The design, performance and application of a novel optical system for high throughput single molecule detection (SMD) configured in a continuous flow format using microfluidics is reported. The system consisted of a microfabricated polymer-based multi-channel fluidic network situated within the optical path of a laser source (λex = 660 nm) with photon transduction accomplished using an electron-multiplying charge coupled device (EMCCD) operated in a frame transfer mode that allowed tracking single molecules as they passed through a large field-of-view (FoV) illumination zone. The microfluidic device consisted of 30 microchannels possessing dimensions of 30 µm (width) × 20 µm (depth) with a 25 µm pitch. Individual molecules were electrokinetically driven through the fluidic network and excited within the wide-field illumination area with the resulting fluorescence collected via an objective and imaged onto the EMCCD camera. The detection system demonstrated sufficient sensitivity to detect single DNA molecules labeled with a fluorescent tag (AlexaFluor 660) identified through their characteristic emission wavelength and the burst of photons produced during their transit through the excitation volume. In its present configuration and fluidic architecture, the sample processing throughput was ![[similar]](http://www.rsc.org/images/entities/char_223c.gif)
4.02 × 105 molecules s–1, but could be increased dramatically through the use of narrower channels and a smaller pitch. The system was further evaluated using a single molecule-based fluorescence quenching assay for measuring the population differences between duplexed and single-stranded DNA molecules as a function of temperature for determining the duplex melting temperature, Tm.
