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Microchip UV Absorbance Detection Applied to Isoelectric Focusing of Proteins

  • Junjie Ou
  • Carolyn L. Ren
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 949)

Abstract

Isoelectric focusing (IEF) is considered as an attractive separation technique for biologically amphoteric compounds (e.g., proteins and peptides) based on their isoelectric point (pI). With the advancement in micromachining technology, microchip format IEF has attracted significant attention. Both single-point and whole column imaging detection (WCID) methods have been employed for analyzing the separation performance in a microchip. WCID is more favorable than single-point detection because the latter requires the focused bands to be mobilized and thus adds more complexity to the design and operation of such microchips. Fluorescence- and UV absorbance-based WCID have been successfully adapted in glass and PDMS microchips. We have developed polydimethylsiloxane (PDMS) microchips for IEF applications where UV-WCID is employed for evaluating the separation performance. The chips are designed for use in the iCE280 analyzer (Convergent Bioscience Inc., Toronto), for capillary-based IEF where UV-WCID is employed for analyzing the separation performance. Three kinds of microchips that have been successfully developed using standard soft lithography technology are described in detail.

Key words

Microfluidic chip Isoelectric focusing Protein separation UV absorption detection Whole channel imaging detection 

Notes

Acknowledgments

This work was supported by the Strategic Project Grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) awarded to Dr. Carolyn L. Ren and her collaborators.

References

  1. 1.
    Tang Q, Harrata AK, Lee CS (1996) High-resolution capillary isoelectric focusing-electrospray ionization mass spectrometry for hemoglobin variants analysis. Anal Chem 68:2482–2487CrossRefGoogle Scholar
  2. 2.
    Shen Y, Berger SJ, Anderson GA, Smith RD (2000) High-efficiency capillary isoelectric focusing of peptides. Anal Chem 72:2154–2159CrossRefGoogle Scholar
  3. 3.
    Silvertand LHH, Torano JS, van Bennekom WP, de Jong GJ (2008) Recent developments in capillary isoelectric focusing. J Chromatogr A 1204:157–170CrossRefGoogle Scholar
  4. 4.
    Hofmann O, Che D, Cruickshank KA, Muller UR (1999) Adaptation of capillary isoelectric focusing to microchannels on a glass chip. Anal Chem 71:678–686CrossRefGoogle Scholar
  5. 5.
    Mao Q, Pawliszyn J (1999) Demonstration of isoelectric focusing on an etched quartz chip with UV absorption imaging detection. Analyst 124:637–641CrossRefGoogle Scholar
  6. 6.
    Guillo C, Karlinsey JM, Landers JP (2007) On-chip pumping for pressure mobilization of the focused zones following microchip isoelectric focusing. Lab Chip 7:112–118CrossRefGoogle Scholar
  7. 7.
    Vlckova M, Kalman F, Schwarz MA (2008) Pharmaceutical applications of isoelectric focusing on microchip with imaged UV detection. J Chromatogr A 1181:145–152CrossRefGoogle Scholar
  8. 8.
    Wu X-Z, Huang T, Liu Z, Pawliszyn J (2005) Whole-column imaging-detection techniques and their analytical applications. Trends Anal Chem 24:369–382CrossRefGoogle Scholar
  9. 9.
    Gotz S, Karst U (2007) Recent developments in optical detection methods for microchip separations. Anal Bioanal Chem 387:183–192CrossRefGoogle Scholar
  10. 10.
    Peng Y, Pallandre A, Tran NT, Taverna M (2008) Recent innovations in protein separation on microchips by electrophoretic methods. Electrophoresis 29:157–178CrossRefGoogle Scholar
  11. 11.
    Richards DP, Stathakis C, Polakowski R, Ahmadzadeh H, Dovichi NJ (1999) Labeling effects on the isoelectric point of green fluorescent protein. J Chromatogr A 853:21–25CrossRefGoogle Scholar
  12. 12.
    Duffy DC, McDonald JC, Schueller OJA, Whitesides GM (1998) Rapid prototyping of microfluidic systems in poly(dimethylsiloxane). Anal Chem 70:4974–4984CrossRefGoogle Scholar
  13. 13.
    Ou J, Ren CL, Pawliszyn J (2010) A simple method for preparation of macroporous polydimethylsiloxane membrane for microfluidic chip-based isoelectric focusing applications. Anal Chim Acta 662:200–205CrossRefGoogle Scholar
  14. 14.
    Ou J, Glawdel T, Samy R, Wang S, Liu Z, Ren CL, Pawliszyn J (2008) Integration of dialysis membranes into a poly(dimethylsiloxane) microfluidic chip for isoelectric focusing of proteins using whole-channel imaging detection. Anal Chem 80:7401–7407CrossRefGoogle Scholar
  15. 15.
    Ou J, Glawdel T, Ren CL, Pawliszyn J (2009) Fabrication of a hybrid PDMS/SU-8/quartz microfluidic chip for enhancing UV absorption whole-channel imaging detection sensitivity and application for isoelectric focusing of proteins. Lab Chip 9:1926–1932CrossRefGoogle Scholar
  16. 16.
    Liu Z, Ou J, Samy R, Glawdel T, Huang T, Ren CL, Pawliszyn J (2008) Side-by-side comparison of disposable microchips with commercial capillary cartridges for application in capillary isoelectric focusing with whole column imaging detection. Lab Chip 8:1738–1741CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media,LLC 2013

Authors and Affiliations

  • Junjie Ou
    • 1
  • Carolyn L. Ren
    • 1
  1. 1.Department of Mechanical and Mechatronics EngineeringUniversity of WaterlooWaterlooCanada

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