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

Molecular biology techniques allow generating combinatorial libraries of large number of enzyme variants. However, there is currently no technology available for screening the enzyme libraries for identifying variants with improved activity for the substrate. We propose to develop a novel, microfluidic, high-throughput platform for determining kinetic constants of enzyme variants and identifying variants with improved affinity for organophosphorus (OP) agents. During Phase I, proof-of-concept will be established by determining the kinetic parameters for the hydrolysis of paraoxon, parathion and diisopropyl fluorophosphate by the catalytic enzyme organophosphorus hydrolase. Integrated on-chip valves will be used for precise metering of reagents. Impedance spectroscopy will be used to monitor reaction progress and completion and ultimately to determine enzyme activity. A preliminary design for the Phase II end-product will be developed for a high-throughput capability. During Phase II, the platform will be further optimized and validated using multiple OP stimulants and for determining enzyme affinity in blood plasma samples. This will be followed by independent third-party testing of the platform with bona fide OP nerve agents. 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.