Increase of Reaction Rate and Sensitivity of Low-Abundance Enzyme Assay using Micro/Nanofluidic Preconcentration Chip

摘要

We report a novel method of increasing both the reaction rate and the sensitivity of low-abundance enzyme assay using a microanofluidic 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 ~10 min compared to ~1 h without preconcentration, which demonstrates 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 ~100 fold enhancement in sensitivity compared to the result without the preconcentration step. This microanofluidic 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.