Microscopic Screening of Cyclodextrin Channel Blockers by DiffusiOptoPhysiology

Blockers of pore-forming toxins (PFTs) limit bacterial virulence by blocking relevant channel proteins. However, screening of desired blockers from a large pool of candidate molecules is not a trivial task. Acknowledging its advantages of low cost, high throughput, and multiplicity, DiffusiOptoPhysiology (DOP), an emerging nanopore technique that visually monitors the states of individual channel proteins without using any electrodes, has shown its potential use in the screening of channel blockers. By taking different α-hemolysin (α-HL) mutants as model PFTs and different cyclodextrins as model blockers, we report direct screening of pore blockers solely by using fluorescence microscopy. Different combinations of pores and blockers were simultaneously evaluated on the same DOP chip and a single-molecule resolution is directly achieved. The entire chip is composed of low-cost and biocompatible materials, which is fully disposable after each use. Though only demonstrated with cyclodextrin derivatives and α-HL mutants, this proof of concept has also suggested its generality to investigate other pore-forming proteins.