Microscopic Screening of Cyclodextrin Channel Blockers by DiffusiOptoPhysiology.

Area of Science:

• Biophysics
• Nanotechnology
• Microscopy

Background:

• Pore-forming toxins (PFTs) are key bacterial virulence factors.
• Blocking PFTs is a strategy to limit bacterial infections.
• Screening PFT blockers is challenging due to cost and throughput limitations.

Purpose of the Study:

• To evaluate DiffusiOptoPhysiology (DOP) as a method for screening PFT blockers.
• To demonstrate the use of fluorescence microscopy for direct single-molecule PFT blocker screening.
• To assess the potential of DOP for high-throughput screening of novel therapeutics.

Main Methods:

• Utilized DiffusiOptoPhysiology (DOP), a nanopore technique using fluorescence microscopy.
• Employed α-hemolysin (α-HL) mutants as model PFTs and cyclodextrins as model blockers.
• Simultaneously evaluated pore-blocker combinations on a disposable DOP chip.

Main Results:

• Achieved direct, single-molecule resolution screening of PFT blockers using fluorescence microscopy.
• Demonstrated successful identification of blockers for α-HL mutants.
• Confirmed the low-cost, biocompatible, and disposable nature of the DOP chip.

Conclusions:

• DiffusiOptoPhysiology (DOP) is a viable and efficient technique for screening PFT blockers.
• The method offers advantages in cost, throughput, and resolution for drug discovery.
• This proof-of-concept study suggests broad applicability for investigating various pore-forming proteins.