A rapid multi-reagents switching microvalve system integrated with a total internal reflection fluorescence microscopy (TIRFM) was developed for real time imaging of a single protein behavior. The binding and dissociation process between a chaperonin GroEL and cochaperonin GroES was observed in this TIRFM microfluidic system. This TIRFM microfluidic system was constructed by a smooth glass in microchannel surface, that is essential for TIR imaging, and the polydimethylesiloxane (PDMS) microvalves that is operatable with a rapid response of around 100 ms. The smooth glass surface was etched with an optimized hydrofluoric acid and nitric acid (HF-HNO3) solution and had the average surface roughness of 3.2 nm. The microvalves were based on a pneumatic PDMS membrane that is beneficial for a rapid (<100 ms) response. This TIRFM microfluidic system contains three inlet channels and one outlet channel and the flow in every inlet channel were precisely controlled by the PDMS microvalves. These microvalves were operated by air pressure pulses of 100 ms to hold, open or closed for switching. This TIRFM microfluidic system enabled a reagent exchange within 100 ms with TIRFM observation. For single molecular imaging in this system, biotinylated GroEL (D490C) was immobilized to the glass microchannel surface through streptavidin and biotinylated BSA. A solution including 1 nM IC5-GroES was introduced for 100 ms into the observation area and then washed out with 2 mM ATP buffer solution. Fluorescence spots of IC5-GroES appeared after rapid solution switching, and disappeared several seconds later. As a result, we succeeded in detecting fluorescence signal from single molecules in TIRFM microfluidic system. The micro-constructed TIRFM microfluidic system can realize dynamic analysis of the single molecule level protein-protein binding and dissociation under a controlled multi-reagent exchange.
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