Use of Dual Optical Tweezers and Microfluidics for Single-Molecule Studies.

Abstract

Visual biochemistry is a powerful technique for observing the stochastic properties of single enzymes or enzyme complexes that are obscured in the averaging that takes place in bulk-phase studies. To achieve visualization, dual optical tweezers, where one trap is fixed and the other is mobile, are focused into one channel of a multi-stream microfluidic chamber positioned on the stage of an inverted fluorescence microscope. The optical tweezers trap single molecules of fluorescently labeled DNA and fluid flow through the chamber and past the trapped beads, stretches the DNA to B-form (under minimal force, i.e., 0 pN) with the nucleic acid being observed as a white string against a black background. DNA molecules are moved from one stream to the next, by translating the stage perpendicular to the flow to enable the initiation of reactions in a controlled manner. To achieve success, microfluidic devices with optically clear channels are mated to glass syringes held in place in a syringe pump. Optimal results use connectors permanently bonded to the flow cell, tubing that is mechanically rigid and chemically resistant, and which is connected to switching valves that eliminate bubbles that prohibit laminar flow.