Hydrodynamic focusing in microstructures: Improved detection efficiencies in subfemtoliter probe volumes

We present a method for improving detection efficiencies in single molecule confocal fluorescence spectroscopy with subfemtoliter probe volumes within microfluidic channels. Our approach is based on hydrodynamically focusing an analyte stream within a microfluidic channel down to a width on the same order of magnitude as that of the confocal probe volume. Experiments are performed in which fluorescently labeled polystyrene microspheres (930 nm diameter) are motivated through a microchannel and passed through a focused laser beam at a variety of flow rates (0.1-11 mu L/min). Hydrodynamic focusing of the analyte stream is achieved by introduction of two sheath flow streams that flank the central analyte stream. Through variation of the relative flow rates in each input stream the analyte stream can be controlled with micron resolution. A maximum hydrodynamic focusing width of 3 mu m was achieved within a 50 mu m wide microfluidic channel; hence, a larger proportion of molecules traveling through the microfluidic device were detected. Furthermore, simple statistical methods are used to investigate the resulting fluorescence bursts and generate single-particle burst width and burst area distributions. From these data, the total detection efficiencies are shown to be an order of magnitude better than those achievable in conventional unfocused systems. (c) 2007 American Institute of Physics.