Mechanism of a Disassembly Driven Sensing System Studied by Stopped-Flow Kinetics

Abstract

<div> Mechanistic studies were carried out on the kinetics for the assembly of a DimerDye (DD12) and the binding of the monomeric DimerDye (DD1) with nicotine in aqueous buffer and artificial saliva. DD12 is non-fluorescent, while monomeric DD1 and DD1-nicotine fluoresce. Binding isotherms were determined from steady-state fluorescence experiments. The report includes measurements of the steady-state fluorescence at pHs 2.2, 6.3 and 12.1, and stopped-flow kinetic data for the homodimerization forming DD12 and DD1-nicotine formation in buffer and artificial saliva. Analysis of the homodimerization kinetics led to the recovery of the association and dissociation rate constants for DD12. These rate constants were used in the global analysis for the coupled kinetics for DD1-nicotine formation, which led to the determination of the association and dissociation rate constants for nicotine binding to DD1.</div>

Mechanism of a Disassembly-Driven Sensing System Studied by Stopped-Flow Kinetics

“…We first characterize the general assembly and photophysical properties of the system using absorbance and steady-state fluorescence. We then used stopped-flow experiments in buffer and artificial saliva to establish the kinetics of individual steps and to reveal how complex media affect the kinetics at the heart of this disassembly driven sensor …”