness and the amount of catalyst are easily controlled. Therefore, this sensing matrix can be miniaturized or adapted to any electrode shape. In this study, the PQQ/PPy sensor has been miniaturized to micrometer dimensions and investigated as a modified electrode detector for CE. The combination of the highly efficient separation capabilities of CE with the excellent sensitivity of the PQQ/PPymodified electrode offers an attractive approach for selective and sensitive thiol detection. Evaluation of the PQQ/PPy-modified (16) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1993, 65, 247-250. (17) Lin, B. L.; Colon, L. A.; Zare, R. N. J. Chromatogr. A 1994, 680, 263-270. (18) Zhong, M.; Lunte, S. M. Anal. Chem. 1999, 71, 251-255. (19) Cox, J. A.; Tess, M. E.; Cummings, T. E. Rev. Anal. Chem. 1996, 15, 173223. (20) O’Shea, T. J.; Lunte, S. M. Anal. Chem. 1994, 66, 307-311. (21) Halbert, M. K.; Baldwin, R. P. Anal. Chem. 1985, 57, 591-595. (22) Huang, X.; Kok, W. T. J. Chromatogr. A 1995, 716, 347-353. (23) Cox, J. A.; Gray, T. J. Anal. Chem. 1989, 61, 2462-2464. (24) Zhou, J.; O’Shea, T. J.; Lunte, S. M. J. Chromatogr. A 1994, 680, 271-277. (25) Zhou, J.; Lunte, S. M. Anal. Chem. 1995, 67, 13-18. (26) Wang, A.; Zhang, L.; Zhang, S.; Fang, Y. J. Pharm. Biomed. Anal. 2000, 23, 429-436. (27) Inoue, T.; Kirchhoff, J. R. Anal. Chem. 2000, 72, 5755-5760.
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EXPERIMENTAL SECTION Reagents. PQQ (>97% purity) was purchased from Fluka (Milwaukee, WI). Pyrrole (Aldrich, Milwaukee, WI) was distilled before use. The thiols L-cysteine, N-acetyl-L-cysteine, and glutathione were obtained from Sigma (St. Louis, MO), and D,Lhomocysteine was obtained from Aldrich. All other chemicals were of analytical reagent grade and no further purification was necessary. Solutions were prepared with distilled deionized water purified to a resistivity of 17-18 MΩ‚cm by a Barnstead B pure water purification system. Apparatus. All electrochemical measurements for the preparation and evaluation of the PQQ/PPy-modified electrodes were conducted in a conventional three-electrode electrochemical cell under anaerobic conditions with a Bioanalytical Systems (BAS, West Lafayette, IN) 100B electrochemical analyzer. Ag/AgCl (3 M NaCl) (BAS, MF-2020) and platinum wires were used as the reference and auxiliary electrodes, respectively. Preliminary separation conditions were developed on a Beckman Instruments P/ACE MDQ capillary electrophoresis system (Fullerton, CA) with UV detection at 214 nm. A 50-cm polyimidecoated fused-silica capillary with an i.d. of 50 µm and an o.d. of 360 µm (Polymicro Technologies Inc., Phoenix, AZ) and a separation voltage of 15 kV were used. Electrophoretic separations with EC detection were then conducted on a laboratory-built CEEC system, which has been previously described.28 In this case, an 80-cm fused-silica capillary with an i.d. of 50 µm and an o.d. of 360 µm was employed. Electrochemical detection for CE also used a three-electrode configuration with a model RE-4 (BAS) Ag/AgCl reference electrode. The detection compartment was shielded in a Faraday cage to minimize contributions from external noise. The working electrode was aligned in an end-column configuration29 with the aid of a capillary-electrode holder described by Fermier et al.30 An on-column fracture decoupler31 was placed 2.5 cm from the capillary outlet and used to isolate the detector from the separation voltage. Potential control and current monitoring were achieved by a BAS LC-4C amperometric detector, which was modified for use with CE. All data were collected by an IBM P166 MHz computer via an A/D converter and analyzed using P/ACE MDQ capillary electrophoresis system software. Preparation of the PQQ/PPy-Modified Electrode Detector. Carbon disk electrodes were constructed from 300-µmdiameter graphite rods (Mitsubishi Pencil),29 which were carefully sanded to a diameter of near 245 µm and then inserted into a 2 cm length of fused silica capillary (360 µm o.d., 245 µm i.d., Polymicro Technologies Inc.) such that the carbon extended from both ends of the capillary. Epoxy glue (Borden Inc., Columbus, OH) was used to seal and secure the carbon rod firmly in place in the capillary. At one end of the carbon rod, electrical contact (28) Smith, A. R.; Kirchhoff, J. R.; Tillekeratne, L. M. V.; Hudson, R. A. Anal. Commun. 1999, 36, 371-374. (29) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. G. Anal. Chem. 1991, 63, 189192. (30) Fermier, A. M.; Gostkowski, M. L.; Colon, L. A. Anal. Chem. 1996, 68, 1661-1664. (31) Linhares, M. C.; Kissinger, P. T. Anal. Chem. 1991, 63, 2076-2078.
was made by using nickel print to attach a piece of copper wire to the exposed carbon. At the other end, the carbon rod was cut flush to the end of the capillary with a razor blade to create a disk electrode surface. Electrodes were polished prior to modification with diamond (1 µm) and alumina (
