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Single Molecule Studies (Publications)

Single Molecule Studies of Cellulose-Binding Enzymes

Enzymatic depolymerization of cellulose is an essential process in the production of biofuels. Single molecule fluorescence studies of this process would facilitate increased understanding of cellulose saccharification and lead to more efficient design of enzymatic cocktails for biofuel production. Fluorescence labeling techniques can hinder the catalytic function of the cellulase protein by modifying active sites. We demonstrate a method for fluorescent-label attachment to cellulase, which retains wild-type functionality of the protein and yields fluorescently bright, high-purity labeling. The spectroscopic characteristics of fluorescently-labeled enzymes are studied using fused-silica sub-micrometer fluidic channels to isolate cellulase molecules during laser induced fluorescence. We show that unbound fluorescent dye is successfully removed using solid-phase purification, providing a highly homogeneous population of cellulases with up to six dyes per cellulase. It is also concluded that the addition of more than six fluorophores per cellulase hinders the total photon output per cellulase. This method can be adapted to label any mixture of labeled proteins and we believe it will lead to improved visualization of cellulase-cellulose interactions at the single molecule level.

Compression and Free Expansion of Single DNA Molecules in Nanochannels

Our goal is to understand the mechanical properties of single DNA strands under high compression forces in confined environments. Characterization of these properties is important for an understanding of DNA packing into chromatin or bacteriophage heads as well as the verification of theoretical biopolymer models. We are currently investigating the compression and subsequent free expansion of DNA molecules in artificial nanofluidic devices. Our method stands in contrast to established techniques for investigating single DNA strands, for example the stretching by an external force, which was used to study the elasticity of DNA molecules.

Nanofluidic Devices for Single Molecule Spectroscopy

Micrometer and sub-micrometer fluidic devices are used in a variety of applications for analytical biochemistry. Previous work describes the benefits of performing single molecule spectroscopy in small engineered focal volumes with regard to uniform illumination intensity and controllable analyte flow. The goal of this project is to further reduce the engineered focal volume with sub-100nm fluidic channel dimensions. Reduction of the focal volume will reduce the probability of multiple molecule occupancy and could lead to spectroscopy with higher accuracy. Biophysical interaction at the channel-fluid interface becomes increasingly dominant at smaller dimensions and therefore warrants comparison of the practical benefits of each design.

A Direct Measurement of the Trapping Time of DNA at an Entropic Barrier

In this work, we bring experimental techniques designed for quantitative single molecule studies to bear on the observation of individual DNA molecules as they are delayed by, probe, and finally overcome entropic barriers. The sizes of DNA molecules and their flow speeds in fluidic channels can be quantified by measuring photon bursts collected when fluorescently labeled molecules pass through a focused laser. Our ability to carefully measure the delay time for individual molecules represents an improvement over the indirect, or bulk, methods previously used to determine the same quantity.