Quantification of receptor targeting aptamer binding characteristics using single-molecule spectroscopy.

Area of Science:

• Biophysics
• Microbiology
• Analytical Chemistry

Background:

• Outer membrane proteins (OMPs) on bacteria like Salmonella typhimurium are crucial targets for diagnostics and therapeutics.
• Aptamers offer high specificity and affinity for molecular recognition, making them promising for biosensor development.
• Quantifying bacterial surface receptors and their binding kinetics at the single-cell level remains challenging.

Purpose of the Study:

• To develop and validate a single-molecule approach for quantifying outer membrane proteins (OMPs) on live Salmonella typhimurium.
• To determine the binding kinetics and affinity of aptamers targeting specific OMPs on S. typhimurium.
• To assess the feasibility of using aptamer-based fluorescence correlation spectroscopy (FCS) for bacterial receptor analysis.

Main Methods:

• Utilized fluorescence correlation spectroscopy (FCS) with fluorescently labeled aptamer probes.
• Applied correlation analysis to study the diffusion dynamics of bound and unbound aptamers on live S. typhimurium.
• Quantified dissociation constants and receptor densities by analyzing aptamer-bacterial interactions.

Main Results:

• Successfully quantified OMPs on live S. typhimurium using aptamer probes.
• Determined dissociation constants for aptamers ranging from 0.1285 to 0.3772 nM.
• Calculated receptor densities on S. typhimurium to be between 42.27 and 49.82 receptors/µm².
• Demonstrated high binding affinity of aptamers to S. typhimurium surface receptors.

Conclusions:

• The developed single-molecule FCS method enables sensitive quantification of bacterial surface receptors and their binding kinetics.
• Aptamers exhibit high affinity for S. typhimurium OMPs, suitable for biosensor applications.
• This methodology can be broadly applied to study live bacterial receptor-ligand interactions in various biological and environmental contexts.