Summary
Plastic microfluidic devices are fabricated with an array of pseudo-valves for two-dimensional (2D) protein separation. The devices are made by compression molding; the mold is created by electroplating on a glass master fabricated by photolithography. Each device consists of one channel for isoelectric focusing (IEF) and multiple parallel channels for polyacrylamide gel electrophoresis (PAGE). The IEF channel (first dimension) is orthogonal to the PAGE channels (second dimension). Microfluidic pseudo-valves are created at the intersections of orthogonal channels by photodefinable, in situ gel polymerization. These valves enable the introduction of two types of separation media into orthogonal channels for performing 2D protein separation in the device. The presence of the pseudo-valves prevents one separation medium from being contaminated by the other medium, although proteins are allowed to transfer from the first to the second dimension under an electric field. Two-dimensional protein separation is achieved in less than 10 min, an improvement of two orders of magnitude compared with the conventional 2D gel electrophoresis using an IEF strip and a PAGE slab.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Petricoin, E. F., Belluco, C., Araujo, R. P., Liotta, L. A. (2006). The blood peptidome: a higher dimension of information content for cancer biomarker discovery. Nat. Rev. Cancer 6, 961–967
Service, R. F. (2001). Gold rush - High-speed biologists search for gold in proteins. Science 294, 2074–2077
Chen, X. X., Wu, H. K., Mao, C. D., Whitesides, G. M. (2002). A prototype two-dimensional capillary electrophoresis system fabricated in poly(dimethylsiloxane). Anal. Chem. 74, 1772–1778
Li, Y., Buch, J. S., Rosenberger, F., DeVoe, D. L., Lee, C. S. (2004). Integration of isoelectric focusing with parallel sodium dodecyl sulfate gel electrophoresis for multidimensional protein separations in a plastic microfludic network. Anal. Chem. 76, 742–748
Tsai, S. W., Loughran, M., Karube, I. (2004). Development of a microchip for 2-dimensional capillary electrophoresis. J. Micromech. Microeng. 14, 1693–1699
Das, C., Zhang, J., Denslow, N. D., Fan, Z. H. (2007). Integration of isoelectric focusing with multi-channel gel electrophoresis by using microfluidic pseudo-valves. Lab Chip 7, 1806– 1812
Koh, C. G., Tan, W., Zhao, M. Q., Ricco, A. J., Fan, Z. H. (2003). Integrating polymerase chain reaction, valving, and electrophoresis in a plastic device for bacterial detection. Anal. Chem. 75, 4591–4598
Fredrickson, C. K., Xia, Z., Das, C., Ferguson, R., Tavares, F. T., Fan, Z. H. (2006). Effects of fabrication process parameters on the properties of cyclic olefin copolymer microfluidic devices. J. Microelectromech. Syst. 15, 1060–1068
Das, C., Fan, Z. H. (2006). Effects of separation length and voltage on isoelectric focusing in a plastic microfluidic device. Electrophoresis 27, 3619–3626
Anderson, R. C., Bogdan, G. J., Puski, A., Su, X. (1998). Genetic analysis systems: improvements and methods. In: Solid-State Sensor and Actuator Workshop, Transducer Research Foundation: Hilton Head Island, SC, pp. 7–10
Lagally, E. T., Medintz, I., Mathies, R. A. (2001). Single-molecule DNA amplification and analysis in an integrated microfluidic device. Anal. Chem. 73, 565–570
Das, C., Fredrickson, C. K., Xia, Z., Fan, Z. H. (2007). Device fabrication and integration with photodefinable microvalves for protein separation. Sens. Actuators A Phys. 134, 271–277
Das, C., Xia, Z., Stoyanov, A., Fan, Z. H. (2005). A laser-induced fluorescence imaging system for isoelectric focusing. Instrum. Sci. Technol. 33, 379–389
Fan, Z. H., Harrison, D. J. (1994). Micromachining of capillary electrophoresis injectors and separators on glass chips and evaluation of flow at capillary intersections. Anal. Chem. 66, 177–184
Rabilloud, T. (2000). Detecting proteins separated by 2-D gel electrophoresis. Anal. Chem. 72, 48A–55A
Unlu, M., Morgan, M. E., Minden, J. S. (1997). Difference gel electrophoresis: a single gel method for detecting changes in protein extracts. Electrophoresis 18, 2071–2077
Hoorn, E. J., Hoffert, J. D., Knepper, M. A. (2006). The application of DIGE-based proteomics to renal physiology. Nephron Physiol. 104, 61–72
Acknowledgement
This work is supported by grants from the Army Research Office (48461-LS, 52924-LS-II) and the startup fund from the University of Florida. The author thanks his students and postdoctoral fellows, including Champak Das, Jiyou Zheng, Alexander Stoyanov, Carl Fredrickson, Zheng Xia, and Andrew Simon, for their work cited in this chapter.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2009 Humana Press, a part of Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Fan, Z.H. (2009). Microfluidic Devices with Photodefinable Pseudo-valves for Protein Separation. In: Foote, R., Lee, J. (eds) Micro and Nano Technologies in Bioanalysis. Methods in Molecular Biology™, vol 544. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-59745-483-4_4
Download citation
DOI: https://doi.org/10.1007/978-1-59745-483-4_4
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-934115-40-4
Online ISBN: 978-1-59745-483-4
eBook Packages: Springer Protocols