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High-Throughput Microfabricated CE/ESI-MS: Automated Sampling from a Microwell Plate Bailin Zhang,† Frantisek Foret,* and Barry L. Karger*
Barnett Institute and Department of Chemistry, Northeastern University, Boston, Massachusetts 02115
A new design for high-throughput microfabricated capillary electrophoresis/electrospray mass spectrometry (CE/ ESI-MS) with automated sampling from a microwell plate is presented. The approach combines a sample-loading port, a separation channel, and a liquid junction, the latter for coupling the device to the MS with a miniaturized subatmospheric electrospray interface. The microdevice was attached to a polycarbonate manifold with external electrode reservoirs equipped for electrokinetic and pressure-fluid control. A computer-activated electropneumatic distributor was used for both sample loading from the microwell plate and washing of channels after each run. Removal of the electrodes and sample reservoirs from the microdevice structure significantly simplified the chip design and eliminated the need both for drilling access holes and for sample/buffer reservoirs. The external manifold also allowed the use of relatively large reservoirs that are necessary for extended time operation of the system. Initial results using this microfabricated system for the automated CE/ESI-MS analysis of peptides and protein digests are presented. The rapid identification and characterization of large numbers of compounds (protein digests, combinatorial libraries, etc.) are major needs in the postgenome era. Mass spectrometry (MS) plays a key role in this area of structure analysis, and critical to the success of this approach is sample handling prior to MS analysis. Microfluidic devices for MS analysis have been of recent interest because they have the potential to meet the increased requirements in handling very small sample volumes without dead volume connections.1-14 Short separation channels on the mi* To whom correspondence should be addressed. † Present address: ArQule, Inc., Woburn, MA, 01801. (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) Xue, Q.; Foret, F.; Dunayevskiy, Y. M.; Foret, F.; Karger, B. L. Rapid Commun. Mass Spectrom. 1997, 11, 1253-1256. (4) Zhang, B.; Liu, H.; Karger, B. L.; Foret, F. Anal. Chem. 1999, 71, 32583264. (5) Zhang, B.; Foret, F.; Karger, B. L. Anal. Chem. 2000, 72, 1015-1022. (6) Liu, H.; Felten, C.; Xue, Q.; Zhang, B.; Jedrzejewski, P.; Karger, B. L.; Foret, F. Anal. Chem. 2000, 72, 3303-3310. (7) Li, J.; Thibault, P.; Bings, N. H.; Skinner, C. D.; Wang, C.; Colyer, C.; Harrison, D. J. Anal. Chem. 1999, 71, 3036-3045. (8) Bings, N. H.; Wang, C.; Skinner, C. D.; Colyer, C. L.; Thibault, P.; Harrison, J. D. Anal. Chem. 1999, 71, 3292-3296. (9) Lazar, I. M.; Ramsey, R. S.; Sundberg, S.; Ramsey, J. M. Anal. Chem. 1999, 71, 3627-3631. 10.1021/ac001432v CCC: $20.00 Published on Web 04/28/2001
© 2001 American Chemical Society
crodevices also provide potential for fast separations. Furthermore, the small size of the device significantly reduces the footprint of the instrumentation, which leads to the potential of high-density parallel processing for high-throughput analysis. The coupling of a microchip to MS using electrospray ionization was initially demonstrated for infusion analysis using a flatedged surface.1-3 More recent reports have demonstrated improved performance with electrospray ESI emitter tips, through the attachment of such tips to the device.4-11 In the latter case, the guiding channel for the capillary ESI tip was created either by a double-etching procedure,4,5 polymer casting6 or hand drilling.7,8 Highly efficient on-chip CE/ESI-MS separations using external capillary ESI tips have been demonstrated.4,5,7,8 Some attempts have also been made to microfabricate the ESI tips;12,13 however, these devices were designed only for infusion analysis. Although current reports have clearly shown high performance CE/ESI-MS analyses, the practical application of the microdevices has been limited. After the analysis, the devices developed todate have to be either discarded or manually cleaned before the next sample. In addition, many of the current protocols for highthroughput sample processing are based on microwell plate technology, (e.g., digestion, preconcentration, desalting, etc.). Transfer of the individual samples onto the current microdevices generally requires an additional manual pipetting step. In the present work, an improved microdevice design for highthroughput MS analysis is introduced. The design strategy is based on maximizing the duty cycle of the analysis, while minimizing unnecessary sample transfer steps. In previous work, we demonstrated high-throughput infusion MS analysis using an automated microwell-plate-positioning system close to the mass spectrometer.15 Both single capillaries and capillary arrays were used for the analysis of samples infused directly from the microwell plate into an ESI-ion trap15 or a MALDI-TOF mass spectrometer.16 To minimize the path length for liquid flow, the sample microwell plate was oriented vertically on a motorized (10) Figeys, D.; Van Oostveen, I.; Ducret, A.; Aebersold, R. Anal. Chem. 1996, 68, 1822-1828. (11) Figeys, D.; Aebersold, R. Anal. Chem. 1998, 70, 3721-3727. (12) Licklider, L.; Wang, X.; Desai, A.; Tai, Y.; Lee, T. Anal. Chem. 2000, 72, 367-375. (13) Schultz, A.; Corso, T. N.; Prosser, S. J.; Zhang, S. Anal. Chem. 2000, 72, 4058-4063. (14) Wen, J.; Lin, Y.; Xiang, F.; Matson, D. W.; Udseth, H. R.; Smith, R. D. Electrophoresis 2000, 21, 191-197. (15) Felten, C.; Foret, F.; Mina´rik, M.; Karger, B. L. Anal. Chem. 2001, 73, 1449-1454. (16) Hu, P.; Rejtar, T.; Preisler, J.; Foret, F.; Karger, B. L. 48th ASMS Conference on Mass Spectrometry and Allied Topics, Long Beach, CA, 2000.
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Figure 1. (A) Photograph of the glass chip (5 × 2 cm) used in the system. (B) Diagram of the microdevice design, including the microchip and polycarbonate manifold. (C) Photograph of the microdevice attached to the polycarbonate manifold and subatmospheric ESI chamber. Also shown is a photograph of the pneumatic controller used for sample loading and washing of the microchip.
translation stage. For sample injection, the microwell plate was moved to the stationary sampling capillary or capillary array so that the sample from the desired well was aspirated for analysis. In this work, we substituted a microdevice with CE separation capability for the above infusion capillary. The microdevice was designed with a polycarbonate device holder that is capable of pneumatic sample manipulation for the automated transfer of samples from the microwell plate onto the microdevice. The holder integrates the microdevice, which contains a sample introduction loop, separation channel, and liquid junction, with an external subatmospheric ESI interface and a manifold of electrode reservoirs. The external pneumatic control system allows direct sample loading from the microwell plate, followed by rapid separation and MS analysis. Both electrokinetic and hydraulic fluid control can be employed, thus allowing use of surface-modified separation channels (to minimize adsorption) without electroosmotic flow and eliminating sample injection bias.17 In this paper, we present initial results with the system for the CE/ESI-MS analysis of standard peptides as well as protein digests. EXPERIMENTAL SECTION Microdevice Fabrication and Instrument Design. The microdevice (Figure 1A) was fabricated using standard photo(17) Schultz, L. L.; Colyer, C. L.; Fan, Z. H.; Roy, K. I.; Harrison, D. J. Electrophoresis 1999, 20, 529-538.
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lithographic/wet chemical etching techniques, as described previously.4,5 The circular separation channel, 75-µm i.d., was 11 cm long, and the loop defining the length of the sample plug was 1.5 mm (sample volume 6.6 nL) long. The liquid junction was cut by a dicing saw as a rectangular channel (1 × 1 mm). The guiding channels for the ESI tip, sample-loading capillary, and outlets on the edge of the microdevice were etched to a circular diameter of 390 µm. The electrospray tip (25-µm i.d., 375-µm o.d., and 3 cm long) was inserted into the guiding hole until it gently touched the end of the separation channel. The tightness of the fit meant that glue did not have to be applied to fix the ESI tip in the hole. Care was exercised to cleave the end of the electrospray capillary to obtain smooth edges without flaking. The gap between the end of the separation channel and the electrospray capillary was measured to be 1 µL of buffer is flushed through the separation channel, the electrolyte reservoirs have to be replaced after half of the microtiter well plate has been analyzed because of the change in liquid levels in the reservoirs. Because the accumulating liquid rises by ∼1 mm/20 µL, the resulting hydrostatic pressure difference can induce liquid flow in the channels of the microdevice, which in turn can degrade separation efficiency. Fortunately, the volume of the external reservoirs can be easily increased to minimize this difference in liquid height buildup during extended operation. For example, by increasing the diameter of the reservoirs from 5 mm (current system) to 16 mm, the volume would increase 10-fold. This increase would essentially eliminate hydrostatic pressure differences during the analysis of the whole plate. Alternatively, the content of the electrode reservoirs could be designed as a flowthrough system15. Nevertheless, the results presented in this paper demonstrate that the design strategy of external electrolyte reservoirs as well as sampling directly from the microtiter plate provide good separation performance with the potential for automated high-throughput CE/ESI-MS. Future work will demonstrate the long-term use of this microdevice approach for automated single- and multiple-channel operation with MS detection.
CONCLUSIONS A new microdevice for automated high-throughput CE/ESIMS, integrating a separation module equipped with a subatmospheric ESI interface and with injection directly from microwell plates has been introduced. Buffer reservoirs external to the device and an electropneumatic distributor were used for automated device washing and sample loading. The removal of the electrode and sample reservoirs from the microdevice significantly simplified manufacture and operation of the system while the system was tested for CE separation. The system can also be employed for other separation modes, for example, capillary liquid chromatography in a single- or a multiple-channel arrangement. The microdevice provides a possible means for high-throughput peptide identification and analysis, as well as analysis of chemical and combinatorial libraries. High-throughput quantitative analysis for pharmacokinetic studies should also be possible using this approach. ACKNOWLEDGMENT The authors gratefully thank NIH (Grant GM 15847) for support of this research and Contribution No. 789 from the Barnett Institute.
Received for review December 5, 2000. Accepted March 20, 2001. AC001432V
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