Ultra-small chamber for single-molecule detection of biological reaction

Abstract

We report here development of ultra-small reaction chamber array from silicone-elastomer sheet and glass plate, and its applications for biological assay. It was shown that single-molecules of protein, or DNA can be enclosed in each hermetic chamber stably. The volumes of chambers are extremely small (6∼10 femtoliters) so that small numbers of molecule (∼2000) can reach to detectable concentrations (∼micro molars). When single enzyme molecules were enclosed in the chambers, its catalytic activity was fluorescently monitored to demonstrate the feasibility of single-molecule assay using the chamber. We also apply this method to access reaction efficiency of protein molecular motor, F1-ATPase. Motor molecules were enclosed and manipulated to rotate reversely using magnetic tweezers. The resultant extremely small amount of synthesized ATP was calculated from the speed of the motor after released from the manipulation. This measurement revealed first time that the motor is designed to tightly couple the chemical reaction and mechanical event. Thus, it was proven that the ultra-small chamber can be a new strong tool for ultra-sensitive bioassay.