IDBR: Nanodroplet reactor arrays and imaging system for biomolecular structure and kinetics

University Of Massachusetts Amherst, Amherst MA

Investigators

Abstract

IDBR: Nanodroplet reactor arrays and imaging system for biomolecular structure and kinetics Technical description. This objective of this project is to develop an instrument capable of high-throughput single-molecule sensitive fluorescence measurement in arrays of aqueous nanodroplets in oil. This instrument will make the observation of single-molecule trajectories more accurate, higher-throughput, and more convenient. It will also facilitate new types of measurements to address some of the most basic but still unanswered questions in molecular biology and biophysics. The following activities are planned: (1) Construction of a microscope with integrated fluidics; (2) The design, prototyping and development of an inexpensive, passive microfluidic device, compatible with single-molecule sensitive imaging, for making nanodroplet arrays; (3) Demonstration and validation of the integrated instrument through a study of nucleic acid structure and structural kinetics; (4) Widespread dissemination through publication, collaboration, and education; (5) Training of students at all levels on the instrument and in microfluidics and single-molecule sensitive techniques; and (6) Outreach and recruitment through the Northeast Alliance for Graduate Education and the Professoriate (NEAGEP) Significance. Single-fluorophore and single-particle sensitive measurements are now commonly used to better understand structural transformations of, and interactions between, biological molecules. The unprecedented detail revealed by these measurements is facilitating new understandings of the biomolecular mechanisms that drive living systems. One requirement of single-molecule observations is that a molecule under study must be made to stay in the detection region of a microscope. For in-vitro measurements, this is most often accomplished by chemically tethering the molecules to a heterogeneous glass surface, or confining them in the pores of a gel. In either case, the presence of heterogeneous surfaces frequently perturbs or inactivates biomolecules and modifies the optical properties of the dyes that are used to study them. These problems, which constitute a significant limitation to in-vitro single-molecule-sensitive measurement, are widely acknowledged but rarely addressed. Droplet arrays provide a compelling alternative to surface attachment or gel confinement for single-molecule measurement and imaging. Nanoscopic aqueous droplets in oil provide a homogeneous environment for the confined molecules. Most biomolecules function well inside these droplets. The droplets function as nanoscopic ?test-tubes,? with each droplet in an array providing a unique environment, so that molecules can be observed under many different conditions simultaneously. The intellectual merit of this work is therefore threefold. (1) A new approach for single-molecule sensitive observations in vitro will be developed that will simplify and improve measurements. (2) Use of this instrument in studies of the flexibility and structural transformations of nucleic acids will result in an improved physical understanding of how these molecules function. (3) Many additional studies will be engendered by a simpler, faster platform for single-molecule measurement; the outcome of these studies will impact fields as diverse as materials science and drug discovery. Broader Impact. In addition to the broad intellectual impact noted in (3) above, educational and societal impacts will result from education and outreach activities. As part of this work, students at all levels and with many backgrounds will be introduced to interdisciplinary research and single-molecule sensitive techniques. Training opportunities through new laboratory units are planned for students at the undergraduate and graduate level. The instrument will be used directly in graduate-level training. To help break societal barriers associated with the participation of women and minority members in the STEM (science, technology, engineering and mathematics) disciplines, recruitment and retention of the best students from the broadest possible pool of applicants will be facilitated by outreach activities through NEAGEP.