Analysis and Discovery of Functional Biomolecules Using a Combination of Fluorescence Microscopy and Micro- and Nano-devices for Single-molecule Detection

Abstract

The author has developed several methodological approaches that use nanophotonic and microfluidic devices to accelerate pharmaceutical research and development. Here, the author describes two of these approaches and provides practical examples. The first is a nanophotonic approach to break the concentration limit of diffusing fluorophore-labeled molecules in single-molecule imaging. Although single-molecule imaging is highly useful in characterizing the kinetics of biomolecular interactions, it requires nanomolar concentrations of labeled molecules in solution. Zero-mode waveguides are nanophotonic structures that reduce the illumination volume by more than three orders of magnitude relative to conventional fluorescence microscopy, thereby allowing single-molecule investigations at micromolar to millimolar concentrations of fluorescent molecules i.e., under near-physiological conditions. The second approach is microfluidic microdroplet-based, allowing the discovery of novel biomolecules with the desired activities. Microfluidics allows the ultrarapid production of monodisperse microdroplets such as water-in-oil microdroplets. Each microdroplet serves as a nano/picoliter-volume test tube, which increases assay sensitivity by increasing the effective concentration of molecules and decreasing the time required to reach detection thresholds. I hope you find this review helpful in your research.