光重合性Phos-tag含有アクリルアミドゲルを用いるリン酸化化合物の高感度検出システムの開発

Abstract

Novel methods were developed for the specific entrapment and separation of phosphorylated compounds using Phos-tag polyacrylamide gels fabricated at the channel crossing points of the microfluidic electrophoresis chips. The channel intersection of the microchip was filled with a solution comprising acrylamide, N,N-methylene-bisacrylamide, Phos-tag acrylamide, and 2,2'-azobis [2-methyl-N-(2-hydroxyethyl)propionamide], which functioned as a photocatalytic initiator. In situ polymerization at the channel crossing point was performed by irradiation with a UV LED laser beam. The electrophoretically trapped phosphorylated compounds were released from the gel by switching the voltage to deliver high concentrations of phosphate and EDTA in a background electrolyte and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the phosphorylated compounds were concentrated by a factor of 100-fold, and the peak resolution was comparable to that obtained by pinched injection that a common microchip electrophoresis analysis method. This method was successfully utilized to preconcentrate and analyze phosphorylated peptides.