Strategy for Allosteric Analysis Based on Protein-Patterned Stationary Phase in Microfluidic Chip

Abstract

An effective method is presented for the on-chip analysis of chiral interactions with a successful depression of nonspecific adsorption. The alumina gel-derived protein network on poly(methyl methacrylate) (PMMA) microchannel was explored to form a protein-stationary phase and then used to carry out electrophoresis for fast enantioseparation coupled with electrochemical detection. On the basis of the chemical modification of a synthesized copolymer containing silane-functionalized scaffold, alumina sol-gel could react readily with the silane groups and form steady microstructure on the chip surface achieving the encapsulation of functional biomolecules. Compared with the native PMMA microchannels, the modified surfaces exhibited much better wettability, more stable and enhanced electroosmotic mobility, and less nonspecific adsorption. The water contact angle and EOF of alumina-gel-derived PMMA substrate were 22 degrees and 4.3 x 10(-4) cm(2) V(-1) s(-1), compared to those of 73 degrees and 1.9 x 10(-4) cm(2) V(-1) s(-1) from the untreated one, respectively. Bovine serum albumin, acting as a target protein, could be stably and homogeneously immobilized in the modified PMMA microchannel to fabricate a protein-stationary phase. Under a mild condition, D- and L-tryptophan were efficiently separated with a resolution of 1.57. The as-prepared microchip can perform chiral separations within short time, indicating that the general protocol has the potential to provide a platform for high throughput screening of enantiomer candidates such as those biochemical drugs with protein targets and the research of receptor interactions.