Rapid single-molecule imaging in cyclic olefin copolymer channels

Rapid preparation of high quality capture surfaces is a major challenge for surface-based single-molecule protein binding assays. Here we introduce a simple method to activate microfluidic chambers made from cyclic olefin copolymer for single-molecule imaging with total internal reflection fluorescence microscopy. We describe a surface coating protocol and demonstrate single-molecule imaging in off-the-shelf microfluidic parts that can be activated for binding assays within a few minutes. As the first example, biotinylated protein directly captured on the neutravidin-coated surface was detected using fluorescently labeled antibody. We then showed detection of a fusion construct containing green fluorescence protein and verified its single fluorophore behavior by observing stepwise photobleaching events. Finally, a target protein was identified in the crude cell lysate using antibody-sandwich complex formation. In all experiments, controls were completed to ensure that nonspecific binding to the surface was minimal. Based on our results, we conclude that the simple surface preparation described in this paper enables single-molecule imaging assays without time-consuming coating procedures.