title: On-chip protein isoelectric focusing using a photoimmobilized pH gradient.
abstract: An immobilized pH gradient was directly constructed on the inner wall of a microfluidic chip channel by photoimmobilizing focused carrier ampholytes onto the wall. A mixture of carbonic anhydrase, myoglobin, and trypsin inhibitor was successfully isoelectric-focused and separated with good linearity between the pI values of proteins and the location of the focused bands. Furthermore, coating methods for the resistance of protein nonselective adsorption and simultaneously for pH gradient photocoupling were screened. The PEG-silane coating method was found to be better than the cross-linked polyacrylamide coating and aminosilane modification methods. Finally, based on the open tubular column mode of carrier ampholytes' immobilization and effective antiadsorption coating, the immobilized pH gradient was reused and the chip was recycled for the first time. By virtue of its remarkable features including simplicity, convenience, high efficiency of protein enrichment and separation, and potential for coupling site-selective IEF with other analytical or separation techniques, this novel method promises to be useful in several applications related with zwitterionic biomolecules.
authors.affiliation: School of Life Science, Beijing Institute of Technology, Beijing, P. R. China.
authors.forename: Lin
authors.initials: L
authors.lastname: Xia
authors.forename: FengMing
authors.initials: F
authors.lastname: Lin
authors.forename: Xin
authors.initials: X
authors.lastname: Wu
authors.forename: Chuanli
authors.initials: C
authors.lastname: Liu
authors.forename: Jianshe
authors.initials: J
authors.lastname: Wang
authors.forename: Qi
authors.initials: Q
authors.lastname: Tang
authors.forename: Shiyong
authors.initials: S
authors.lastname: Yu
authors.forename: Kunjie
authors.initials: K
authors.lastname: Huang
authors.forename: Yulin
authors.initials: Y
authors.lastname: Deng
authors.forename: Lina
authors.initials: L
authors.lastname: Geng
mesh_terms: D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D011506:Proteins / Q000737:chemistry*
chemical_list: D011506:Proteins
keywords: Isoelectric focusing; Microfluidic chip; Photoimmobilized pH gradient; Protein separation