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On‐chip protein isoelectric focusing using a photo‐immobilized pH gradient
Author(s) -
Xia Lin,
Lin FengMing,
Wu Xin,
Liu Chuanli,
Wang Jianshe,
Tang Qi,
Yu Shiyong,
Huang Kunjie,
Deng Yulin,
Geng Lina
Publication year - 2014
Publication title -
journal of separation science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.72
H-Index - 102
eISSN - 1615-9314
pISSN - 1615-9306
DOI - 10.1002/jssc.201470191
Subject(s) - chemistry , isoelectric focusing , immobilized ph gradient , chromatography , adsorption , coating , ethylene glycol , isoelectric point , protein purification , polyacrylamide , biomolecule , polymer chemistry , organic chemistry , biochemistry , enzyme
(J. Sep. Sci. 2014, 37 (21), 3174–3180) DOI: 10.1002/jssc.201400795 An immobilized pH gradient was directly constructed on the inner wall of a microfluidic chip channel by photo‐immobilizing focused carrier ampholytes (CAs) onto the wall. Amixture 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 non‐selective adsorption and simultaneously for pH gradient photo‐coupling were screened. The poly (ethylene glycol)‐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 CA immobilization and effective anti‐adsorption 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 isoelectric focusing with other analytical or separation techniques, this novel method promises to be useful in several applications related with zwitterionic biomolecules.