Gold Nanoparticle Assembly Microfluidic Reactor for Efficient On-line Proteolysis*S
Gold Nanoparticle Assembly Microfluidic Reactor for Efficient On-line Proteolysis*S
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DOI：
10.1074/mcp.t600055-mcp200
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发表时间：
2007-08
影响因子：
7
通讯作者：
Yun Liu;Yan Xue;Ji Ji-Ji;Xian Chen;J. Kong;Pengyuan Yang;H. Girault;Baohong Liu
中科院分区：
文献类型：
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作者：
Yun Liu;Yan Xue;Ji Ji-Ji;Xian Chen;J. Kong;Pengyuan Yang;H. Girault;Baohong Liu
设计了包埋胰蛋白酶的金纳米颗粒网络的微芯片反应器，用于有效地在线蛋白水解低水平的蛋白质和来自小鼠巨噬细胞的复杂提取物。纳米结构的表面涂层组装通过聚（二烯丙基二甲基氯化铵）和金纳米粒子的逐层静电结合。组装过程进行了监测，紫外-可见光谱，原子力显微镜，石英晶体微天平。根据Langmuir等温模型对胰蛋白酶的控制吸附进行了理论研究，拟合的最大Γ和K值分别为1.2 × 10−7 mol/m2和4.1 × 105 m−1。酶动力学分析证实，胰蛋白酶，这是包埋在具有高负载能力的生物相容性的金纳米粒子网络，保持其生物活性。吸附胰蛋白酶的最大蛋白水解速率为400 mm/（min·μg）。使用微芯片反应器消化每次分析的微量蛋白质，并通过MALDI-TOF MS/MS鉴定所得到的胰蛋白酶产物。通过在线消化和LC-ESI-MS/MS分析有效地鉴定了从小鼠巨噬细胞提取的蛋白质混合物。
A microchip reactor coated with a gold nanoparticle network entrapping trypsin was designed for the efficient on-line proteolysis of low level proteins and complex extracts originating from mouse macrophages. The nanostructured surface coating was assembled via a layer-by-layer electrostatic binding of poly(diallyldimethylammonium chloride) and gold nanoparticles. The assembly process was monitored by UV-visible spectroscopy, atomic force microscopy, and quartz crystal microbalance. The controlled adsorption of trypsin was theoretically studied on the basis of the Langmuir isotherm model, and the fitted Γmax and K values were estimated to be 1.2 × 10−7 mol/m2 and 4.1 × 105 m−1, respectively. An enzymatic kinetics assay confirmed that trypsin, which was entrapped in the biocompatible gold nanoparticle network with a high loading capacity, preserved its bioactivity. The maximum proteolytic rate of the adsorbed trypsin was 400 mm/(min·μg). Trace amounts of proteins down to femtomole per analysis were digested using the microchip reactor, and the resulting tryptic products were identified by MALDI-TOF MS/MS. The protein mixtures extracted from the mouse macrophages were efficiently identified by on-line digestion and LC-ESI-MS/MS analysis.