Lee Jeong Hoon, Song Yong-Ak, Tannenbaum Steven R, Han Jongyoon
Department of Electrical Engineering and Computer Science, Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, Massachusetts 02139, USA.
Anal Chem. 2008 May 1;80(9):3198-204. doi: 10.1021/ac800362e. Epub 2008 Mar 22.
We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a micro/nanofluidic preconcentration chip. The disposable preconcentration device made out of PDMS with a surface-patterned ion-selective membrane increases local enzyme/substrate concentrations for rapid monitoring of enzyme activity. As a model system, we used trypsin as the enzyme and BODIPY FL casein as the fluorogenic substrate. We demonstrated that the reaction rate of trypsin-BODIPY FL was significantly enhanced by increasing the local concentrations of both trypsin and BODIPY FL casein in the preconcentration chip. The reaction time required to turn over substrates at 1 ng/mL was only approximately 10 min compared to approximately 1 h without preconcentration, which demonstrates a significantly higher reaction rate through the increase of the concentrations of both the enzyme and substrate. Furthermore, trypsin activity can be measured down to a concentration level of 10 pg/mL, which is a approximately 100 fold enhancement in sensitivity compared to the result without the preconcentration step. This micro/nanofluidic preconcentrator chip could be used as a generic micro reaction platform to study any enzyme-substrate systems, or other biochemical reaction systems in low concentration ranges.
我们报道了一种使用微纳流体预浓缩芯片提高低丰度酶检测反应速率和灵敏度的新方法。由聚二甲基硅氧烷（PDMS）制成的带有表面图案化离子选择性膜的一次性预浓缩装置能够提高局部酶/底物浓度，以快速监测酶活性。作为模型系统，我们使用胰蛋白酶作为酶，硼二吡咯氟化物（BODIPY FL）酪蛋白作为荧光底物。我们证明，通过提高预浓缩芯片中胰蛋白酶和BODIPY FL酪蛋白的局部浓度，胰蛋白酶 - BODIPY FL的反应速率显著提高。与未进行预浓缩时约1小时相比，将底物浓度为1 ng/mL时转化所需的反应时间仅约为10分钟，这表明通过提高酶和底物的浓度，反应速率显著提高。此外，胰蛋白酶活性的检测下限可低至10 pg/mL，与未进行预浓缩步骤的结果相比，灵敏度提高了约100倍。这种微纳流体预浓缩芯片可作为通用的微反应平台，用于研究任何酶 - 底物系统或低浓度范围内的其他生化反应系统。
