Microfluidic High-throughput Platform for Determining Kinetic Constants of Enzyme Variants

Current high-throughput platforms rely on labeled substrates that produce detectable spectroscopic signals upon reaction for monitoring enzyme catalytic activity. Add to that the high price tag for these instruments, ranging from several hundred thousand to millions of dollars, and there is a clear need for alternate solutions for a label-free, low-cost, high-throughput enzyme screening platform. We propose to develop a novel, microfluidic, high-throughput platform for determining enzyme kinetics. Specifially, the platform will utilize impedance measurements for identifying enzyme variants with improved affinity for organophosphorus (OP) agents. The platform features multiple parallel processors that generate a gradient of reagent concentrations while seamlessly integrating sample metering, mixing and incubation functionalities using on-chip pneumatic valving. Proof-of-concept was provided during Phase I, by monitoring the hydrolysis of OP agent simulants using organophosphorus hydorlase. During Phase II, we will refine and optimize the platform using multi-physics based simulations. Prototypes will be fabricated using state-of-art microfabrication techniques and detailed experimental characterization would be carried out to demonstrate platform operation. We have assembled an interdisciplinary team of engineers and scientists from CFDRC and Auburn University with expertise in design, modeling, fabrication and experimental characterization of microfluidic systems for a successful development of the proposed platform.