(266c) Quantifying Transcription of Clinically Relevant Immobilized DNA within a Continuous Flow Microfluidic Reactor

We present a novel microreactor that contains H5 influenza cDNA immobilized directly to the reactor walls to study the kinetics and reagent dependence of in vitro transcription reactions in a microfluidic reactor. Enzyme and rNTP substrate continuously flow over the cDNA and create RNA, which flows to a downstream collection well. Using nanogram quantities of cDNA, we found that enzyme limiting conditions caused by preconcentration of the cDNA in a low volume microreactor channel can be overcome as the enzyme binds and concentrates near the channel wall. Kinetics confirm this phenomenon, and show that the time scale for the enzyme binding can be approximated by . Surprisingly, on chip transcription reactions have a strong dependence on rNTP concentration from 5mM to 9mM, despite a low consumption rate of rNTP molecules that is largely independent of flow rate. An electrostatic interaction between the tightly packed negatively charged cDNA and the negatively charged rNTP molecules can explain this behavior. Faster flow rates decrease the time it takes to fill DNA promoter sites with enzyme, while additionally refreshing rNTP and MgCl2 to allow greater consumption of rNTP. These two effects cause reactions with higher concentrations of cDNA in the reactor channel to have a greater dependence on flow rate. At high flow rates (>0.37nL/s) the reaction rate begins to drop, likely due to the release and escape of enzyme molecules from the cDNA layer. This critical flow rate can be predicted by a new modified Péclet number. Together, these insights can inform reactor design for a variety of applications.