Currently, the two prominent schemes for single-molecule fluorescence detection (SMFD), confocal microscopy and camera-based total-internal-reflection or wide-field microscopy, are ultimately limited in their ability to combine the detection of many molecules with obtaining data at sufficiently high time resolution, necessary for resolving fast dynamics with single-molecule fluorescence resonance energy transfer (smFRET). In particular, monitoring enzymatic reactions using smFRET is extremely challenging and remains to a large extent unexplored.
Here, I propose a novel nanofluidic device to overcome these limitations by using nanochannels, which provide a well-defined flow path for a
fluorescent species through the excitation/detection focus of a conventional wide-field microscope. Using an array of nanochannels offers several advantages: First, the geometrical confinement enables long observation times of non-immobilized molecules. Second, the residence time of molecules in the channels is easily controlled by the flow velocity set by the syringe pump. Third, faster flow rates together with using a CCD camera in ‘streaking mode’ enable a sub-millisecond
time resolution. Fourth, a high-throughput detection is achieved by using a parallel array of channels. Fifth, enzymatic reactions can be directly triggered by mixing necessary components on-the-fly using an additional inlet.
The described device will pave the way for high-throughput single-molecule detection, which will greatly expand the possibilities for researchers to apply single-molecule methods in the area of Life Sciences.