Li Yan, Yan Bo, Deng Chunhui, Yu Wenjia, Xu Xiuqing, Yang Pengyuan, Zhang Xiangmin
Department of Chemistry, Institute of Biomedical Science, Fudan University, Shanghai, China.
Proteomics. 2007 Jul;7(14):2330-9. doi: 10.1002/pmic.200700112.
An easily replaceable enzymatic microreactor has been fabricated based on the glass microchip with trypsin-immobilized magnetic silica microspheres (MS microspheres). Magnetic microspheres with small size (approximately 300 nm in diameter) and high magnetic responsivity to magnetic field (68.2 emu/g) were synthesized and modified with tetraethyl orthosilicate (TEOS). Aminopropyltriethoxysilane (APTES) and glutaraldehyde (GA) were then introduced to functionalize the MS microspheres for enzyme immobilization. Trypsin was stably immobilized onto the MS microspheres through the reaction of primary amines of the proteins with aldehyde groups on the MS microspheres. The trypsin-immobilized MS microspheres were then locally packed into the microchannel by the application of a strong field magnet to form an on-chip enzymatic microreactor. The digestion efficiency and reproducibility of the microreactor were demonstrated by using cytochrome c (Cyt-C) as a model protein. When compared with an incubation time of 12 h by free trypsin in the conventional digestion approach, proteins can be digested by the on-chip microreactor in several minutes. This microreactor was also successfully applied to the analysis of an RPLC fraction of the rat liver extract. This opens a route for its further application in top-down proteomic analysis.
基于带有固定化胰蛋白酶的磁性二氧化硅微球（MS微球）的玻璃微芯片，制备了一种易于更换的酶促微反应器。合成了尺寸小（直径约300 nm）且对磁场具有高磁响应性（68.2 emu/g）的磁性微球，并用正硅酸四乙酯（TEOS）进行了改性。然后引入氨丙基三乙氧基硅烷（APTES）和戊二醛（GA）对MS微球进行功能化，以固定酶。通过蛋白质的伯胺与MS微球上的醛基反应，将胰蛋白酶稳定地固定在MS微球上。然后通过施加强磁场将固定有胰蛋白酶的MS微球局部填充到微通道中，形成片上酶促微反应器。以细胞色素c（Cyt-C）作为模型蛋白，证明了该微反应器的消化效率和重现性。与传统消化方法中游离胰蛋白酶12小时的孵育时间相比，片上微反应器可在几分钟内消化蛋白质。该微反应器还成功应用于大鼠肝脏提取物的反相液相色谱（RPLC）馏分分析。这为其在自上而下的蛋白质组学分析中的进一步应用开辟了一条途径。
