Fluorescence-lifetime imaging of DNA-dye interactions within continuous-flow microfluidic systems.

Recent years have seen significant progress in the development of microfabricated systems for use in the chemical and biological sciences. Much of this development has been driven by a need to perform rapid measurements on small sample volumes in areas such as chemical synthesis, DNA analysis, drug discovery, pharmaceutical screening, proteomics, and medical diagnostics. It is well recognized that, when compared to macroscale instruments, microfluidic systems engender a number of distinct advantages with respect to speed, analytical throughput, reagent usage, process control, automation, and operational and configurational flexibility. Although all these advantages are directly facilitated by system downscaling (and the associated improvements in both mass and thermal transfer), the instantaneous-reaction volumes that characterize microfluidic systems typically range from a few picoliters to hundreds of nanoliters. This means that analyte detection and identification is a significant challenge and often defines the principal limitations of a microfluidic system. Despite this problem, a variety of detection methods have been successfully transferred and integrated with microfluidic systems. The use of fluorescence as a readout mechanism in microfluidic systems is advantageous because of the high sensitivity, noninvasiveness, and ease of implementation of this method. Accordingly, fluorescence methods are routinely used in a wide range of analyses in single-point or imaging modalities. Although useful and easy to implement, steadystate (time-integrated) fluorescence methods are limited with respect to quantitative analysis of fluidic samples because of their sensitivity to experimental parameters such as optical pathlength, radiation scattering, inhomogeneous excitation, and fluorophore bleaching. In contrast, time-resolved fluorescence techniques are particularly advantageous for quantifying fluorescence emission because their ratiometric nature makes them largely independent of instrumentationbased artifacts associated with excitation and detection efficiencies, background scattered radiation, and autofluorescence. The enhanced quantification available with fluorescence-lifetime imaging (FLIM) has recently been used by Benninger et al. to quantitatively map fluidic temperatures in microfluidic environments with micrometer spatial resolution. Furthermore, FLIM has been used to perform threedimensional spatial mapping of solvent viscosity and mixing and to evaluate laminar flow in microfluidic systems. Herein we extend the utility of time-resolved fluorescence imaging as a detection modality for microfluidic systems by reporting the study of DNA–dye binding interactions in continuous-flow microfluidic reactors. In contrast to typical bulk studies of DNA–dye interactions where only the end point of a reaction is accessible, the use of a continuousflow microfluidic reactor enables the extraction of kinetic information through a time-to-space conversion, that is, multiple time points can be accessed by measuring at different positions along the mixing channel. Microfluidic systems therefore provide an ideal platform for studying the temporal variation in fluorescence kinetics during and after DNA–dye binding, while the application of timeand polarizationresolved fluorescence imaging facilitates the resolution of multiple reaction species through their respective decay kinetics and rotational mobility. Crucially, the combination of microfluidics and FLIM generates novel information that cannot be extracted from bulk measurements or timeintegrated fluorescence signals alone. Specifically, in this study we demonstrate the resolution of emitting states of the DNA-intercalating dye Hoechst 33258 (H33258) as it binds to large DNA plasmids in the nonequilibrium conditions provided by a simple microfluidic reactor. Time-resolved fluorescence imaging of the microfluidic environment was achieved by using a quasi-widefield multi[*] Dr. R. K. P. Benninger, I. Munro, Dr. C. Dunsby, Dr. M. A. A. Neil, Prof. P. M. W. French Department of Physics Imperial College London Exhibition Road, South Kensington, London, SW72AZ (UK) Fax: (+44)207-594-7714 E-mail: paul.french@imperial.ac. uk