Fully Microfabricated and Integrated SU-8-Based Capillary

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Anal. Chem. 2007, 79, 9135-9144

Fully Microfabricated and Integrated SU-8-Based Capillary Electrophoresis-Electrospray Ionization Microchips for Mass Spectrometry Tiina Sikanen,†,‡ Santeri Tuomikoski,§ Raimo A. Ketola,‡,⊥ Risto Kostiainen,‡ Sami Franssila,§ and Tapio Kotiaho*,†,‡

Laboratory of Analytical Chemistry, Department of Chemistry, P.O. Box 55, FI-00014 University of Helsinki, Finland, Division of Pharmaceutical Chemistry, Faculty of Pharmacy, P.O. Box 56, FI-00014 University of Helsinki, Finland, Micro and Nanosciences Laboratory, Department of Electrical and Communications Engineering, P.O. Box 3500, FI-02015 Helsinki University of Technology, Finland, and Drug Discovery and Development Technology Center, P.O. Box 56, FI-00014 University of Helsinki, Finland

We present a fully microfabricated and monolithically integrated capillary electrophoresis (CE)-electrospray ionization (ESI) chip for coupling with high-throughput mass spectrometric (MS) analysis. The chips are fabricated fully of a negative photoresist SU-8 by a standard lithographic process which enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, reproducible analytical performance from chip to chip. As the coaxial sheath flow interface is patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free. No significant peak broadening occurs so that very narrow peak widths (down to 2-3 s) are obtained. The sheath flow interface also enables comprehensive optimization of both the CE and the ESI conditions separately so that the same chip design is adaptable to diverse analytical conditions. Plate numbers of the order of 105 m-1 and good resolution are routinely reached for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. In addition, a limit of detection of 100 nM corresponding to a total amount of only 4.5 amol (per injection volume of 45 pL) and excellent quantitative linearity (R2 ) 0.9999; 100 nM to 100 µM) were obtained in small-molecule analysis using verapamil as a test compound. The quantitative repeatability was proven good (8.5-21.4% relative standard deviation, peak area) also for the other drug substances and peptides tested. Electrospray ionization (ESI)1-3 mass spectrometry (MS) provides a universal and highly sensitive detection tool, especially * Corresponding author. E-mail: [email protected]. Phone: 358-919159168. Fax: 358-9-19159556. † Laboratory of Analytical Chemistry, Department of Chemistry, University of Helsinki. ‡ Division of Pharmaceutical Chemistry, University of Helsinki. § Micro and Nanosciences Laboratory, Department of Electrical and Communications Engineering, Helsinki University of Technology. ⊥ Drug Discovery and Development Technology Center, University of Helsinki. (1) Dole, M.; Mach, L. L.; Hines, R. L.; Mobley, R. C.; Ferguson, L. P.; Alice, M. B. J. Chem. Phys. 1968, 49, 2240-2249. (2) Yamashita, M.; Fenn, J. B. J. Chem. Phys. 1984, 88, 4451-4459. 10.1021/ac071531+ CCC: $37.00 Published on Web 10/31/2007

© 2007 American Chemical Society

for bioanalysis. In recent years, numerous chip-based designs4-6 have been presented for miniaturization of the ESI source in order to reach more efficient ionization through nanospray.7,8 Most importantly, nanospray ionization provides not only increased sensitivity but also direct coupling with microfluidic separation devices due to congruent flow rates between the two. Chip-based ESI sources have been implemented on various materials, including silicon, glass, and polymers.4-6 Depending on the material of choice, many different emitter designs have been proposed, yet the advantages of sharp-pointed emitter tips are widely acknowledged in order to avoid sample spillage and to enable small Taylor cone volume and high electric field strength at the tip. The first fully microfabricated systems integrating liquid chromatographic (LC) separation and ESI source on a single chip have already been published9,10 and commercialized.10 However, the combination of chip-based capillary electrophoresis (CE) and ESI remains challenging in order to provide similarly reproducible systems with respect to fabricational as well as analytical properties. The most well-established fabrication techniques are available for silicon processing so that sharp-featured silicon ESI emitters can be realized in both in-plane11-13 and out-of-plane14,15 direction. (3) Alexandrov, M. L.; Gall, L. N.; Krasnov, N. V.; Nikolaev, V. I.; Pavlenko, V. A.; Shrukov, V. A. Dokl. Akad. Nauk SSSR 1984, 277, 379-383. (4) Foret, F.; Kusy´, P. Electrophoresis 2006, 27, 4877-4887. (5) Lazar, I. M.; Grym, J.; Foret, F. Mass Spectrom. Rev. 2006, 25, 573-594. (6) Sung, W. C.; Makamba, H.; Chen, S. H. Electrophoresis 2005, 26, 17831791. (7) Wilm, M. S.; Mann, M. Int. J. Mass Spectrom. Ion Processes 1994, 136, 167180. (8) Wilm, M. S.; Mann, M. Anal. Chem. 1996, 68, 1-8. (9) Xie, J.; Miao, Y.; Shih, J.; Tai, Y. C.; Lee, T. D. Anal. Chem. 2005, 77, 69476953. (10) Yin, H.; Killeen, K.; Brennen, R.; Sobek, D.; Werlich, M.; van der Goor, T. Anal. Chem. 2005, 77, 527-533. (11) Sainiemi, L.; Nissila¨, T.; Jokinen, V.; Sikanen, T.; Kotiaho, T.; Kostiainen, R.; Ketola, R. A.; Franssila, S. Sens. Actuators, B, in press, 2007. (12) Legrand, B.; Ashcroft, A. E.; Buichaillot, L.; Arscott, S. J. Micromech. Microeng. 2007, 17, 509-514. (13) Kim, W.; Guo, M.; Yang, P.; Wang, D. Anal. Chem. 2007, 79, 3703-3707. (14) Schultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063. (15) Sjo ¨dahl, J.; Melin, J.; Griss, P.; Emmer, Å.; Stemme, G.; Roeraade, J. Rapid Commun. Mass Spectrom. 2003, 17, 337-341.

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Silicon emitters have also been arrayed16 or integrated with a silicon micropillar pattern11,17 to enable on-chip LC separation. However, the use of semiconductive silicon devices is unfeasible in applications requiring high internal potential differences, such as CE. In contrast to silicon, glass microdevices enable highly efficient CE separations18,19 but suffer from relatively tedious processing required for realization of sharp-featured ESI emitters. Thus, the first CE-ESI chips were fabricated of glass and operated by direct spraying from the microchannel outlet on a planar chip edge.20,21 Unfortunately, the hydrophilic nature of glass subjects this approach to large Taylor cone volumes and sample spillage at the microchannel outletsand results in loss of the previously achieved separation. The wettability of glass can be reduced, for instance, through application of hydrophobic coatings around the planar spraying orifice,21 yet the best performance has been achieved by coupling the glass-based separation devices with MS through transfer capillaries or nanospray needles.5,6 Nevertheless, integration of an external ESI emitter requires manual postprocessing in addition to microfabrication and may easily result in large dead volumes and reduced separation efficiency. Recently, Hoffmann et al.22 published a glass-based CE-ESI chip with a monolithically integrated, sharp-featured nanospray emitter. This improved, dead-volume-free design was postprocessed from a commercial Borofloat glass chip by milling a cone structure to the end of a CE channel. In the next step, the cone was drawn to a sharp tip shape by methods adapted from the fabrication of conventional nanospray needles. Although complete separation of some common pharmaceuticals was accomplished through sheathless ESI/MS detection, the chips had to be postprocessed one-by-one so that the processing cycle time was over 10 min for each tip. Polymer microfabrication technology at its best enables batchwise patterning of sharp emitter structures as an integral part of separation microdevices. In comparison to silicon and glass processing, patterning of polymer substrates is relatively rapid and inexpensive. Although various polymer-based ESI emitter designs have been described,4-6 only a few have been successfully coupled with on-chip separation.23 Instead, also polymer separation chips are often described with external spraying capillaries and needles. This is because enclosed, accurately defined, and sharpfeatured emitter structures can be achieved with only a few polymer microfabrication methods. For instance, sharp-featured emitters with enclosed fluidic connections have been realized using poly(dimethylsiloxane) (PDMS),24 parylene,25 parylene and (16) Dethy, J. M.; Ackermann, B. L.; Delatour, C.; Henion, J. D.; Schultz, G. A. Anal. Chem. 2003, 75, 805-811. (17) Eghbali, H.; De Malsche, W.; Clicq, D.; Gardeniers, H.; Desmet, G. LC‚GC Eur. 2007, 20, 208-222. (18) Manz, A.; Harrison, D. J.; Verpoorte, E. M. J.; Fettinger, J. C.; Paulus, A.; Lu ¨ di, H.; Widmer, H. M. J. Chromatogr. 1992, 593, 253-258. (19) Jacobson, S. C.; Culbertson, C. T.; Daler, J. E.; Ramsey, M. Anal. Chem. 1998, 70, 3476-3480. (20) Ramsey, R. S; Ramsey, J. M. Anal. Chem. 1997, 69, 1174-1178. (21) Xue, Q.; Foret, F.; Dunayevski, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (22) Hoffmann, P.; Ha¨usig, U.; Schulze, P.; Belder, D. Angew. Chem., Int. Ed. 2007, 46, 4913-4916. (23) Thorslund, S.; Lindberg, P.; Andre´n, P. E.; Nikolajeff, F.; Bergquist, J. Electrophoresis 2005, 26, 4674-4683. (24) Kim, J. S.; Knapp, D. R. J. Am. Soc. Mass Spectrom. 2001, 12, 463-469.

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cyclo-olefin copolymer,26 or polyimide and poly(ethyleneterephthalate) (PET).27 In addition, lidless polymer emitters have been described with negative photoresist SU-828 or PET29 as structural material. Although successful in direct infusion ESI/MS experiments, none of these emitter designs was integrated with on-chip separation. Thorslund et al.23 have realized a sheathless CE-ESI interface in PDMS that consists of a chip-based CE unit integrated with a graphite-coated PDMS emitter.30 In addition, a two-dimensional solid-phase extraction unit was integrated with the PDMS CEESI chip by the same group.31 Unfortunately, hydrophobic PDMS suffers from a high degree of nonspecific adsorption as well as limited chemical stability in organic solvents, which easily gives rise to abundant interfering background ions originating from the polymer.32 Moreover, accurate PDMS emitters are somewhat difficult to realize batchwise and the surface charge on native PDMS surfaces is often prone to aging effects33,34 so that surface modification is required for stable electroosmotic flow (EOF). We have recently introduced a three-layered, fully SU-8-based ESI emitter design incorporating a fluidic inlet, an enclosed microchannel, and a sharp-pointed emitter tip.35,36 Our approach for aligning of SU-8 layers exploits lithographic accuracy and results in accurately defined emitter structures. In addition, we have characterized the analytical performance of SU-8 microchannels for chip-based CE with fluorescence detection.37,38 Both fabricational26,35,39-42 and analytical37,38 advantages of SU-8 as a structural material in microfluidic devices are renowned. Briefly, SU-8 microstructures of almost any desired shape and size can be fabricated by standard UV lithography, and more complex, multilayered structures through bonding. SU-8 is also a chemically stable material with favorable, glasslike surface properties with (25) Licklider, L.; Wang, X. Q.; Desai, A.; Tai, Y. C.; Lee, T. D. Anal. Chem. 2000, 72, 367-375. (26) Kameoka, J.; Craighead, H. G.; Zhang, H.; Henion, J. Anal. Chem. 2001, 73, 1935-1941. (27) Gobry, V.; van Oostrum, J.; Martinelli, M.; Rohner, T. C.; Reymond, F.; Rossier, J. S.; Girault, H. H. Proteomics 2002, 2, 405-412. (28) Arscott, S.; LeGac, S.; Druon, C.; Tabourier, P.; Rolando, C. J. Micromech. Microeng. 2004, 14, 310-316. (29) Ek, P.; Sjo ¨dahl, J.; Roeraade, J. Rapid Commun. Mass Spectrom. 2006, 20, 3176-3182. (30) Dahlin, A. P.; Wetterfall, M.; Liljegren, G.; Bergstro ¨m, S.; Andre´n, P.; Nyholm, L.; Markides, K. E.; Bergquist, J. Analyst 2005, 130, 193-199. (31) Dahlin, A. P.; Bergstro ¨m, S. K.; Andre´n, P. E.; Markides, K.; Bergquist, J. Anal. Chem. 2005, 77, 5356-5363. (32) Huikko, K.; O ¨ stman, P.; Grigoras, K.; Tuomikoski, S.; Tiainen, V. M.; Soininen, A.; Puolanne, K.; Manz, A.; Franssila, S.; Kostiainen, R.; Kotiaho, T. Lab Chip 2003, 3, 67-72. (33) Kim, M. S.; Cho, S. I.; Lee, K. N.; Kim, Y. K. Sens. Actuators, B 2005, 107, 818-824. (34) Ren, X.; Bachman, M.; Sims, C.; Li, G. P.; Allbritton, N. J. Chromatogr., B 2001, 762, 117. (35) Tuomikoski, S.; Sikanen, T.; Ketola, R. A.; Kostiainen, R.; Kotiaho, T.; Franssila, S. Electrophoresis 2005, 26, 4691-4702. (36) Sikanen, T.; Tuomikoski, S.; Ketola, R. A.; Kostiainen, R.; Franssila, S.; Kotiaho, T. J. Mass Spectrom., submitted for publication. (37) Sikanen, T.; Tuomikoski, S.; Ketola, R. A.; Kostiainen, R.; Franssila, S.; Kotiaho, T. Lab Chip 2005, 5, 888-896. (38) Sikanen, T.; Heikkila¨, L.; Tuomikoski, S.; Ketola, R. A.; Kostiainen, R.; Franssila, S.; Kotiaho, T. Anal. Chem., 2007, 79, 6255-6263. (39) Tuomikoski, S.; Franssila, S. Sens. Actuators, A 2005, 120, 408-415. (40) Jackman, R.; Floyd, T.; Ghodssi, R.; Schmidt, M.; Jensen, K. J. Micromech. Microeng. 2001, 11, 263-269. (41) Blanco, F. J.; Agirregabiria, M.; Garcia, J.; Berganzo, J.; Tijero, M.; Arroyo, M. T.; Ruano, J. M.; Aramburu, I.; Mayora, K. J. Micromech. Microeng. 2004, 14, 1047-1056. (42) Zhang, J.; Tan, K. L.; Gong, H. Q. Polym. Test. 2001, 20, 693-701.

Figure 1. Schematic diagram of the CE-ESI microchip (dimensions not to scale): the three structural SU-8 layers in the patterning order (left), the ready-made microchip released from the silicon substrate and flipped over (middle), and the unilateral chip design accompanied by typical voltage arrangements during injection and separation steps (right). The separation voltages given for the separation correspond to electric field strength of 1000 V/cm. The flow streams during injection and separation steps are indicated with arrows (right).

respect to surface charge and EOF.37 In addition to ESI emitters and CE microchannels, SU-8 has been used for realization of pumps, valves, and filters42 as well as monolith filled LC microchannels.43 In this study, we present two fully microfabricated and monolithically integrated CE-ESI chip designs that are capable for high-throughput MS analysis and hold repeatable analytical properties from run to run. The CE-ESI chips presented here are fabricated fully of negative photoresist SU-8 by a standard lithographic process. The presented fabrication process enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, with reproducible analytical performance from chip to chip. Furthermore, the SU-8 chips are ready for use directly after microfabrication without manual postprocessing or surface treatment and can be easily filled by capillary forces before use. The presented SU-8 chip design enables high separation efficiency for small molecules and peptides within a typical cycle time of only 30-90 s. EXPERIMENTAL SECTION Materials and Reagents. SU-8 negative photoresist (SU-8 50, Microchem Corporation, Newton, MA) and SU-8 developer (mrDev 600) were purchased from Micro Resist Technologies GmbH (Berlin, Germany). Single-side polished silicon wafers with 〈100〉 orientation (Okmetic, Vantaa, Finland) were used as substrates for SU-8 structures. The thickness and diameter of the silicon wafers were 625 ( 25 µm and 100 mm, respectively. Hydrogen fluoride (50%, VLSI Selectipur grade, Merck, Darmstadt, Germany) was used for wet etching of the silicon oxide, and 2-propanol (VLSI Selectipur grade, BASF Electronic Materials GmbH, Ludwigshafen, Germany) was used for rinsing of the SU-8 chips. Tetraethylammonium iodide was purchased from SigmaAldrich (St. Louis, MO), and tetrapropylammonium iodide, tetrabutylammonium iodide, and tetrapentylammonium iodide were from Fluka (Buchs SG, Switzerland). Tetrahexylammonium bromide and phenazon (antipyrine) were purchased from Aldrich Chemical (Milwaukee, WI), and verapamil hydrochloride and metoprolol were from ICN Biomedicals (Aurora, OH). Ranitidine, angiotensin I, angiotensin II, substance P, horse heart myoglobin (HHM), fluorescein isothiocyanate, and 1-methoxy-2-propyl acetate (PMA) were from Sigma-Aldrich (Steinheim, Germany). Methanol and acetic acid were from J. T. Baker (Deventer, Holland), and (43) Carlier, J.; Arscott, S.; Thomy, V.; Fourrier, J. C.; Caron, F.; Camart, J. C.; Druon, C.; Tabourier, P. J. Micromech. Microeng. 2004, 14, 619-624.

ammonium acetate was from Merck. Water was purified with a Milli-Q water purification system (Millipore, Molsheim, France). All reagents and solvents were of analytical or HPLC grade. All solutions were filtered (0.2 µm) and degassed by sonication for 10 min before use. Microchip Fabrication. Monolithically integrated CE-ESI microchips were fabricated fully of negative photoresist SU-8 using thermally oxidized silicon wafers (oxide thickness 1.0 µm) as carrier substrates in the fabrication process. At the end of the fabrication process, the silicon wafer is released providing freestanding, fully SU-8 microchips that are ready for use directly after the microfabrication. The fabrication process is based on the methods we have earlier described for fabrication of microfluidic channels38,39 and for sharp ESI emitters.35 The three structural SU-8 layers and the final microdevice are shown schematically in Figure 1. First, a 70 µm thick SU-8 layer was spin-coated on top of the silicon wafer and processed according to the standard SU-8 fabrication parameters. Inlets of the microfluidic chip (2 mm in diameter) were patterned to this first layer. The second SU-8 layer was spin-coated on top of the first one and patterned to form the microchannel walls. The thickness of the second layer and thus the microchannel depth was 50 µm. After postexposure bake of the second layer, the first two layers were both developed simultaneously. Next, a 70 µm thick SU-8 layer was first applied on top of a 100 µm thick, flexible polymer substrate followed by bonding with the previously patterned SU-8 layers. The bonding process was similar as earlier described by Tuomikoski and Franssila,39 except that in this study the Pyrex bonding substrate was replaced by a flexible polymer sheet and the bonding temperature was decreased from 68 to 63 °C. Exposure of the third SU-8 layer was done through the transparent polymer substrate. After postexposure bake of the third SU-8 layer, the polymer substrate was peeled away and the third SU-8 layer was developed. In addition to inlets and microchannel pattern, all three SU-8 layers were UV exposed to tip shape and aligned using a LOMO EM-5006 mask aligner. In the final step, the microchips were released from the silicon substrate by sacrificial etching of the oxide layer and rinsed with deionized water and 2-propanol. A typical etching time was approximately 2 h for the current chip design having a 2 cm separation channel. In this study, two different microchip layouts were used. Both layouts had a 25 mm long separation channel intersected by a 10 mm long injection channel through a simple cross intersection so that the effective separation length was 20 mm. In addition, Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 2. (A) Micrograph of the bilateral chip design at the emitter tip area with two auxiliary channels intersecting the separation channel on both sides. (B) A scanning electron micrograph of the accurately defined emitter tip of an SU-8 CE-ESI microchip.

the first (unilateral) layout had a 10 mm long auxiliary channel intersecting the separation channel just before the ESI tip (Figure 1), whereas the second (bilateral) layout had two congruent auxiliary channels intersecting the separation channel on both sides (Figure 2A). The microchannel cross-sectional dimensions were 30 µm × 50 µm (w × h) and 100 µm × 50 µm (w × h) for the separation and auxiliary channels, respectively. The distance between the intersection and the tip apex was approximately 800 µm. The microchannel dimensions at the tip area after intersection were 100 µm × 50 µm (w × h) or 150 µm × 50 µm (w × h) for the unilateral or the bilateral design, respectively. Mass Spectrometry. The SU-8 microchips were coupled to an API3000 triple-quadrupole mass spectrometer (Perkin-Elmer Sciex, Toronto, Canada) using a modified nanospray source (Proxeon Biosystems, Odense, Denmark), equipped with an xyzaligning stage and CCD cameras. The instrument was operated in positive ion mode using nitrogen generated by a Whatman 75720 nitrogen generator (Haverhill, MA) as curtain gas. An external power supply (Micralyne Inc., Edmonton, Canada) was used to apply the separation and ES voltages, as specified. The voltages were applied through external platinum wires placed to the liquidfilled inlets. Before use, a block of PDMS (Sylgard 184, Dow Corning, Midland, MI) with 2 mm inlet holes was attached on top of an SU-8 chip to increase the inlet volume. A solution of methanol/ water/acetic acid 80/19/1 was fed as sheath liquid through the auxiliary channel(s) to the separation buffer just before the ES tip. The ES voltage of +3500 V, corresponding +2500 V relative to the MS, was provided through the sheath liquid flow so that the same voltage also served as counter voltage for the CE separation (Figure 1). The distance between the emitter tip and the MS sampling orifice was typically between 4 and 8 mm. The samples were introduced electrokinetically (20.0-30.0 s) through the simple cross injector in semipinched injection mode, i.e., injection voltages of +800 and +600 V were applied to the injection and separation channel inlets, respectively, while the injection channel outlet was grounded (Figure 1). The sheath flow inlet was left floating during the injection. The CE separations were performed in cathodic mode using separation voltages between +4500 and +5000 V corresponding to electric field strengths 9138

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between 500 and 1000 V/cm (with respect to the ES voltage). In addition, small antileakage voltages were applied to the injection channel ends (Figure 1). The separation current was typically between 10 and 15 µA and was divided at the intersection into the electrospray (100-300 nA) and the auxiliary channel. The excess current was led to ground from the auxiliary channel through a 100 MΩ resistor coupled in parallel with the ES voltage supply. Both a current monitoring feature of the power supply and an additional amperometer coupled in series with the resistor were employed to monitor the currents. Data were recorded using either a full-scan MS mode with a dwell time of 250 ms per scan or selected reaction monitoring (SRM) in MS/MS mode with a dwell time of 150 ms per selected precursor/product ion pair. The detection sensitivity was measured for verapamil using SRM with two precursor/product ion pairs (m/z 455.4/165.2 and m/z 455.4/303.2) and a dwell time of 200 ms per selected pair. Fluorescence Microscopy. An Eclipse TE300 inverted microscope equipped with a TE-FM epifluorescence attachment (Nikon Instruments, Badhoevedorp, The Netherlands) was used for fluorescence imaging of the intersection of the separation and auxiliary channels. To visualize the flows, fluorescein isothiocyanate (200 µM) was added to the separation buffer, while a grounded aluminum plate served as the counter electrode instead of MS. Otherwise, the separation and ES conditions and voltages were the same as in MS measurements. The fluorescent micrographs were captured using a CoolSNAP-Pro color CCD camera (CHEOS, Espoo, Finland) and an exposure time of 4 s. RESULTS AND DISCUSSION Microchip Fabrication. The SU-8 microchips presented here were fabricated with a three-layer fabrication process so that the microchannels were implemented in the middle layer (Figure 1). As the inlets are patterned directly on the silicon substrate in the first SU-8 layer, developer or non-cross-linked SU-8 cannot enter the enclosed microchannels after bonding of the third layer and no prolonged development times are needed for SU-8 residual removal. The same process also enables patterning of several (n > 3) SU-8 layers and, thus, fabrication of ever more complex chip designs with multilevel functions.

The free-standing SU-8 microchips used in this study were achieved by releasing the SU-8 structures from the silicon substrate by sacrificial oxide etching. After etching, the released silicon wafers can be reused for another process cycle so that the fabrication costs are due SU-8 alone and, thus, remarkably low. Furthermore, no special equipment is required besides a standard mask aligner and the total fabrication cycle time is relatively fast thanks to the simple lithographic process. With the use of the current CE-ESI chip design, 12 microchips are obtained from one 4 in. wafer, with an average yield of >90%. In addition, several wafers can be processed simultaneously in the lithographic process. Both the structural complexity and the ease of batch fabrication attainable through SU-8 processing are the most distinct advantages over previously published fabrication procedures of CEESI microchips. In our design, no manual postprocessing is required and all fluidic connections and microchannel intersections are patterned with lithographic accuracy and, thus, without hidden dead volumes. Moreover, the SU-8 fabrication process presented here enables not only complex fluidic designs but also straightforward integration of the accurately defined, sharp-pointed emitter tip directly at the end of the separation channel (Figure 2B). The alignment accuracy for the three SU-8 layers is based on lithography and is therefore within a few micrometers for all structures. Well-controlled geometry at the emitter tip area is especially crucial for stable and reproducible ESI/MS performance from chip to chip. In our earlier study,36 we have shown that runto-run repeatabilities of 4-11% relative standard deviation (RSD) and chip-to-chip repeatability of 14% RSD can be reached in smallmolecule analysis using SU-8-based direct infusion ESI emitters that resemble the tip structure implemented on the current CEESI chip design. Fluidic Design. Owing to the similarity of flow rates feasible for microfluidic separation devices and those providing optimal sensitivity in nanospray ionization, the coupling of the two is often considered relatively straightforward. However, establishment of an adequate potential difference to control EOF and to reach sufficient separation efficiency is not a trivial task. The common solution is twofold: the counter potential for the separation is either applied through a liquid junction interface44-46 or by integrating the counter electrode in the end of the microchannel.23,30,31 The former solution is more widely applied and typically accomplished through external sprayers attached to the microchannel outlet. This inevitably generates dead volume at the interface area and may result in peak broadening. Integrated electrodes for the latter solution, in turn, are often more difficult to realize and suffer from limited lifetime. In this study, electric field for the CE separation was established by applying the potential difference between the separation channel inlet (anode) and the sheath flow inlet (cathode). Such configuration resembled a conventional coaxial sheath flow interface47 except that the sheath flow channel (i.e., the auxiliary (44) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 32583264. (45) Deng, Y.; Henion, J.; Li, J.; Thibault, P.; Wang, C.; Harrison, J. Anal. Chem. 2001, 73, 639-646. (46) Tachibana, Y.; Otsuka, K.; Terabe, S.; Arai, A.; Suzuki, K.; Nakamura, S. J. Chromatogr., A 2003, 1011, 181-192. (47) Smith, R. D.; Olivares, J. A.; Nguyen, N. T.; Udseth, H. R. Anal. Chem. 1988, 60, 436-441.

channel) was patterned as an integral part of the SU-8 chip. Owing to high fabricational accuracy, the on-chip interface was deadvolume-free (Figure 2A) and likely to have negligible contribution to peak broadening. Somewhat similar CE-ESI chip designs with on-chip coaxial sheath flow channels have been earlier reported by Zhang et al.44 and Razunguzwa et al.48 However, their CEESI chips were realized in glass and necessitated the use of nebulizer gas44 or an external spraying capillary48 in order to achieve efficient ionization from a planar chip edge. In addition to highly accurate and straightforward processing, SU-8 polymer is a useful material due to its favorable, glasslike surface properties. We have earlier shown that fully SU-8 microchannels provide high cathodic EOF, on the average 80-90% of that of glass,37,38 and hold repeatable EO properties as well as fairly low degree of surface interactions compared to many other polymers. However, at acidic pH, a relatively exceptional switch in the SU-8 surface charge occurs from negative to positive with a pI value of 3-4. Here, we took advantage of such surface chemistry in order to introduce the sheath liquid flow electrokinetically to the ESI emitter tip. Commonly, the EOF in the negatively charged auxiliary channels is toward the inletsnot the tipsaccording to the applied electric field. This easily leads to sample leakage from the intersection area into the auxiliary channel after separation. The problem is typically solved by eliminating the surface charge through coating of the auxiliary channel, for instance, with polyacrylamide.20,44 In the case of SU-8, no such coating was required, since the cathodic EOF could be eliminated and even reversed by simply adjusting the sheath liquid pH below 3. In this study, a methanol/ water/acetic acid 80/19/1 solution was used to evoke anodic EOF in the auxiliary channel, albeit, both the hydrodynamic flow and the electrospray aspiration were also contributing to the overall flow rate in the auxiliary channel. However, the contribution of these pressure-induced flows to peak broadening in the separation channel was considered insignificant as the fluidic resistance of the separation channel was nearly 7-fold to that of the auxiliary channel. Thus, the flow rate in the separation channel was solely determined by the applied electric field strength and the buffer composition. For instance, using a solution of methanol/water 50/ 50 with 20 mM ammonium acetate and electric field strength of 500 V/cm, the average flow rate was 24 nL/min determined by the migration rate of an uncharged analyte (i.e., phenazon having a pKa value of 1.4). On the basis of our earlier experience,36 the average flow rate in the auxiliary channel was approximately 300 nL/min measured with an SU-8 emitter design resembling the tip structure used in the current CE-ESI chip. To demonstrate the efficiency of the presented chip design, two different layouts were tested: a unilateral chip design that had one auxiliary channel and a bilateral chip design that had two auxiliary channels intersecting the separation channel just before the ESI tip. As a result, the separation performance of both designs was shown to be somewhat similar so that five different tetraalkylammonium halides were well resolved within 30-40 s (Figure 3, parts A and B). The resolution was 0.9-1.8 or 0.8-2.0, and the measured plate numbers were (0.69-4.0) × 104/m or (4.2-6.2) × 104/m in CE-ESI separations corresponding to (48) Razunguzwa, T. T.; Lenke, J.; Timpermann, A. T. Lab Chip 2005, 5, 851855.

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Figure 3. (A and B) Separation of five tetraalkylammonium halides (12 µM each) and (C and D) confluence of the separation buffer and sheath liquid flows at the interface of (A and C) a bilateral and (B and D) a unilateral SU-8 CE-ESI chip. The separation buffer was methanol/ water/1-methoxy-2-propyl acetate 50/45/5 with 20 mM ammonium acetate, and the injected amount of tetraalkylammonium halides was 540 amol (Vinj ) 45 pL). The applied electric field strength during separation was 1000 V/cm. The detection was performed (A and B) in full-scan MS mode between m/z 120 and 700 or (C and D) through fluorescence microscope with 200 µM fluorescein isothiocyanate added in the separation buffer. The microchannel walls are indicated with white lines in parts C and D.

Figure 3, parts A and B, respectively. The peak widths at baseline were extremely narrow, between 2.4 and 6.0 s, for both designs. Thus, no significant peak broadening occurs that might be attributed to the introduction of the sheath flow in general terms, although in case of tetrahexylammonium (C6H13)4N+ a somewhat broader peak was obtained using the bilateral than the unilateral chip design (Figure 3, parts A and B). In addition, we examined the ionization efficiency by determining the total ion current (TIC) stabilities under various separation conditions. In our earlier study, TIC stabilities as low as 2% RSD could be maintained in direct infusion ESI/MS for timescales of 30 min36 using methanol/water/acetic acid 80/19/1 solutions and SU-8 emitters of similar shape as the ESI tip in the current CEESI design. In this study, the same solution was constantly used as sheath liquid composition with the same applied ES voltage (+2.5 kV), but the separation buffer composition was altered. As the TIC stability is largely dependent on the total surface tension and viscosity of the solution to be electrosprayed, the separation buffer composition was also shown to affect the stability when the two flows merged at the ESI tip. With the use of the unilateral chip design, the ion current (in)stability was shown to decrease fairly linearly from 22.8% to 5.2% when the amount of organic 9140 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

solvent (methanol) in the separation buffer was increased from 30% to 70%. This indicated that a certain amount of organic solvent has to be added to the separation buffer for efficient ionization. However, the amount of organic solvent will also affect the separation efficiency and has to be adaptable toward purely aqueous conditions. For such purposes, we developed the bilateral chip design. With the use of this design, good TIC stability of 10.8% was obtained even with purely aqueous separation solutions (i.e., no added methanol in the separation buffer) indicating more efficient confluence of the separation buffer and sheath flow. A CE-ESI/MS analysis under these purely aqueous conditions is demonstrated later on in Figure 5B. In conclusion, as the separation buffer and sheath liquid are both laminar flows, only diffusive mixing takes places at the intersection as shown by fluorescence micrographs in Figure 3, parts C and D. Thus, no significant sample dilution or peak broadening occurs due to introduction of the sheath flow through the auxiliary channel(s). However, the organic sheath flow enhances the ES ionization process, especially when aqueous separation buffers are used, and also provides means for establishing an adequate potential difference for the CE separation.

Figure 4. (A) A CE separation of three drug substances (100 µM metoprolol, verapamil, and phenazon) measured with the unilateral SU-8 CE-ESI chip design and (B-D) the mass spectra corresponding to the observed peaks. The separation buffer was methanol/water/1-methoxy2-propyl acetate 50/45/5 with 20 mM ammonium acetate, and the injected amounts of drugs were 4.5 fmol (Vinj ) 45 pL). The applied electric field strength during separation was 500 V/cm. The detection was performed in full-scan MS mode between m/z 180 and 465.

Figure 5. (A) Six consecutive CE separations of three peptide standards (250 µM substance P, angiotensin I, and angiotensin II) under optimized conditions in methanol/water 60/40 with 20 mM ammonium acetate. (B) CE separation of the same peptides in purely aqueous 20 mM ammonium acetate. The injected amount of peptides was 11.3 fmol (Vinj ) 45 pL). The separations were performed using (A) the unilateral or (B) the bilateral SU-8 chip design. The applied electric field strength during separation was (A) 1000 or (B) 500 V/cm. The MS/MS detection was performed in SRM mode with one precursor/product ion pair per peptide.

Analysis of Drugs and Biomolecules. The performance of SU-8 microchips was demonstrated in the CE-ESI/MS analysis of selected drug substances (small molecules), peptides, and a protein. The separation conditions were optimized with respect to separation voltage and the separation buffer composition, while the sheath liquid composition was the same in all analysis, i.e., methanol/water/acetic acid 80/19/1. The CE-ESI/MS analyses under optimized conditions are presented for the drugs, peptides, and the protein in Figures 4A, 5A, and 6A, respectively. For both chip designs tested, the injection volume was determined by the microchannel geometry at the simple cross intersection. Defined by the intersecting injection and separation channels, both 30 µm × 50 µm (w × h), the volume of the sample plug was as low as 45 pL constituting only 0.15% of the effective

separation channel volume (30 nL). Owing to this minute injection volume, the sample consumption in CE-ESI/MS analysis was in the low-femtomole range for all analytes, more precisely 4.5, 11.3, and 3.2 fmol for the drug substances, the peptides, and the protein, respectively. The total amount of sample solution applied to the sample inlet was typically 2.0-5.0 µL per analysis. However, several consecutive measurements could be performed with only a single sample application, as shown for the peptide analysis in Figure 5A. Both the selected drug substances (metoprolol, verapamil, and phenazon) and the selected peptide standards (substance P (111), angiotensin I, and angiotensin II) were well resolved within 60-80 s under optimized conditions. The drug substances were detected as protonated molecules [M + H]+ in full-scan MS mode, Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

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Figure 6. (A) Total ion chromatogram (TIC) of the HHM (1 mg/mL) analysis and (B) the corresponding mass spectrum at 1.31 min after injection. The injected amount of HHM was 45 pg corresponding to 3.2 fmol (Vinj ) 45 pL). The separation buffer was methanol/water/1methoxy-2-propyl acetate 50/45/5 with 1% acetic acid, and the applied electric field strength 500 V/cm. The detection was performed in full-scan MS mode between m/z 800 and 1600.

whereas SRM was used for detection of the peptides with doubly protonated molecules [M + 2H]2+ as precursor ions. The drug substances were most efficiently ionized and sufficiently resolved with elevated (50%) methanol concentrations in the separation buffer (Figure 4A-D). In peptide analysis, most efficient ionization was observed from purely aqueous separation buffer, though at the cost of the overall resolving power (Figure 5B). As a compromise, a higher (60%) methanol concentration in the separation buffer was considered to provide optimal conditions for the peptide separation, i.e., high resolution (RS values of 3.3 and 2.0) and a moderate ionization efficiency with signal-to-noise ratio (S/N) between 93 and 132 (Figure 5A). To demonstrate the feasibility of SU-8 microchips for protein analysis, a CE-ESI/MS analysis was carried out on a low molecular weight protein, horse heart myoglobin (HHM, MW 16 953.0 Da). In our earlier study, we have shown that unwanted adsorption of proteins on SU-8 walls during CE experiments can be reduced by adding a small amount of commercial SU-8 developer to the separation buffer.38 The developer is a watersoluble, relatively inert solvent and tailored for use in SU-8 fabrication to dissolve un-cross-linked resin. In this study, we used the developer base, i.e., PMA, instead of the commercial developer for elimination of surface interactions. Although the same result can likewise be reached with other commonly used dynamic modifiers, such sodium dodecyl sulfate (SDS) or Tween 20,38 PMA was considered more appropriate for MS analysis due to its lower surface activity and lesser impact on the electrospray process. Thus, PMA was added to the separation buffer to a final concentration of 5% to reduce the adsorption of HHM on SU-8 walls. As a result, HHM was eluted as a nicely symmetric peak (AS ) 0.83) with a migration time of 1.31 min under acidified separation conditions with negligible EOF (Figure 6A). With a view of demonstrating that the use of PMA will not deteriorate the separation efficiency, a similar PMA concentration (5%) was also employed in the analysis of tetraalkylammonium halides (Figure 3, parts A and B) and drugs (Figure 4A). As there were no significant interactions between SU-8 walls and the selected small molecules, addition of PMA did not affect the peak shapes. The resolving power was also unchanged indicating that PMA does not significantly interact with the analytes as such, but rather with the SU-8 surface. 9142 Analytical Chemistry, Vol. 79, No. 23, December 1, 2007

Analytical Performance and Repeatability. The analytical performance of chip-based separation devices is often compromised due to limited control of external pressure effects and injection anomalies49 or poorly understood surface properties, such as nonspecific adsorption or origin of the EOF in the case of polymer materials.50 The SU-8 chip presented here enables good control on the surface properties as well as comprehensive optimization of both the CE separation and ES ionization conditions separately thanks to the integral sheath flow interface. These are the most distinct advantages over the previously published, monolithically integrated sheathless CE-ESI interfaces implemented either on glass22 or PDMS.23 With these CE-ESI microchips, the separation voltage as well as the separation buffer composition can only be altered within the limits that provide sufficient ES stability and ionization efficiency. However, baseline resolution of some common pharmaceuticals has been demonstrated with the glass-based CE-ESI microchip.22 Zhang et al.44 have described a glass chip incorporating an integrated planar-edged nebulizer as well as an on-chip sheath flow interface. In their work, plate numbers of 7.0 × 104 m-1 were obtained within a total analysis time of 4 min using a 10 cm long separation channel. In addition, efficient CE separations with plate numbers >105 m-1 have been demonstrated with a few microchips that employ external emitter needles or transfer capillaries for ESI/MS coupling.26,44,51-53 However, manual postprocessing required for the integration of needles and capillaries renders these systems somewhat different from the fully microfabricated and monolithically integrated CE-ESI chip design presented here and, thus, less attractive from the viewpoint of batch fabrication. Moreover, the microdevices with transfer capillaries44,52 make use of further separation occurring “off-chip” in the long capillary. Here, plate numbers as high as 0.4-1.2 × 105 m-1 were obtained within a total analysis time of only 30-90 s and a 2 cm long separation channel. Moreover, the repeatability of the CEESI/MS analysis of drugs and peptides (Figures 4A and 5A) was (49) Crabtree, H. J.; Cheong, E. C. S.; Tilroe, D. A.; Backhouse, C. J. Anal. Chem. 2001, 73, 4079-4086. (50) Beattie, J. K. Lab Chip 2006, 6, 1409-1411. (51) Li, J.; Thibault, P.; Bings, N. H.; Skinner, C. D.; Wang, C.; Colyer, C.; Harrison, J. Anal. Chem. 1999, 71, 3036-3045. (52) Li, J.; Kelly, J. F.; Chernushevich, I.; Harrison, J. D.; Thibault, P. Anal. Chem. 2000, 72, 599-609. (53) Zhang, B.; Foret, F.; Karger, B. L. Anal. Chem. 2000, 72, 1015-1022.

Table 1. Analytical Parameters Determined for the Repeated CE Separations of the Drug Substances (n ) 4) and the Peptide Standards (n ) 6) Presented in Figures 4A and 5A, Respectively drug substance [M + H]+

migration time (tR), s (% RSD)

peak width (wh), s

metoprolol (268.2) verapamil (455.4) phenazon (189.4)

38.8 (10.0%)

4.2 ( 0.7

43.8 (10.0%)

4.5 ( 1.0

75.5 (10.3%)

7.2 ( 1.1

peptide (precursor/product ion)

migration time (tR), s (% RSD)

peak width (wh), s

substance P (1-11) (674.5/600.6) angiotensin I (649.0/269.2) angiotensin II (523.9/263.3)

30.5 (4.3%)

2.5 ( 0.2

45.8 (8.1%)

2.7 ( 0.2

55.4 (8.1%)

2.7 ( 0.3

resolution (RS)a 1.2 ( 0.3 5.5 ( 0.3

resolution (RS) 3.3 ( 0.7 2.0 ( 0.2

plate number (N), m-1 b (0.72 ( 0.25) × 105 (0.84 ( 0.34) × 105 (0.92 ( 0.28) × 105

peak area, counts (% RSD)c 5.37 × 107 (8.7%) 2.13 × 108 (8.5%) 9.60 × 107 (21.4%)

plate number (N), m-1

peak area, counts (% RSD)d

(0.41 ( 0.06) × 105

1.19 × 105 (10.9%)

(0.83 ( 0.18) × 105 (1.2 ( 0.2) × 105

2.98 × 105 (10.5%)

a Calculated using peak widths at baseline (w ) according to R ) 2(t b b S R2 - tR1)/(wb1 + wb2). Calculated using peak width at half-height (wh) according to N ) 5.545(tR/wh)2 (separation path 2 cm). c Calculated using ranitidine [M + H]+ at m/z 315.1, comigrating with metoprolol, as an internal standard. d Calculated using angiotensin I as an internal standard for substance P (1-11) and angiotensin II.

proven good as described in Table 1. The peak widths were between 2.5 and 7.2 s repeatedly from run to run, and the migration time repeatabilities of 4.3-10.3% were obtained without any internal calibration. Moreover, thanks to the narrow and symmetric peak shape, the separation resolution was generally good (RS 1.2-5.5). The detection sensitivity of the chip-based CE-ESI/MS was determined using the unilateral chip design. The limit of detection (LOD) was measured with verapamil injections under SRM using two selected precursor/product ion pairs of m/z 455.4 f 165.2 and 455.4 f 303.2. The selectivity of the method was first examined through the analysis of blank samples after which the measurements were repeated 3-4 times at each concentration level between 50 nM and 100 µM verapamil in methanol/water 40/60 with 20 mM ammonium acetate. The measured LOD (S/N ) 3) was found to be approximately 100 nM corresponding to a total amount of only 4.5 amol of verapamil in the injected volume of 45 pL. The quantitative linearity of the chip-based CE-ESI/ MS was determined with verapamil injections at a concentration range of 100 nM to 100 µM under SRM. As a result, an excellent correlation coefficient of R2 ) 0.9999 was obtained establishing good linearity over a range of 3 orders of magnitude. These results are fairly similar (with respect to sample concentration) to those previously reported for other chip-based CE-ESI44 and LC-ESI54 interfaces. However, here the concentration level of 10-4 M was reached with a 10-fold smaller injection volume than used, for instance, by Zhang et al.44 Most importantly, the attomole detection level was reached without any sample concentration that automatically occurs during LC analysis. Moreover, the same LOD and similar quantitative linearity were reached with three individual SU-8 chips indicating good reproducibility and feasibility for quantitative CE-ESI/MS analysis. The quantitative repeatability (peak area) was separately measured for the three drug substances (Figure 4) in full-scan (54) Mandel, F.; Coˆte´, L.; Vollmer, M. Determination of Low Femtogram Drug Levels In Serum By HPLC-Chip/MS. Proceedings of the 53rd ASMS Conference on Mass Spectrometry and Allied Topics, San Antonio, TX, June 5-9, 2005.

MS mode at concentration level of 100 µM. For this purpose, another drug substance, i.e., ranitidine [M + H]+ at m/z 315.1, comigrating with metoprolol was added as an internal standard to the sample solution to a final concentration of 100 µM. The repeatabilities (peak area) were calculated from the extracted ion chromatograms for repeated measurements (n ) 4) and were 8.7%, 8.5%, and 21.4% for metoprolol, verapamil, and phenazon, respectively (Table 1). Similarly, the quantitative repeatability (peak area) was measured for six consecutive separations of the three peptides (substance P, angiotensin I, and angiotensin II) in SRM mode (Figure 5B). The repeatabilities (peak area) were calculated using angiotensin I, middle in the migration order, as an internal standard for substance P and angiotensin II. The values were accordingly 10.9% for substance P and 10.5% for angiotensin II. These results suggest good quantitative repeatability in both full-scan MS and SRM modes and are consistent with those of the previously published CE-ESI chips,51,52,55 most of which employ external needles/capillaries. CONCLUSIONS In this study, fully microfabricated and monolithically integrated CE-ESI interfaces were developed using a standard lithography process to pattern three-layered, free-standing SU-8 chips. The chips were coupled to an MS to enable high-throughput analysis of small molecules and peptides. As a result, very narrow peak widths (down to 2-3 s), plate numbers of the order of 105 m-1, and high resolution were routinely obtained for small molecules and peptides within a 2 cm separation length and a typical cycle time of only 30-90 s per sample. The feasibility of the SU-8 chip for protein analysis was also demonstrated. As the sheath flow ESI interface was patterned as an integral part of the SU-8 chip, the fluidic design is dead-volume-free so that peak broadening during analyses is negligible. In addition, the presented chip design enables comprehensive optimization of both the CE separation and the ES ionization conditions separately and (55) Vrouwe, E. X.; Gysler, J.; Tjaden, U. R.; van der Greef, J. Rapid Commun. Mass Spectrom. 2000, 14, 1682-1688.

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can thereby be adapted to CE-ESI/MS analysis of diverse samples, even under purely aqueous separation conditions. Both the analytical and quantitative repeatabilities from run to run were also proven good with LOD in the low attomole level for small molecules. In addition, the presented fabrication process enables straightforward batch fabrication of multiple chips with precisely controlled dimensions and, thus, of reproducible analytical performance from chip to chip. Moreover, the same process enables

instance, on-chip sample preparation units can be integrated with the current CE-ESI/MS design. ACKNOWLEDGMENT The authors gratefully acknowledge the Academy of Finland (project 211019), University of Helsinki Research Funds, and the Finnish Cultural Foundation for financial support of the work.

patterning of several (n > 3) SU-8 layers and, thus, fabrication of

Received for review July 19, 2007. Accepted September 25, 2007.

more complex chip designs with multilevel functions so that, for

AC071531+

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