Anal. Chem. 2003, 75, 3301-3307
A Microchip Electrophoresis Device with Integrated Electrochemical Detection: A Direct Comparison of Constant Potential Amperometry and Sinusoidal Voltammetry Nicole E. Hebert,† Werner G. Kuhr,‡ and Sara A. Brazill*,†
Department of Chemistry, University of California, Riverside, California 92521-0403
A microchip electrophoresis system with integrated electrochemical detection is described in this work. The hybrid device utilizes poly(dimethylsiloxane) as the electrophoresis channel substrate and a planar gold electrode lithographically fabricated onto a glass slide for electrochemical detection. The system is characterized by the separation and detection of various neurotransmitters. The gold working electrode is placed just inside the separation channel without adverse effects on the detection sensitivity, due to the electrical decoupling of the detection and electrophoresis systems. The close proximity of the working electrode to the exit of the separation channel results in symmetric peak shapes and efficient separations (50 000-100 000 plates/m). A direct comparison between the frequency-based electrochemical technique, sinusoidal voltammetry, and the more commonly used constant potential (DC) amperometry is made. Sinusoidal voltammetry is found to be roughly an order of magnitude more sensitive than DC amperometry, with calculated mass detection limits (S/N ) 3) of 12 amol and 15 amol for dopamine and isoproterenol, respectively. The area of microfluidics has seen much growth since its beginnings over a decade ago as a result of its promise of faster, less expensive, and truly portable measurement capabilities. By far, miniaturized electrophoresis devices have been the most widely developed as a result of their scalability and compatibility with the substrates available for device construction. Electrophoresis has been used on-chip not only as a separation technique, but also for fluid transport and mixing, which has allowed for the design and implementation of numerous integrated functions, such as polymerase chain reaction (PCR),1 enzymatic derivatization reactions,2 and assays.3,4 * To whom correspondence should be addressed. Phone (909) 787-3485, Fax: (909) 787-4713. E-mail:
[email protected]. † University of California, Riverside. ‡ Current address: ZettaCore Inc., Denver, CO 80222. (1) Hong, J. W.; Fujii, T.; Seki, M.; Yamamoto, T.; Endo, I. Electrophoresis 2001, 22, 328-333. (2) Wang, J.; Chatrathi, A. P.; Ibanez, A.; Escarpa, A. Electroanalysis 2002, 14, 400-404. (3) Wang, J.; Chatrathi, M. P.; Tian, B. M. Anal. Chem. 2001, 73, 1296-1300. 10.1021/ac0262457 CCC: $25.00 Published on Web 05/20/2003
© 2003 American Chemical Society
Whereas initial studies were carried out almost exclusively on glass-based devices, many polymeric materials have been investigated because of advantages such as decreased cost and ease of fabrication. These materials include poly(dimethylsiloxane) (PDMS),1,5-12 polycarbonate,13,14 and poly(methyl methacrylate).15-18 Additionally, the majority of the work performed with microfluidic devices has involved the use of laser-induced fluorescence (LIF) detection, which has demonstrated high detection performance characteristics (e.g., sensitivity and separation efficiency).19-22 Unfortunately, LIF detection conventionally requires relatively bulky (orders of magnitude larger than the microchip device) and expensive instrumentation, and the on-chip integration is not straightforward. The shortcomings of LIF detection have led to considerable interest in electrochemical detection, which is sensitive, inexpen(4) Wang, J.; Chatrathi, M. P.; Tian, B. M.; Polsky, R. Anal. Chem. 2000, 72, 2514-2518. (5) Backofen, U.; Matysik, F. M.; Lunte, C. E. Anal. Chem. 2002, 74, 40544059. (6) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (7) Gawron, A. J.; Martin, R. S.; Lunte, S. M. Electrophoresis 2001, 22, 242248. (8) Liu, Y.; Fanguy, J. C.; Bledsoe, J. M.; Henry, C. S. Anal. Chem. 2000, 72, 5939-5944. (9) Martin, R. S.; Gawron, A. J.; Lunte, S. M.; Henry, C. S. Anal. Chem. 2000, 72, 3196-3202. (10) Martin, R. S.; Gawron, A. J.; Fogarty, B. A.; Regan, F. B.; Dempsey, E.; Lunte, S. M. Analyst 2001, 126, 277-280. (11) Martin, R. S.; Ratzlaff, K. L.; Huynh, B. H.; Lunte, S. M. Anal. Chem. 2002, 74, 1136-1143, 1144-1207. (12) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H. K.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40. (13) Liu, Y. J.; Ganser, D.; Schneider, A.; Liu, R.; Grodzinski, P.; Kroutchinina, N. Anal. Chem. 2001, 73, 4196-4201. (14) Olsen, K. G.; Ross, D. J.; Tarlov, M. J. Anal. Chem. 2002, 74, 1436-1441. (15) Wang, J.; Pumera, M.; Chatrathi, M. P.; Escarpa, A.; Konrad, R.; Griebel, A.; Dorner, W.; Lowe, H. Electrophoresis 2002, 23, 596-601. (16) Lee, G. B.; Chen, S. H.; Huang, G. R.; Sung, W. C.; Lin, Y. H. Sens. Actuators, B 2001, 75, 142-148. (17) Grass, B.; Neyer, A.; Johnck, M.; Siepe, D.; Eisenbeiss, F.; Weber, G.; Hergenroder, R. Sens. Actuators, B 2001, 72, 249-258. (18) Grass, B.; Siepe, D.; Neyer, A.; Hergenroder, R. Fresenius’ J. Anal. Chem. 2001, 371, 228-233. (19) Haab, B. B.; Mathies, R. A. Anal. Chem. 1999, 71, 5137-5145. (20) Fister, J. C.; Jacobson, S. C.; Davis, L. M.; Ramsey, J. M. Anal. Chem. 1998, 70, 431-437. (21) Ocvirk, G.; Tang, T.; Harrison, D. J. Analyst 1998, 123, 1429-1434. (22) Culbertson, C. T.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 2000, 72, 5814-5819.
Analytical Chemistry, Vol. 75, No. 14, July 15, 2003 3301
sive to implement, has low power requirements, and is easily miniaturized, with respect to both the supporting electronics and the electrodes themselves. In a recent publication by Baldwin et al., all the electrodes required for both application of the electrophoresis voltage and electrochemical detection were produced through photolithography onto a planar substrate,23 which furthered the development toward a rugged device with few to no moving parts. Microfluidic electrophoretic separation with electrochemical detection has been used to detect numerous compounds, such as nitroaromatic explosives and organophosphate nerve agents,24,25 DNA restriction fragments,26 carbohydrates,27,28 copper(II) peptide complexes,7,10 and most notably, neurotransmitters.5,7-9,11,15,23,26,27,29-34 Detection is typically accomplished through the use of DC amperometry, which simply involves holding the working electrode at a particular potential sufficient to oxidize or reduce the compounds of interest and recording the resulting current response. Although this technique has been shown to be very sensitive, this work will explore the enhancement in sensitivity gained through the use of sinusoidal voltammetry (SV) and data analysis in the frequency domain. SV is an electrochemical detection technique that is very similar to fast scan cyclic voltammetry, except that a large amplitude sine wave is used as the excitation waveform, and data analysis is performed in the frequency domain. In SV, the raw time domain is collected from the electrochemical cell and converted into the frequency domain in order to better decouple the faradaic signal from the background components, as well as to generate a unique “fingerprint” frequency spectrum to aid in identification and isolation of the chemical species. We have demonstrated the use of the frequency spectrum to selectively isolate oligonucleotides tagged with four different ferrocene derivatives35 as well as to null out each signal from a pair of chromatographically unresolved peaks (resolution
