Studying reaction kinetics by simultaneous FRET and cross-correlation analysis in a miniaturized continuous flow reactor

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Abstract
In this study we present a refined optical detection technique for investigation of fast reactions based on confocal fluorescence spectroscopy in a miniaturized continuous flow (μCF) reactor. The special setup allows for simultaneous observation of the reaction on the basis of fluorescent resonance energy transfer (FRET) as an indicator of the reaction progress. Determination of the flow velocity via spatial fluorescence cross-correlation enables the conversion of spatial information, i.e. the position of the detection point, into the respective temporal information. To overcome the disadvantage of conventional continuous flow reactors of high sample consumption, we used a microfluidic chip allowing an economical expenditure of sample solution at low concentration, while the silicon/glass chip is perfectly adaptable to the confocal setup. Inside these channels, rapid, diffusion-based mixing under laminar flow conditions is performed at the crossing of three channels by squeezing the solutions into thin layers. Using this μCF device, we investigated the irreversible cleavage reaction of a double stranded DNA oligomer by the enzyme exonuclease III. The complementary DNA strands are stained with TAMRA and Cy 5 dye molecules, respectively, undergoing an energy transfer if both strands are annealed. The reduction of the Cy 5 fluorescence directly corresponds to the progress of the cleavage reaction.