Final Report Summary - NANOFLUIDIC-SMFD (Nanofluidic devices for high-throughput single-molecule-fluorescence detection) The ability to observe and manipulate single molecules allows researchers to study the function and structure of biomolecular systems beyond spatiotemporal averaging effects inherent to conventional biochemical techniques. Detection of single entities can be, for example, achieved by chemically attaching small fluorophores to proteins or DNA of interest. Upon excitation of the fluorophores, the emitted light is then being used for reporting on the position or the conformational state of these labelled molecules. Currently, the two prominent schemes for single-molecule fluorescence detection (SMFD), confocal microscopy and camera-based epifluorescence 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 and interactions between the biomolecules. In particular, using a technique called single-molecule fluorescence resonance energy transfer (smFRET), we are able to monitor small conformational changes within single enzymes with nanometre resolution. However, monitoring enzymatic reactions, such as DNA synthesis, using smFRET is extremely challenging and remains to a large extent unexplored. In the CIG proposal, we proposed novel nanofluidic devices to overcome these limitations by using nanochannels that provide a well-defined flow path for a fluorescent species through the excitation/detection focus of a conventional epi-fluorescence microscope. Using this special geometry, fluorescent molecules enter the field of view, for example, from the top and, as the height of the channels is made smaller than the diffraction-limited detection volume of the microscope objective, the molecules remain in focus until they exit the field of view thereby extending the observation span without requiring the immobilisation of molecules on a surface.During the second period of the project, we submitted a patent application on the working principle of our nanofluidic devices and published a preprint on bioRxiv (https://doi.org/10.1101/201079) summarising our results: We successfully demonstrated the high-throughput detection of fluorescently labelled DNA molecules interconverting between two conformations, we showed the salt-induced stabilisation of the open conformation of a DNA hairpin in the mixing devices and, finally, we were able to monitor enzymatically driven DNA synthesis in the mixing devices.The CIG has been very important for the career development of the supported researcher who is soon expected to become tenured after starting as an assistant professor in 2012. The awarded grant has been used to finance two years of his first PhD student, who recently defended his PhD thesis. As a direct consequence of the CIG grant allowing the researcher to build and establish up his group within the Laboratory of Biophysics at Wageningen UNiversity & Research, he successfully acquired further funding including, but not limited to, further applications of nanofluidic devices in the life sciences.
