Development of one-dimensional multipoint detection lifetime spectroscopy for monitoring single molecule binding kinetics within fluidic channels

There is a significant need across research and medical communities for highly sensitive spectroscopic techniques. Perhaps one of the most significant challenges facing physical and biological scientists is the accurate detection and identification of single molecules in free-solution environments for extended periods with high speed timing resolution. Currently such technologies do not exist. More specifically, we seek to drastically revamp conventional single molecule detection approaches by improving the performance in terms of timing, sensitivity, and spatial resolution via the integration of a novel multipoint detection spectrometer with nanofluidics. This technology will be used for tracking molecules in real time down the length of a fluidic channel. This tool will be used for studying molecular binding of Calmodulin (CaM) in the presence of calcium ions. This technology will function by flowing single molecules within a fluidic chip and monitoring changes in CaM conformations via fluorescence resonance energy transfer lifetimes in real time. This approach utilizes conventional semiconductor processing techniques to fabricate the fluidic devices and the detection technology builds on aspects of highly sensitive avalanche photodiode array detectors. We expect this work to have major impact and open up new possibilities for nano-analytical tools in the chemical and biological sciences. Importantly fundamental questions regarding different conformational states explored by a molecule, quantification of transition rates, and identification of transition pathways will be addressed.