Microchip Electrophoresis Utilizing In Situ Photopolymerized Thrombin-Immobilized Preconcentrator Gels for Specific Entrapment and Analysis of Thrombin Aptamers

A method was developed for the specific entrapment and separation of thrombin aptamers using a thrombin-immobilized polyacrylamide gel fabricated at the channel crossing point of a microfluidic electrophoresis chip. The channel intersection of the poly(methyl methacrylate) (PMMA) microchip was filled with a solution comprising thrombin, acrylamide, N,N-methylene-bis-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 an LED laser beam. The fabricated thrombin-immobilized gel (100 ×100 × 30 µm) contained approximately 40 fmol of thrombin and therefore could entrap thrombin aptamers at the femtomolar level. The electrophoretically trapped thrombin aptamers were released from the gel by switching the voltage, which delivered high concentrations of phosphate ions in a background electrolyte. The broad sample band eluted from the gel was effectively reconcentrated at the boundary of a pH junction generated by sodium ions delivered from the outlet reservoir. The reconcentrated sample components were then separated and fluorometrically detected at the end of the separation channel. Under the optimized conditions, the thrombin aptamers were concentrated by a factor of 1,000-fold, and the peak resolution was comparable to that obtained by pinched injection. This method was successfully utilized to preconcentrate and analyze thrombin aptamers.