Assembly-controlled biocompatible interface on a microchip: Strategy to highly efficient proteolysis
Assembly-controlled biocompatible interface on a microchip: Strategy to highly efficient proteolysis
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DOI：
10.1002/chem.200501622
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发表时间：
2006-08-25
影响因子：
4.3
通讯作者：
Liu, Baohong
中科院分区：
文献类型：
--
作者：
Liu, Yun;Zhong, Wei;Liu, Baohong
通过使用掺有蛋白酶的带电多糖的逐层（LBL）组装来实现高效蛋白水解，在微芯片上构建了生物相容性界面。使用石英晶体微天平和原子力显微镜（AFM）监测天然聚电解质的受控组装和酶吸附步骤。这种多层组装膜提供了具有高酶负载能力的生物相容性互连网络。与溶液中的胰蛋白酶消化相比，微通道中吸附的胰蛋白酶的最大消化速率显着加快至1600 mm min(-1) mu g(-1)。根据Langmuir等温线模型计算出吸附热力学常数K为1.6×10(5)M-1，最大吸附量Gamma(max)为3.6×10(-6)molm(-2)，是天然表面单层胰蛋白酶的30倍。采用含有胰蛋白酶的可调接口构建微芯片反应器，用于消化飞摩尔蛋白质，并通过 MALDI-TOF 质谱分析产生的肽。在几秒钟内实现了高效的芯片上蛋白水解，并且生物样品的鉴定是可行的。
A biocompatible interface was constructed on a microchip by using the layer-by-layer (LBL) assembly of charged polysaccharides incorporating proteases for highly efficient proteolysis. The controlled assembly of natural polyelectrolytes and the enzyme-adsorption step were monitored by using a quartz-crystal microbalance and atomic force microscopy (AFM). Such a multilayer-assembled membrane provides a biocompatible interconnected network with high enzyme-loading capacity. The maximum digestion rate of the adsorbed trypsin in a microchannel was significantly accelerated to 1600 mm min(-1) mu g(-1), compared with the tryptic digestion in solution. Based on the Langmuir isotherm model, the thermodynamic constant of adsorption K was calculated to be 1.6 x 10(5) M-1 and the maximum adsorption loading Gamma(max) was 3.6 x 10(-6) mol m(-2), 30 times more than a monolayer of trypsin on the native surface. The tunable interface containing trypsin was employed to construct a microchip reactor for digestion of femtomoles of proteins and the produced peptides were analyzed by MALDI-TOF mass spectroscopy. The efficient on-chip proteolysis was obtained within a few seconds, and the identification of biological samples was feasible.