Anal. Chem. 2005, 77, 527-533
Microfluidic Chip for Peptide Analysis with an Integrated HPLC Column, Sample Enrichment Column, and Nanoelectrospray Tip Hongfeng Yin,* Kevin Killeen, Reid Brennen, Dan Sobek, Mark Werlich,† and Tom van de Goor†
Agilent Laboratories, 3500 Deer Creek Road, Palo Alto, California 94304 and 5301 Stevens Creek Boulevard, Santa Clara, California 95051-7201
Current nano-LC/MS systems require the use of an enrichment column, a separation column, a nanospray tip, and the fittings needed to connect these parts together. In this paper, we present a microfabricated approach to nano-LC, which integrates these components on a single LC chip, eliminating the need for conventional LC connections. The chip was fabricated by laminating polyimide films with laser-ablated channels, ports, and frit structures. The enrichment and separation columns were packed using conventional reversed-phase chromatography particles. A face-seal rotary valve provided a means for switching between sample loading and separation configurations with minimum dead and delay volumes while allowing high-pressure operation. The LC chip and valve assembly were mounted within a custom electrospray source on an ion-trap mass spectrometer. The overall system performance was demonstrated through reversed-phase gradient separations of tryptic protein digests at flow rates between 100 and 400 nL/min. Microfluidic integration of the nano-LC components enabled separations with subfemtomole detection sensitivity, minimal carryover, and robust and stable electrospray throughout the LC solvent gradient. Recent advances in proteomic research have brought increasing demands on the sensitivity, throughput, and robustness of instrumentation for protein analysis. Despite the complexity of its instrumentation, ultrasmall-bore liquid chromatography coupled to nanoelectrospray mass spectrometry (i.e., nano-LC/MS) is the technology of choice for analyzing proteomic samples. In addition, typical nano-LC protocols incorporate an enrichment/desalting column for preconcentrating large sample volumes diluted in saline solutions. The best mass spectrometer detection sensitivity is achieved at flow rates below 300 nL/min; however, the use of such low flow rates exacerbates delay volume issues and complicates the troubleshooting of leakage and flow blockage problems at the flow connections. In addition, dead volumes downstream from the LC column contribute to band broadening. Even dead volumes in a traditional “zero dead volume” connector can * To whom correspondence should be addressed. E-mail: hongfeng_yin@ agilent.com, fax: (650) 485-7839. † Santa Clara. 10.1021/ac049068d CCC: $30.25 Published on Web 12/09/2004
© 2005 American Chemical Society
be significant when the peak LC elution volume is smaller than 100 nL. Typical proteomic samples from 2-D gel spots are usually diluted to low concentrations in microliter amounts of solvents. Employment of an enrichment column in conjunction with a sixport rotary valve allows injection of such samples at flow rates up to 20 µL/min. Nevertheless, the incorporation of a valve and an enrichment column into a nano-LC system greatly increases the number of connections and the potential for flow leak and blockage problems. In the past few years, researchers have tried to address the above-described problems through advances in miniaturization technologies using different approaches. The most critical microfluidic device elements for a LC chip MS system are the on-chip liquid phase separation columns and the nanoelectrospray interface to the mass spectrometer. Initial work on miniaturized devices coupled to MS did not include chromatography, but instead, focused only on the use of a microfabricated device to perform sample infusion with ESI-MS from a flat device surface.1,2 Later designs focused on microdevices with fine nanoelectrospray tips. Several groups have reported the use of preformed fused-silica nanoelectrospray tips glued to the glass microdevices.3-7 To address the critical alignment and dead volume issues between the nanoelectrospray tip and the separation channel, as well as chemical background noise caused by the glue in the joints, a liquid junction interface between the nanoelectrospray tip and separation channel has also been developed.8 There have also been developments on microfabricated electrospray tips and coupling of such tips to micromachined microfluidic structures. One way of microfabricating the tip is through deep silicon etching, creating an array of electrospray tips perpendicular to the plane of the silicon surface.9,10 Alternatively, surface micro(1) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Zavracky, P. M.; McGruer, N. E.; Karger, B. L. Anal. Chem. 1997, 69, 426-430. (2) Ramsey R. S.; Ramsey, J. M. Anal. Chem. 1997, 69, 1174-1178. (3) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 32583264. (4) Xiang, F.; Lin, Y. H.; Wen, J.; Matson, D. W.; Smith, R. D. Anal. Chem. 1999, 71, 1485-1490. (5) Figeys, D.; Ning, Y.; Aebersold, R. Anal. Chem. 1997, 69, 3153-3160. (6) Lazar, I. M.; Ramsey, R. S.; Sundberg, S.; Ramsey, J. M. Anal. Chem. 1999, 71, 3627-3631. (7) Li, J.; Thibault, P.; Bing, N.; Skinner, C. D.; Wang, C.; Coyer, C.; Harrison, J. Anal. Chem. 1999, 71, 3036-3045. (8) Zhang, B.; Foret, F.; Karger, B. L. Anal. Chem. 2000, 72, 1015-1022. (9) Shultz, G. A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063.
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machining has been used, as well, to create a parylene tip.11 The integrated tip in another microfabricated polymer device was mechanically machined.12 The majority of research of on-chip separation technologies has focused on electrophoretic over chromatographic separations. Electroosmotic flow as well as electrophoretic migration can be controlled by the application of electrical voltages across the terminals of microchannels.13 The advances in such microfluidic technologies allow flow switching between cross-interconnected channels by adjusting electrical potentials at various channel terminals. Chromatographic separations have been electrokinetically driven14,15 but for such a device to function, the LC stationary phase must serve as a generator of the electrokinetic flow by having a high surface charge, yet at the same time must be capable of providing good chromatographic separation. It is difficult to find an optimum surface to achieve both, especially for reversed-phase separations. In addition, changing solvent composition in a gradient will cause a change in the pumping flow, which makes the technique difficult to implement. Efforts to separate these two functional aspects have so far shown limited success. For stationary-phase material in microfluidic devices, research has mainly focused on in-situ-created structures, such as microfabricated posts16 and monolithic polymer structures.17,18 These materials offer the advantage that they can be fixed in place in the channel and do not move due to the fluid flow. There are also indications that ordered porous posts have increased efficiency over packed-bed columns,19 but the surface area available for retention of analytes on nonporous microfabricated posts is relatively low, which limits their sample capacity, and as with monolithic polymer structures, porous posts require redefinition of optimal separation conditions. However, there are many wellestablished methods already available on LC systems using conventional LC media which can be applied to microfluidic systems. A few other microfluidic systems have used these media. For example, LC-like sample preparation for MALDI has also been performed in parallel in a microfluidic system on a polymer disc.20 This system uses a 10-nL volume packed with 15-µm particles for each of its 96 channels. There is a demanding need for high-performance separation of complex mixtures of peptides in a sample matrix that often involves high concentrations of salt, urea, and surfactant and that requires gradient liquid chromatography separation achieved (10) Van Pelt, C. K.; Zhang, S.; Henion, J. D. J. Biomol. Techn. 2002, 13, 7284. (11) Licklider, L.; Wang, X.; Desai, A.; Tai, Y.; Lee, T. D. Anal. Chem. 2000, 72, 367-375. (12) Wen, J.; Lin, Y.; Xiang F.; Matson, D. W.; Udseth, H. R.; Smith, R. D. Electrophoresis 2000, 21, 191-197. (13) Seiler, K.; Harrison, D. J.; Manz, A. Anal. Chem. 1993, 65, 1481-1488. (14) Oleschuk, R. D.; Shultz-Lockyear, L. L.; Ning, Y.; Harrison, D. J. Anal. Chem. 2000, 72, 585-590. (15) Gottschlich, N.; Jacobson, S. C.; Culbertson, C. T.; Ramsey, J. M. Anal. Chem. 2001, 73, 2669-2674. (16) He, B.; Tait, N.; Regnier, F. Anal. Chem. 1998, 70, 3790-3797. (17) Yu, C.; Davey, M. H.; Svec, F.; Frechet, J. M. J. Anal. Chem. 2001, 73, 5088-5096. (18) Throckmorton, D. J.; Shepodd, T. J.; Singh, A. K. Anal. Chem. 2002, 74, 784-789. (19) Gzil, P.; Vervoort, N.; Baron, G. V.; Desmet, G. Anal. Chem. 2003, 75, 62446250. (20) Gustafsson, M.; Hirschberg, D.; Palmberg, C.; Jornvall, H.; Bergman, T. Anal. Chem. 2004, 76, 345-350.
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under optimum LC conditions. Our research has, therefore, focused on utilizing known separation media, such as 3-5-µm reversed-phase particles and mechanical switching of hydrodynamic flow for the purpose of LC sample injection. EXPERIMENTAL SECTION Chemicals and Materials. HPLC grade 2-propanol, acetonitrile, and formic acid were purchased from Sigma (St. Louis, MO). Tryptic digest of bovine serum albumin was purchased from Michrom Bioresources (Auburn, CA). Deionized water was prepared using a Milli-Q system from Millipore (Bedford, MA). Zorbax SB C18 HPLC 3.5- and 5-µm packing materials were obtained from Agilent Technologies (Delaware). Device Fabrication. The laser-ablation fabrication method using polyimide films has been used extensively for the fabrication of inkjet printheads, and we have modified this method to form microfluidic features for a microfluidic “HPLC chip”.21 The superior properties of polyimide films, such as DuPont’s Kapton, make them ideal for use in microfluidic devices, since they are compatible with most organic solvents and across a wide pH range. Furthermore, these films are commercially available with a co-extruded thermoplastic polyimide adhesive layer that bonds to polyimide when heat and pressure are applied, resulting in a laminated all-polyimide device. The fabrication process consists of, in order: laser ablation of polyimide film to form the microfluidic channels, ports, chambers, and columns; cleaning to remove the laser-ablation residues; deposition of electrical contacts for the electrospray; lamination of the polyimide film layers; using laser ablation to trim the chip to its final shape and to form the electrospray tip; and packing the sample enrichment column and LC column with retention media. The laser ablation of the polyimide film was performed using a direct write process with a nonlinear upconverted diode-pumped, solid state laser (Coherent Avia 355-1500) operating at 355 nm in combination with a fixed optics train and a high-precision x-y table controlled from a personal computer. The laser beam was focused onto the polyimide film, and it ablated the irradiated area while the intensity and stage velocity were varied to change the width and depth of the ablated features created in the film. The microfluidic feature dimensions varied from 5 µm in width and depth to 250 µm wide and 50 µm deep. Three layers of filmstop, middle, and bottomswere used to fabricate each chip. Holes (200µm diameter) were ablated through the top and bottom layers of the device to provide access to the channels and chambers inside the finished device. The channels and chambers were laser-ablated either in the inward side of the two outer film layers or in the middle layer. The cross sections of these features were essentially trapezoidal. Each layer had a thickness of between 50 and 125 µm. After laser ablation, the film layers were cleaned using mechanical abrasion in the presence of a solvent solution and then laminated between flat metal plates in a vacuum at 340 °C and a pressure of 200 psi. Laser ablation was also used to trim the structure to its final shape and to cut the electrospray tips using a circular path around the desired laminated tip channel. The final tip shape was conical with a circular end with dimensions from 35 to 100 µm o.d. and up to 2 mm in length. (21) Kaltenbach, P.; et al. U.S. Patent 5500071, March 19, 1996.
Figure 1. The chip. The holes were used for alignment purposes. The dark pattern on the right end of the chip is the electrodeposited metal for contact to the fluid flow channel near the electrospray tip. The inlet ports are visible ∼15 mm from the left end of the chip.
Electrical contacts for the electrospray tip voltage bias were applied by vacuum evaporation or sputtering of a thin (
