A Biosensor Platform for Detecting the Dissipation of Transmembrane Gradients in Single Liposomes

Area of Science:

• Biochemistry
• Molecular Biology
• Biophysics

Background:

• Pore forming proteins create transmembrane pores, disrupting cellular gradients and function.
• Current detection methods like dye release assays lack sensitivity and single-molecule resolution.
• Single molecule detection is sensitive but challenged by intensity variations.

Purpose of the Study:

• To develop a sensitive and general biosensor for detecting pore forming activity.
• To measure transmembrane gradient dissipation at the single liposome level.
• To detect specific toxins like Botulinum neurotoxin without protein-specific reagents.

Main Methods:

• Utilized Förster Resonance Energy Transfer (FRET) biosensors with ligand-responsive oligonucleotides.
• Encapsulated biosensors within liposomes to sustain chemical gradients.
• Employed Total Internal Reflection Fluorescence (TIRF) microscopy for single liposome analysis.

Main Results:

• Demonstrated single liposome resolution detection of transmembrane gradient dissipation.
• Achieved picomolar concentration detection of Botulinum neurotoxin.
• Showcased the biosensor's ability to function independently of pore formation mechanism or protein-specific antibodies.

Conclusions:

• The FRET biosensor platform provides a sensitive and versatile method for detecting pore forming proteins.
• This approach enables the study of transmembrane solute movement and transport mechanisms.
• The technology offers a general platform for detecting toxic substances and understanding their mechanisms of action.