Abstract: As a kind of recognition molecule, aptamer has been studied and applied widely in numerous science fields in recent years. Immobilized enzymatic reactor has drawn much attention because of its striking advantages, such as high digestion efficiency and ease in coupling with the separation and detection systems. In this study, a novel microfluidic enzymatic chip, which immobilized trypsin based on aptamer, was prepared and proposed. An online analysis platform, which consisted of an aptamer-based chip and high performance liquid chromatography tandem mass spectrometry, was established by using a 6-port valve and applied to protein analysis. The enzymatic capacity and stability performance of chip reactor were characterized by using mixed protein sample, which consisted of bovine serum albumin (BSA), myoglobin (Mb) and cytochrome c (Cyt.c). The sample digestion time of the chip reactor was about 5.76 s while 1 μL/min of flow rate was adopted; and moreover, 5 ng of Mb was identified successfully with the sequence coverage of 37%. Furthermore, the sequence coverages and the relative standard deviations were 44.3% and 6.5% for BSA, 65.0% and 2.7% for Mb, 62.0% and 5.6% for Cyt.c respectively when 500 ng digest of mixed proteins were analyzed in three runs. According to experimental results, the online analysis platform possesses the ability of high sensitivity and good stability, which can provide a promising tool for rapid and high-throughput proteomics study in the near future.

Key words: aptamer, chip immobilized enzymatic reactor (chip IMER), protein, trypsin, high performance liquid chromatography tandem mass spectrometry (HPLC-MS/MS)