Development of a Microfabricated Palladium Decoupler

The electrode plates were developed in AZ 300 MIF developer. .... The voltage program that was used for the electrochemical detection experiments was ...
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Anal. Chem. 2004, 76, 2482-2491

Development of a Microfabricated Palladium Decoupler/Electrochemical Detector for Microchip Capillary Electrophoresis Using a Hybrid Glass/ Poly(dimethylsiloxane) Device Nathan A. Lacher and Susan M. Lunte*

Department of Pharmaceutical Chemistry, University of Kansas, 2095 Constant Avenue, Lawrence, Kansas 66047 R. Scott Martin*

Department of Chemistry, Saint Louis University, 3501 Laclede Avenue, St. Louis, Missouri 63103

The fabrication and evaluation of a palladium decoupler and working electrode for microchip capillary electrophoresis (CE) with electrochemical detection is described. The use of the Pd decoupler allows the working electrode to be placed directly in the separation channel and eliminates the band-broadening characteristic of the endchannel configuration. The method used for fabrication of the decoupler and working electrode was based on thinlayer deposition of titanium followed by palladium onto a glass substrate. When employed as the cathode in CE, palladium absorbs the hydrogen gas that is generated by the hydrolysis of water. The effect of the decoupler size on the ability to remove hydrogen was evaluated with regard to reproducibility and longevity. Using boric acid and TES buffer systems, 500 µm was determined to be the optimum decoupler size, with effective voltage isolation lasting for ∼6 h at a constant field strength of 600 V/cm. The effect of distance between the decoupler and working electrode on noise and resolution for the separation of dopamine and epinephrine was also investigated. It was found that 250 µm was the optimum spacing between the decoupler and working electrode. At this spacing, laser-induced fluorescence detection at various points around the decoupler established that the band broadening due to pressure-induced flow that occurs after the decoupler did not significantly affect the separation efficiency of fluorescein. Limits of detection, sensitivity, and linearity for dopamine (500 nM, 3.5 pA/µM, r2 ) 0.9996) and epinephrine (2.1 µM, 2.6 pA/µM, r2 ) 0.9996) were obtained using the palladium decoupler in combination with a Pd working electrode. Since the introduction of miniaturized capillary electrophoresis (CE) systems over a decade ago,1 their fabrication has become much more routine.2,3 Microchip CE is becoming increasingly popular because of some of its advantages over conventional liquid chromatography and CE, especially for high-throughput and small* Corresponding authors. E-mail: [email protected]. Tel.: +1-785-864-3811. Fax: +1-785-864-5736. E-mail: [email protected]. Tel.:+1-314-977-3307. Fax: +1314-977-2521. (1) Manz, A.; Graber, N.; Widmer, H. M. Sens. Actuators, B 1990, 1, 244-248.

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volume samples. These advantages include reduction in reagent usage and waste production, disposability, portability, integration of pre- and postseparation processes, multiplexed analysis, and, more recently, valving capabilities.4-8 As a result of continued research, many important applications having sample pretreatment requirements have led to the development of a multifunctional microchip.2,3,9 Microfluidic chips now have the ability to perform pumping,10,11 extraction,12,13 mixing,14,15 immunoassays,16,17 digestions,18,19 labeling,20 separation,2,3,5,6 and detection,2,3,5,6 making this platform a very powerful analytical tool. Most microfluidic chips are constructed using well-established techniques that have been used for the fabrication of microelectronics. The first microchips were produced from glass or quartz, (2) Reyes, D. R.; Iossifidis, D.; Auroux, P. A.; Manz, A. Anal. Chem. 2002, 74, 2623-2636. (3) Auroux, P. A.; Iossifidis, D.; Reyes, D. R.; Manz, A. Anal. Chem. 2002, 74, 2637-2652. (4) Freemantle, M. Chem. Eng. News 1999, 77 (8), 27-36. (5) Lacher, N. A.; Garrison, K. E.; Martin, R. S.; Lunte, S. M. Electrophoresis 2001, 22, 2526-2536. (6) Vandaveer, W. R., IV; Pasas, S. A.; Martin, R. S.; Lunte, S. M. Electrophoresis 2002, 23, 3667-3677. (7) Unger, M. A.; Chou, H. P.; Thorsen, T.; Scherer, A.; Quake, S. R. Science 2000, 288, 113-116. (8) Ng, J. M.; Gitlin, I.; Stroock, A. D.; Whitesides, G. M. Electrophoresis 2002, 23, 3461-3473. (9) Verpoorte, E. Electrophoresis 2002, 23, 677-712. (10) Lemoff, A. V.; Lee, A. P. Sens. Actuators, B 2000, 63, 178-185. (11) Song, Y. J.; Zhao, T. S. J. Micromech. Microeng. 2001, 11, 713-719. (12) Xu, N.; Lin, Y.; Hofstadler, S. A.; Matson, D.; Call, C. J.; Smith, R. D. Anal. Chem. 1998, 70, 3553-3556. (13) Xiang, F.; Lin, Y.; Wen, J.; Matson, D. W.; Smith, R. D. Anal. Chem. 1999, 71, 1485-1490. (14) Veenstra, T. T.; Lammerink, T. S. J.; Elwenspoek, M. C.; van den Berg, A. J. J. Micromech. Microeng. 1999, 9, 199-202. (15) Liu, R. H.; Sharp, K. V.; Olsen, M. G.; Stremler, M. A.; Santiago, J. C.; Adrian, R. J.; Aref, H.; Beebe, D. J. Transducers ’99; Sendai, Japan, 1999; Vol. 1, pp 730-733. (16) Hadd, A. G.; Jacobson, S. C.; Ramsey, J. M. Anal. Chem. 1999, 71, 52065212. (17) Hatch, A.; Kamholz, A. E.; Hawkins, K. R.; Munson, M. S.; Schilling, E. A.; EWeigl, B. H.; Yager, P. Nat. Biotechnol. 2001, 19, 461-465. (18) L’Hostis, E.; Michel, P. E.; Fiaccabrino, G. C.; Strike, D. J.; de Rooij, N. F.; Koudelka-Hep, M. Sens. Actuators, B 2000, 64, 156-162. (19) Gottschlich, N.; Culbertson, C. T.; McKnight, T. E.; Jacobson, S. C.; Ramsey, J. M. J. Chromatogr., B 2000, 745, 243-249. (20) Liu, Y. J.; Foote, R. S.; Jacobson, S. C.; Ramsey, R. S.; Ramsey, J. M. Anal. Chem. 2000, 72, 4608-4613. 10.1021/ac030327t CCC: $27.50

© 2004 American Chemical Society Published on Web 03/24/2004

but more recently alternative substrates, such as plastics and lowtemperature ceramics, have been explored.21-23 Initially, the fabrication of microchips using substrates that were optically transparent was necessary, as laser-induced fluorescence (LIF) detection was employed. LIF detection has been very popular for microchip CE because of the relative ease with which a laser can be directed onto a microchannel and the sensitivity with which the resulting fluorescence can be collected for a given compound. However, unless the analyte of interest exhibits native fluorescence, this method of detection requires the analyte to be derivatized prior to analysis. Optical detection methods that do not require labeling, such as absorbance, refractive index, and Raman detection, have been explored for microchip CE.21,23-26 Recently, mass spectrometric detection has been used in more research applications with microchip CE, as there is a need for increased throughput and sensitivity with proteomic studies.23,27 In contrast to some optical methods, electrochemistry is unique because miniaturization of electrodes does not diminish the analytical performance28 and opaque chip substrates can be utilized.29 Electrochemical (EC) detection for microchip CE is becoming more popular and has recently been reviewed.5,6,26 Applications of microchip CEEC include neurotransmitter analysis, enzyme assays/immunoassays, clinical diagnostics, and environmental monitoring. Many of the same methods used for the fabrication of microfluidic channels can also be employed to construct electrodes. A fully integrated microchip containing electrodes for both electrophoresis and EC detection has been demonstrated.30 With the ability to also miniaturize power supplies31 and a potentiostat,31,32 it is possible to envision a complete micro total analysis system. Although the advantages of employing EC detection are numerous and applications detailing its use are on the rise, LIF is still the most popular detection method. One reason for this is the lower separation efficiencies, higher detection limits, and peak tailing that are exhibited with EC detection compared to LIF. A major cause of these difficulties is that EC detection requires isolation of the separation field from the potentiostat. The type of isolation or alignment scheme that is chosen to decouple the electric field from the detector can have a dramatic effect on the efficiency and peak skew exhibited in a separation, as was recently demonstrated by Martin et al.32 The most common configuration for electrochemical detection is the end-channel configuration, as initially described for conventional CE in 1991.33 In this mode, the working electrode is aligned 10-20 µm from the end of the (21) Bruin, G. J. M. Electrophoresis 2000, 21, 3931-3951. (22) Becker, H.; Ga¨rtner, C. Electrophoresis 2000, 21, 12-26. (23) Dolnik, V.; Liu, S.; Jovanovich, S. Electrophoresis 2000, 21, 41-54. (24) McDonald, J. C.; Duffy, D. C.; Anderson, J. R.; Chiu, D. T.; Wu, H.; Schueller, O. J. A.; Whitesides, G. M. Electrophoresis 2000, 21, 27-40. (25) Swinney, K.; Markov, D.; Bornhop, D. J. Anal. Chem. 2000, 72, 26902695. (26) Schwarz, M. A.; Hauser, P. C. Lab Chip 2001, 1, 1-6. (27) Limbach, P. A.; Meng, Z. J. Analyst 2002, 127, 693-700. (28) Wightman, R. M. Anal. Chem. 1981, 53, 1125A-1134A. (29) Henry, C. S.; Zhong, M.; Lunte, S. M.; Kim, M.; Bau, H.; Santiago, J. Anal. Commun. 1999, 36, 305-308. (30) Baldwin, R. P.; Roussel, T. J. J.; Crain, M. M.; Bathlagunda, V.; Jackson, D. J.; Gullapalli, J.; Conklin, J. A.; Pai, R.; Naber, J. F.; Walsh, K. M.; Keynton, R. S. Anal. Chem. 2002, 74, 3690-3697. (31) Jackson, D. J.; Naber, J. F.; Roussel, T. J. J.; Crain, M. M.; Walsh, K. M.; Keynton, R. S.; Baldwin, R. P. Anal. Chem. 2003, 75, 3643-3649. (32) Martin, R. S.; Ratzlaff, K. L.; Huynh, B. H.; Lunte, S. M. Anal. Chem. 2002, 74, 1136-1143.

separation channel to enable the separation voltage to ground prior to reaching the working electrode. This has been the most frequently reported method for decoupling in microchip CEEC5,6,34,35 but leads to peak dispersion due to the laminar flow produced as the peak moves from the end of the channel to the large reservoir containing the working electrode. In the study by Martin et al., end-channel versus in-channel detection was compared by using an electrically isolated “floating” potentiostat. Because the potentiostat was electrically isolated, it allowed the working electrode to be placed in the separation channel. Compared to an end-channel alignment, placing the electrode in the separation channel resulted in a 4.6-fold increase in the number of theoretical plates and a 1.3-fold decrease in peak skew (signifying a more symmetrical peak) for catechol.32 This study showed that, in comparison to end-channel detection, placing the electrode in the channel minimizes peak dispersion and improves separation efficiencies. Off-channel detection for conventional CE with EC detection was initially described by Wallingford and Ewing.36 In this case, the working electrode is placed in the capillary and the separation voltage is grounded at a decoupler prior to reaching the working electrode. The decoupler usually consists of a small fracture in the separation capillary just prior to the working electrode. This fracture is placed in a reservoir and provides a path to ground. Several decouplers for microchip CE have been reported recently.37-40 Rossier et al. reported a microchip composed of two different polymers, poly(ethylene terephthalate) and polyethylene (PE).37 Holes were produced in the PE layer covering the channel, connecting the separation field to ground and preventing bulk fluid flow through the holes due to the hydrophobicity of PE. More recently Osbourn and Lunte used a CO2 laser to etch small scores in a glass coverslip.38 This glass coverslip was sealed on top of a piece of poly(dimethylsiloxane) (PDMS) containing the separation channel. The holes in the glass coverslip were then coated with cellulose acetate to ensure electrical conductivity and minimize sample leakage. Limits of detection for dopamine of 25 nM were obtained with this approach. The last decoupling methods for microchip CEEC that have been described employ a decoupling electrode in the separation channel, just prior to the working electrode.39,40 One of the drawbacks of this approach is that grounding the separation field at a cathode leads to the hydrolysis of H2O, causing the formation of hydrogen gas:

2H2O + 2e- f H2 + 2OH-

(1)

If an electrode is placed in the separation channel and the H2 is (33) Huang, X.; Zare, R. N.; Sloss, S.; Ewing, A. E. Anal. Chem. 1991, 63, 189192. (34) Martin, R. S.; Gawron, A. J.; Lunte, S. M. Anal. Chem. 2000, 72, 31963202. (35) Gawron, A. J.; Martin, R. S.; Lunte, S. M. Electrophoresis 2001, 22, 242248. (36) Wallingford, R. A.; Ewing, A. E. Anal. Chem. 1987, 59, 1762-1766. (37) Rossier, J. S.; Ferrigno, R.; Girault, H. H. J. Electroanal. Chem. 2000, 492, 15-22. (38) Osbourn, D. M.; Lunte, C. E. Anal. Chem. 2003, 75, 2710-2714. (39) Chen, D.-c.; Hsu, F.-L.; Zhan, D.-Z.; Chen, C.-h. Anal. Chem. 2001, 73, 758762. (40) Wu, C.-C.; Wu, R.-G.; Huang, J.-G.; Lin, Y.-C.; Chang, H.-C. Anal. Chem. 2003, 75, 947-952.

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not dissipated, a bubble will be produced in the channel and the separation will cease. Therefore, it is necessary to use a cathode material that has the ability to absorb H2. Metals from the platinum group have this capability and have been used in conventional and microchip CE.41 Palladium was first used as a decoupler for conventional CEEC in 1993 by Kok’s group.41 Palladium is able to absorb H2 produced at a cathode up to a Pd/H ratio of 0.6.41,42 Recently, a palladium decoupler for microchip CEEC was described by Chen et al.39 Because this group did not have access to microfabrication facilities, the dimensions of the electrodes and separation channels were large compared to most microfabricated systems. In particular, the device employed separation channels that were 200 µm × 75 µm along with a 3-mm Pd decoupler and a 1-mm working electrode (1 mm from the decoupler). Although the chip design was not optimal, it still demonstrated that the use of the decoupler led to lower limits of detection than end-channel detection. More recently, Wu et al. used platinum for the fabrication of a decoupler.40 However, platinum was shown to be effective for decoupling only at low separation field strengths (up to 90 V/cm), because it does not absorb H2 as efficiently as Pd. In this paper, we describe the fabrication and evaluation of a fully integrated palladium decoupler/electrochemical detector for microchip CEEC using a hybrid glass/PDMS device. This microchip system is amenable to mass production by traditional fabrication techniques. Both a Pd decoupler and working electrode are deposited onto a glass plate prior to reversibly sealing the glass layer to a PDMS substrate containing the separation channel network. The effect of the Pd decoupler size on the ability to decouple separation fields up to 1200 V/cm was investigated. The optimal distance between the decoupler and working electrode was also determined in terms of background noise and separation efficiency. The optimized configuration was evaluated with LIF detection to investigate the effect of the pressure-induced flow that predominates after the decoupler on the peak shape of fluorescein. To the best of our knowledge, this is the first in-depth report concerning the optimization of a metal-based decoupler for microchip CE with regard to electrode dimensions and distance from the working electrode. Finally, it was shown that, compared to what is typically obtained for an end-channel alignment, the optimized design leads to an improved separation for neurotransmitters. EXPERIMENTAL SECTION Chemicals and Materials. The following chemicals and materials were used as received: SU-8 10 photoresist and Nano SU-8 developer (MicroChem Corp., Newton, MA); AZ 1518 photoresist and 300 MIF developer (AZ Resist, Somerville, NJ); 100-mm Si wafers (Silicon, Inc., Boise, ID); Sylgard 184 (Ellsworth Adhesives, Germantown, WI); 1 N NaOH (Aldrich, Milwaukee, WI); boric acid, N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES), epinephrine, and dopamine (Sigma, St. Louis, MO); fluorescein disodium salt dihydrate (Kodak Eastman Fine Chemicals, Rochester, NY); 0.22-µm Teflon filters (Osmonics, Inc., Minnetonka, MN); 1-mL syringes (Becton-Dickenson, Franklin Lanes, NJ); acetone, isopropyl alcohol, 30% H2O2, H2SO4, HNO3, and HCl (Fisher Scientific); Pd (99.95% purity) and Ti (99.97% (41) Kok, W. T.; Sahin, Y. Anal. Chem. 1993, 65, 2497-2501. (42) Czerwinski, A. Pol. J. Chem. 1995, 69, 699-706.

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purity) targets (2-in. diameter × 0.125 in. thick; Kurt J. Lesker Co. Clairton, PA); Ti etchant (TFTN; Transene Co., Danvers, MA); single-strength glass (4 in. × 2 in. × 0.25 in.; Kennedy Glass, Lawrence, KS); Cu wire (22 gauge; Westlake Hardware, Lawrence, KS); silver colloidal paste (Ted Pella, Inc., Redding, CA); JB weld epoxy (J.B. Weld, Sulfur Springs, TX); NANOpure H2O (Labconco, Kansas City, MO); 10 kΩ resistor (Radio Shack, Lawrence, KS); and He gas (Airgas, Inc., Radnor, PA). PDMS Fabrication. PDMS channel structures were produced based on previously published methods.43,44 Briefly, masters for the production of PDMS microchip separation channels were made by coating a 100-mm silicon wafer with SU-8 10 negative photoresist using a spin coater (Brewer Science, Rolla, MO) operating with a spin program of 2000 rpm for 30 s. The photoresist was prebaked at 95 °C for 5 min prior to UV exposure (210 mJ/cm2) with a near-UV flood source (Autoflood 1000, Optical Associates, Milpitas, CA) through either a negative film (transparency; Jostens, Topeka, KS) or a negative photoplotted film (Laser Lab, San Diego, CA), which contained the desired channel structures. The transparency was made from a computer design drawn in Freehand (PC version 8.0, Macromedia, Inc. San Francisco, CA). This design was transferred onto a transparency using an image setter with a resolution of 2400 dpi by a printing service (Jostens). The photoplotted film was made from a computer design drawn using AutoCAD LT 2004 (Autodesk, Inc., San Rafael, CA). With this technique for making masks, we were able to obtain a resolution of 8000 dpi (with lines as small as 12.5 µm).45 Following this exposure, the wafer was postbaked at 95 °C for 5 min and developed in Nano SU-8 developer. The thickness of the photoresist was measured with a profilometer (Alpha Step200, Tencor Instruments, Mountain View, CA), which corresponded to the channel depth of the PDMS structures. A 10:1 mixture of Sylgard 184 elastomer (10.4 g) and curing agent (1.6 g) was poured onto the silicon wafer and cured at 70 °C for ∼2 h. These amounts were chosen so that surface tension held the mixture onto the wafer surface and no mold was needed. The PDMS layer was then pealed off the master. The average thickness (n ) 11) of the PDMS was 1.23 ( 0.15 mm. The separation channel designs are depicted in Figure 1A-C. Design A was used for the determination of effective decoupler size and proper working electrode spacing from the decoupler. Designs B and C were used for the separation aspects discussed in this work. Electrode Plate Fabrication. Single-strength glass was used as a substrate for the electrodes in these experiments. The glass was first cleaned with piranha solution (7:3 H2SO4/H2O2) for ∼30 min to remove organic impurities. The glass was rinsed thoroughly with NANOpure H2O and dried with N2. The glass was placed into a deposition system (Thin Film Deposition System, Kurt J. Lesker Co., Clairton, PA) for subsequent deposits of Ti (adhesion layer) and Pd (electrode layer). The thicknesses of the metals were monitored using a quartz crystal deposition monitor (Inficon XTM/2, Leybold Inficon, Syracuse, NY). Titanium was (43) Duffy, D. C.; McDonald, J. C.; Schueller, O. J. A.; Whitesides, G. M. Anal. Chem. 1998, 70, 4974-4984. (44) Duffy, D. C.; Gillis, H. L.; Lin, J.; Sheppard, N. F.; Kellogg, G. J. Anal. Chem. 1999, 71, 4669-4678. (45) Culbertson, C. T.; Tolley, L. T.; Ramsey, J. M.; Gonda, S. R. Presented at the 26th International Symposium on Capillary Chromatography and Electrophoresis, Las Vegas, NV, May 2003.

Figure 1. Microchip channel and electrode designs. For both designs A and B, the alignment mark is 1.2 mm from the end of the separation channel, the width of the channel is 30 µm, and the depth is 16.5 µm. The remaining channel and electrode dimensions are given in millimeters in the figure. (A) Chip used for Ohm’s law and noise studies; (B) chip used for separations with various electrode spacings; (C) chip used for separations using the optimized decoupler design; (D) decoupler with working electrode. The length of the decoupler varied throughout the study, with the optimized size being 500 µm; the length of the working electrode was 40 µm.

deposited from a Ti target at a rate of ∼2.3 Å/s to a depth of 200 Å. Palladium was deposited from a Pd target at a rate of ∼1.9 Å/s to a depth of 2000 Å. Positive resist (AZ 1518) was dynamically dispensed onto the metal-covered plate at a rate of 100 rpm for 20 s. The spin coater was then spun at 2000 rpm for 23 s. The photoresist was prebaked at 100 °C for 1 min prior to UV exposure through a positive transparency (Jostens). The electrode plates were developed in AZ 300 MIF developer. A postexposure bake at 100 °C for 1 min followed the developing step. The remaining photoresist protects the underlying metal, which will serve as the decoupler/working electrode, from the subsequent chemical etch. Aqua regia (8:7:1 H2O/HCl/HNO3) was used to etch excess Pd metal from the glass plate. Ti etchant (Transene) was used to remove the remaining Ti after the first etch step. The remaining photoresist was removed with acetone. The glass plate was then rinsed thoroughly with H2O and dried with N2. Cu wire was epoxied (J.B. Weld) onto the glass plates, and Ag colloidal paste (Ted Pella) was used to make contact between the electrodes and the Cu wire.

Chip Designs and Construction. As shown in Figure 1, three different channel designs were evaluated for this work. The designs included an alignment mark near the end of the channel. This mark was aligned with the bottom of the decoupler and ensured that a constant distance of 1.2 mm between the end of the decoupler and the beginning of the buffer waste reservoir was maintained. The microchip design shown in Figure 1A is just a single channel connected by two reservoirs. The length of this microchip is 30 mm. Parts B and C of Figure 1 show microchip designs employed for carrying out injections and separations. In Figure 1B, the length of the main channel in this case is 37.5 mm, with each of the side channels having a length of 7.5 mm, leading to an effective separation length of 30 mm. For Figure 1C, the length of the main channel in this case is 57.5 mm, with each of the side channels having a length of 7.5 mm, leading to an effective separation length of 50 mm. For Figure 1A-C, the channel width is 30 µm and the depth is 16.5 µm. Figure 1D shows the design of the decoupler and working electrode used in these experiments. For these experiments, several different sizes of Analytical Chemistry, Vol. 76, No. 9, May 1, 2004

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decoupler were used: 40, 200, 500, 800, and 1000 µm. A working electrode with a length of 40 µm was used for all experiments. The spacing between the working electrode and the decoupler was also varied. The distances between the decoupler and the working electrode studied in these experiments were 50, 70, 125, 250, 500, and 1000 µm. The separation channel layer (PDMS) was reversibly sealed to the glass electrode plate by bringing the two substrates into conformal contact with one another after extensive cleaning of the surface with IPA and drying with N2. The alignment mark included on the PDMS layer was easily aligned with the bottom of the decoupler with the aid of a light microscope. The effective size of the decoupler and working electrode was determined by the area where the separation channels intersected the electrodes. In the case of the optimized decoupler, the exposed surface area was 30 µm (width of separation channel) × 500 µm. For the working electrode, the exposed surface area was 30 µm × 40 µm. Electrophoresis Procedures. Separation voltage was applied using a Spellman CZE 1000R high-voltage power supply (Spellman High Voltage Electronics, Hauppauge, NY). Only one power supply was needed for design A (Figure 1A). The field strength was changed from 0 to 1200 V/cm in 100 V/cm increments. To measure the current produced during electrophoresis, a 10-kΩ resistor was placed in series between the cathode and grounding port. The voltage that passed across the resistor was measured with an analog-to-digital converter (DA-5, Bioanalytical Systems, West Lafayette, IN) and plotted with Chromgraph software (Bioanalytical Systems). The current was calculated from the measured voltage and known resistance using Ohm’s law and plotted versus field strength to obtain an Ohm’s law plot. Capillary zone electrophoresis separations were carried out using a gated injection scheme with two Spellman CZE 1000R high-voltage power supplies (design 1B and 1C). Gated injections were performed by applying a high voltage (1800 V) at the buffer reservoir (b, in Figure 1B or C) and a fraction of this high voltage (1435 V) at the sample reservoir (s), with the sample waste (sw) and buffer waste (bw) reservoirs being grounded. Injection was achieved by momentarily (1 s) floating the high voltage at the buffer reservoir. The CE buffers consisted of 25 mM boric acid, pH 9.2, or 25 mM TES, pH 7.5. The buffer was degassed daily by bubbling He gas through it and was also filtered with a 0.22-µm Teflon filter prior to being introduced into the microchip. The microchips were used without any preconditioning. Stock solutions of dopamine (10 mM) and epinephrine (10 mM) in water were prepared daily. The necessary dilutions were made in buffer prior to use. Electrochemical detection was accomplished using a potentiostat (LC-4CE, Bioanalytical Systems) and the aforementioned DA-5. The electrodes consisted of an integrated 40-µm Pd working electrode, an external Pt wire auxiliary electrode (22 gauge), and an external Ag/AgCl reference electrode (CH Instruments Inc., Austin, TX). Imaging. To study the flow profile that occurs around the decoupler, fluorescein (100 µM in 25 mM boric acid, pH 9.2) was placed in the sample reservoir (Figure 1C) and electrophoretically pumped down the separation channel at 1800 V. Imaging was accomplished by fluorescence microscopy using a Zeiss Axiolab microscope (Carl Zeiss, Thorngood, NY) that was equipped with a 50-W Hg arc lamp assembly and appropriate fluorescein line 2486

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filters. The images were captured by a Microimage Video Systems color video camera (A209, Boyertown, PA) with camera control unit (CCU 209, Microimage Video Systems), video capture card (CG-7 RGB color PC1, Scion Corp., Frederick, MD), and frame grabber software (Scion Image, Scion Corp.) coupled to a personal computer. Monitoring of the H2 absorption at the decoupler was accomplished with the imaging system described above except that no line filters were used. The chip (Figure 1A) and 25 mM boric acid, pH 9.2, were used, and the field strength was varied from 0 to 1000 V/cm in 200 V/cm increments. Bright-field images were captured for each field strength. LIF Experiments. A similar LIF detection setup was described earlier.32 Briefly, a beam from a 25-mW argon ion laser (Omnichrome, Chino, CA) was directed into an optics cube (C6W, Thor Labs, Newton, NJ) using three mirrors (BB1-E02, Thor Labs). The optics cube contained a dichroic mirror (505DRLP, Omega Optical, Brattleboro, VT) that directed the beam 90° into a long working distance objective (NA ) 0.6, 40× LWD Plan Fluorite LCP LFL; Olympus America, Melville, NY), which was translatable in the X-Y planes. This objective focused the beam to a spot size of ∼20 µm onto the separation channel of the microchip, which was secured on a X-Y-Z translatable stage. Fluorescence was collected with the same microscope objective and was passed through the dichroic mirror and reflected 90° with a mirror toward a photomultiplier tube (R1477, Hamamatsu, Bridgewater, NJ) contained in a tube housing (70680, Thermo Oriel, Stratford, CT). Spectral filtering and circular polarizing film (37% neutral; Edmund Scientific, Barrington, NJ) were used to decrease background. Specifically, a piece of polarizing film and a discriminating filter for the excitation light (497DF10, Omega Optical) were placed in the light path before the dichroic; after the fluorescence collection, a second piece of polarizing film and a discriminating filter for the fluorescence (520DF20, Omega Optical) were placed before the PMT. This second piece of polarizing film was rotated to further minimize the original laser light and thus reduce our background. Analog signal filtering was accomplished with the use of a Stanford Research Systems preamplifier (SR570, Sunnyvale, CA) and converted into digital output using a Chrom Perfect (Tigre III) data acquisition system from Justice Systems (40-5000-7202, Melbourne, FL). The A/D converter was interfaced to a PC with Chrom Perfect software (Justice Systems). Sample was injected for 0.5 s via a gated injection scheme using a Microfluidic Power Supply (model µF PSU, Jenway, Essex, England). The voltage program that was used for the electrochemical detection experiments was also used here. Data obtained during the LIF experiments were processed with the Chrom Perfect software (Justice Systems). This software package calculates the number of theoretical plates (N) in the conventional manner using the migration time and the width at half-height. Peak skew, which provides a measure of the asymmetry of a peak, was also calculated. This is defined by the software program as the ratio of the back width to the front width at 10% of the peak height so that a symmetrical peak has a skew of 1.0 and a tailing peak has a skew of >1.0. This formula for calculating peak skew is slightly different from that in the previous report of in-channel detection for microchip CEEC,32 so direct comparisons to those results are not valid.

RESULTS AND DISCUSSION Determination of Decoupler Size. To produce a reliable and reproducible microchip CEEC system, a decoupler capable of absorbing all of the hydrogen that is produced at the cathode must be fabricated. In a previous study by Chen et al., the authors developed a palladium decoupler that had an active cross-sectional area of 3 mm × 200 µm.39 This electrode was able to decouple field strengths up to 714 V/cm. There was no information given regarding the decoupler performance and lifetime with continuous use at this high-field strength. The Pd decoupler described here was evaluated for field strengths up to 1200 V/cm. Another important parameter to investigate is the separation efficiency and band broadening at the decoupler once the flow changes from electrokinetically driven flow to pressure-induced flow. It is well known that pressure-induced flow leads to a parabolic flow profile and increased band broadening in CE.46 In this work, a change of flow regimes could lead to band broadening from a number of possible factors including the parabolic flow profile, the fact that the pressure-induced flow does not have the same flow rate as electrokinetically driven flow, and back pressure from the length of channel past the decoupler.36 Therefore, to minimize the distance a separation band has to travel via pressureinduced flow before reaching the working electrode, the decoupler was made as small as possible but still large enough to effectively decouple the separation voltage and not lead to bubble formation in the channel. Five different decoupler widths (40, 200, 500, 800, and 1000 µm) were evaluated at voltages up to 1200 V/cm. An Ohm’s law plot (current vs field strength) was constructed by varying the field strength and measuring the resulting current. Two different buffer systems, boric acid (25 mM, pH 9.2) and TES (25 mM, pH 7.5), were studied to ensure that the decoupler system was compatible with different buffers and pH values. The chip design depicted in Figure 1A was utilized in these studies. For comparison purposes, conventional platinum wire electrodes were first tested. The platinum wire placed in the inlet reservoir was connected to the high-voltage supply, and a platinum wire connected to ground was placed in the outlet reservoir. The field strength was varied from 0 to 1200 V/cm. The data obtained from 0 to 600 V/cm were found to be very linear (r2 > 0.999) and were used to plot a bestfit line. Figure 2A shows the Ohm’s plots obtained for the boric acid buffer system. With the conventional Pt wire electrodes at field strengths above 600 V/cm there is a deviation from the bestfit line, signifying Joule heating. Next, the Pd decouplers were evaluated by connecting the ground electrode to the decoupler electrode, instead of placing it in the outlet reservoir. The Ohm’s plots obtained for all of the decoupler widths using the boric acid buffer system are also shown in Figure 2A. The 40-µm-wide decoupler functioned linearly up to a field strength of 600 V/cm before bubble formation in the channel occurred. The other decoupler sizes (200, 500, 800, and 1000 µm) functioned up to field strengths of 1200 V/cm without bubble formation. As was witnessed with a conventional platinum electrode setup, deviation from linearity was evident with all of the decoupler widths at separation field strengths above 600 V/cm, signifying Joule heating. (46) Paul, P. H.; Garguilo, M. G.; Rakestraw, D. J. Anal. Chem. 1998, 70, 24592467.

Figure 2. Ohm’s plot obtained using a conventional ground ([), a 40 (+), 200 (9), 500 (2), 800 (×), and 1000 µm (b) decoupler. (A) 25 mM boric acid buffer, pH 9.2; (B) 25 mM TES buffer, pH 7.5.

Figure 2B shows the Ohm’s plots obtained with TES as the buffer system. With TES, the 40-µm decoupler functioned up to 1000 V/cm before bubble formation was seen. In this case, the zwitterionic TES buffer exhibited a lower current,47 which allows the 40-µm decoupler to function at higher field strengths. Once again, the other decouplers, which are wider, were better at decoupling the separation field and functioned up to 1200 V/cm. Deviation from linearity was evident with all of the decouplers at separation field strengths above 600 V/cm, as was the case with the boric acid buffer system. It is possible to visually monitor the absorption of H2 by the Pd decoupler as shown in Figure 3. The H2 absorbs into the thin Pd film, forming a cone shape that gets larger as the separation field strength is increased. It is very interesting to note that the decoupler reverts back to its native state after the voltage is shut off. Most likely the H2 dissipates through the gas-permeable PDMS layer.43,44 Alternatively, it could dissipate through the rest of the Pd film. The final experiment that was performed to determine the optimum width for the Pd decoupler was to determine the lifetime of the decoupler. These tests were carried out to ascertain how long a decoupler could function at a constant current and before bubble formation was observed at a constant field strength of 600 V/cm (limit of linearity before Joule heating becomes a factor). The chip design (Figure 1A) used to obtain the Ohm’s plots was also used in this study. The separation voltage was continuously applied, with buffer being changed every 15 min to minimize changes in the buffer composition. It was found that the 40-µm decoupler functioned for 2 h, while the 200-µm decoupler functioned for ∼3 h before bubbles were produced. The 500- and 800-µm decouplers both functioned for ∼6 h before bubble formation occurred. Since the goal of this work was a rugged and reproducible decoupler, the 500-µm decoupler was chosen as the optimum decoupler width for subsequent work. The dimensions of the separation channel may also play an important role in the operation of the Pd decoupler. To test this (47) Zhou, J.; Lunte, S. M. Electrophoresis 1995, 16, 498-503.

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Figure 3. Bright-field images taken at different separation field strengths showing the absorption of H2 at the Pd decoupler using chip C. The arrow in the first image shows the location of the microchannel; the Pd decoupler is perpendicular to the microchannel and is metallic in color. In each case, a dashed line has been added to aid in visualization of the absorption.

hypothesis, two different masks were used for the fabrication of PDMS masters. Masters made from a lower resolution transparency led to PDMS channels 65 µm wide and 16.5 µm deep. Masters fabricated by higher resolution photoplotting films afforded PDMS channels that were 30 µm wide and 16.5 µm deep. All of the other studies described in this paper utilized masters made from the photoplotted films. As is commonly known in CE, smaller channels are better able to dissipate current.33 When a lower current is obtained, less H2 should be produced from hydrolysis, and given a constant size, a given decoupler should perform longer with smaller channels. This was indeed the case, as the 200-µm decoupler functions at 1200 V/cm with the photoplotted channels using both buffer systems, but with the transparency-defined channels, the decoupler functions only until 200 V/cm using boric acid and 500 V/cm using TES. Overall, performance was substantially improved by reducing the current present at the decoupler by having narrower separation channels. Since the current is higher for boric acid than that of TES, boric acid was used for the rest of the experiments. Boric acid will provide a better example for general decoupler applications although better results can be expected using a lower current buffer such as TES. Determination of Optimal Spacing between the Decoupler and the Working Electrode. Once the optimum decoupler size was determined, the proper spacing between the decoupler and the working electrode was investigated. This determination is important, as the closer the working electrode is placed to the decoupler, the more noise will be observed.39 Conversely, once pressure-induced flow dominates (after the grounding takes place), resolution between analytes with similar migration times can be lost due to band broadening from the parabolic flow. Therefore, in terms of resolution, it is advantageous to place the working electrode as close as possible to the decoupler. The effect of the spacing on both the background noise and resolution of two closely eluting peaks was used to determine the optimum spacing distance. The distance between the bottom end of the decoupler and top of the working electrode was varied from 50, 70, 125, 250, 2488 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004

Figure 4. Peak-to-peak noise as a function of distance of the working electrode from the decoupler with different separation field strengths, 300 (2) and 500 V/cm (9). An enlarged region of the graph is inset to show how background noises continued to be reduced at the larger decoupler/working electrode spacing.

500, and 1000 µm. A standard three-electrode electrochemical cell was used to monitor the peak-to-peak noise levels at the working electrode. A potentiostat was used to apply +500 mV (vs Ag/ AgCl) to the working electrode. The noise levels were determined for each of the different electrode spacings at both 300 and 500 V/cm using the 25 mM boric acid buffer system. The peak-topeak noise values obtained with both 300 and 500 V/cm are shown in Figure 4. Peak noise is ∼400 pA with a 50-µm spacing, decreasing to 14 pA for 250-µm spacing, and finally to a value of less than 1 pA for the noise with a spacing of 1000 µm. As expected, the noise decreased as the spacing between the working electrode and the decoupler increased. Increasing the spacing between the decoupler and the working electrode did substantially reduce noise at the working electrode. However, as a consequence of the pressure-induced flow that occurs after the decoupler, this loss of noise could also be accompanied by a loss in resolution. The catecholamine neurotransmitters dopamine and epinephrine were employed as model compounds to determine the effect of electrode spacing on the resolution of two closely resolved analytes. Hydrodynamic voltammograms for both compounds were obtained using the 250µm spacing (data not shown). It was found that both compounds

Figure 5. Electrochemical detection using a palladium working electrode. The sample was injected via a gated injection scheme (B, +1800 V; S, +1535 V) for 1.0 s. Eapp ) +600 mV (vs Ag/AgCl). Concentration of dopamine and epinephrine is 200 µM. The decoupler width is 500 µm, while the working electrode width is 40 µm. (A) Effect of decoupler/working electrode spacing on peak shape and resolution using the separation channel design depicted in Figure 1B; (B) separation of dopamine and epinephrine with 250-µm spacing using the separation channel design depicted in Figure 1C.

exhibited a maximum current response at an oxidation potential of +600 mV (vs Ag/AgCl) using the Pd working electrode. The potential used for dopamine is similar to that employed for oncapillary detection in conventional CEEC.48 This implies that the working electrode is effectively isolated from the separation field. Incomplete isolation of the separation field is an issue that is often encountered with end-channel detection schemes.32 On the basis of findings from the noise study, three different microchips were fabricated with different working electrode and decoupler spacings (250, 500, and 1000 µm) to study the effect of spacing on resolution using the microchip separation channel design depicted in Figure 1B. Sample was injected for 1 s via a gated injection scheme to yield the electropherograms overlaid in Figure 5A. It is very evident in this figure that resolution between the two closely related neurotransmitters diminishes as the working electrode is moved farther away from the decoupler. From these studies it was concluded that a 250-µm spacing between the bottom of the decoupler and the working electrode gave the best compromise of noise and resolution. This spacing was used in subsequent studies. Optimized Decoupler and Working Electrode Spacing. LIF detection was used to study the band broadening that occurs after the decoupler with the optimized decoupler design (500-µm decoupler, 250-µm spacing) and the separation channel design depicted in Figure 1C, using 25 mM boric acid, pH 9.2. Three different detection points were used, with the laser beam being focused 200 µm prior to the decoupler, 30 µm after the decoupler, and 30 µm after the working electrode. For this study, 10 µM fluorescein was injected via a gated detection scheme by floating the high voltage to the buffer reservoir for 0.5 s. At this pH, fluorescein is doubly negative charged; therefore, in terms of band broadening, it ensures a challenging test of the system, as its low

electrophoretic mobility leads to longer migration times and a greater amount of band broadening due to molecular diffusion.49 Figure 6A shows overlaid electropherograms for each of the different laser spot alignments. There are only small differences between the electropherograms, with only slight changes in peak height, peak width, and migration time observed. Table 1 lists the values obtained for migration time, peak width, peak skew, peak height, and number of theoretical plates. Inspection of these values shows a general trend of a slight increase in peak width and a slight decrease in peak height. Considering these data, it seems that the 250-µm spacing may be the maximum distance that can be used to separate and detect two analytes with very similar migration times. Adequate resolution will only become more difficult the further the working electrode is placed from the decoupler. The increase in migration with detection distance shown in Figure 6A implies that either the pressure-induced flow is slower than electrokinetically driven flow or that back pressure arising from the length of channel after the decoupler may be playing a role. The migration time of fluorescein detected after the working electrode is drastically increased compared to when the flow is transitional between electrokinetically driven and pressure driven (Table 1). In addition, in both Figures 4 and 5A, it is possible to see the reduction in noise as the working electrode is moved farther away from the electrode. Therefore, with the Pd decoupler, there is a tradeoff between noise and resolution, so it is important to determine which is more important for a given application before designing the microchip. Finally, there did not appear to be any visual change in the fluid flow once the separation voltage was grounded (Figure 6B). It is possible that as one switches from electrically driven to pressure-induced flow, a vortex or turbulent flow could result. Figure 6B shows that this is not the case here.

(48) Wallenborg, S. R.; Nyholm, L.; Lunte, C. E. Anal. Chem. 1999, 71, 544549.

(49) Weinberger, R. Practical Capillary Electrophoresis, 2nd ed.; Academic Press: San Diego, CA, 2000.

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Figure 6. Characterization of the device with fluorescein using chip C. (A) LIF detection of 10 µM fluorescein at various points around the decoupler. The sample was injected via a gated injection scheme (B, +1800 V; S, +1535 V) for 0.5 s; (B) fluorescence image depicting flow profile. Table 1. Separation Performance for Fluorescein at Various Points around the Decoupler (n ) 3) placement of beam

av migration time (s)

av width (50% height) (s)

av skew (10% height) (s)

av height (AFU)

av no. of plates

200 µm before decoupler 30 µm after decoupler 30 µm after electrode

92.90 ( 0.38 93.73 ( 0.54 99.87 ( 1.27

2.28 ( 0.01 2.35 ( 0.01 2.49 ( 0.03

1.34 ( 0.07 1.36 ( 0.10 1.30 ( 0.01

39.2 ( 0.3 40.8 ( 1.9 32.7 ( 1.3

9174 ( 37 8779 ( 69 8916 ( 14

Low noise is important, but having good resolution is critical when studying closely related neurotransmitters, as shown in Figure 5B. A microchip with the optimized decoupler size (500 µm) and electrode spacing (250 µm) was used to determine the limits of detection for two closely eluting compounds, dopamine and epinephrine. The longer separation channel depicted in Figure 1C was used to improve resolution between the neurotransmitters. Using a Pd decoupler and a 40-µm Pd working electrode resulted in LODs, sensitivities, and linearities for dopamine of 500 nM, 3.5 pA/ µM, and r2 ) 0.9996 (5-500 µM), respectively. The slightly later eluting epinephrine exhibited LODs, sensitivities, and linearities of 2.1 µM, 2.6 pA/ µM, and r2 ) 0.9996 (5-500 µM), respectively. The values obtained for the LODs and sensitivities were much higher than expected considering the low noise levels that are obtained with this decoupled system. However, this is most likely due to the use of Pd as a working electrode, which is not optimal for the detection of neurotransmitters. Electrode materials such as platinum or carbon have been most commonly employed for the determination of neurotransmitters.5 Efficiencies obtained for the separation shown in Figure 5B are 12 400 (248 000/m) for dopamine and 13 700 (274 000/m) for epinephrine. These values 2490 Analytical Chemistry, Vol. 76, No. 9, May 1, 2004

begin to rival the performance of all glass microchip CEEC devices (N for dopamine ) 21 000)50 and are impressive when one considers the fact that these separations are in PDMS-based devices. Furthermore, these separations were made by diluting concentrated stock solutions (10 mM) with run buffer, so that the ionic strengths of the buffer and sample are essentially matched; therefore, no type of sample stacking is occurring in these devices, as has been used previously.34,35 CONCLUSIONS The fabrication and optimization of a fully integrated palladium decoupler/electrochemical detector for microchip CEEC has been described. This decoupler allows the working electrode to be placed directly in the separation channel and improves the separation performance of the hybrid glass/PDMS device. Furthermore, the device is fabricated with lithographic procedures, is amenable to mass production, and avoids the specialized nature of many decouplers described for conventional CEEC. A detailed study to determine the smallest but most effective decoupler size was performed, and the minimal distance necessary for the spacing between the decoupler and working electrode was (50) Woolley, A. T.; Lao, K.; Glazer, A. N.; Mathies, R. A. Anal. Chem. 1998, 70, 684-688.

determined. Studies with LIF detection showed that the optimized designs do not significantly affect the separation performance achievable by the device. Overall, it has been shown that this approach to electrochemical detection for microchip CE improves what typically is obtained with an end-channel alignment. Since palladium is not an optimal working electrode material, LODs for dopamine and epinephrine were higher than expected, especially considering the low noise levels. To provide better detection performance, future work will focus on integrating a more useful working electrode material, such as platinum or carbon, with the palladium decoupler. Once this is accomplished, these microchip devices will be used for monitoring neuropeptide transport and metabolism in cell culture systems.

ACKNOWLEDGMENT The authors thank Bryan Huynh for his assistance with the LIF experiments and Professor Chris Culbertson (Kansas State University) for his advice on the use of photoplotted films for fabricating PDMS masters. This research was supported by grants from the National Science Foundation (CHE-0111618) and the National Institutes of Health (RO1 NS42929), as well as a National Cancer Institute Training Grant (N.A.L.; T32 CA09242). We also thank Nancy Harmony for her assistance in the preparation of the manuscript. Received for review September 10, 2003. Accepted January 5, 2004. AC030327T

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