Mobility-based wall adsorption isotherms for comparing capillary electrophoresis with single-molecule observations

Area of Science:

• Biophysics
• Analytical Chemistry
• Surface Science

Background:

• R-phycoerythrin (RPE) is an autofluorescent protein with potential applications in bioseparation.
• Understanding protein adsorption on surfaces is critical for optimizing analytical techniques.
• Fused-silica is a common material used in capillary electrophoresis (CE).

Purpose of the Study:

• To investigate the adsorption properties of R-phycoerythrin (RPE) on a fused-silica surface.
• To quantify adsorption and desorption kinetics at the single-molecule level.
• To determine adsorption isotherms and capacity factors for RPE on fused-silica.

Main Methods:

• Capillary electrophoresis (CE) was used to measure band shapes and migration times.
• Total internal reflection fluorescence microscopy (TIRFM) enabled single-molecule imaging of adsorption/desorption events.
• ImageJ software was utilized for counting adsorbed RPE molecules and estimating parameters.
• Computer simulations were performed to construct mobility-based adsorption isotherms.

Main Results:

• Adsorption and desorption events of RPE on fused-silica were directly observed and quantified.
• The capacity factor and desorption rate of RPE were estimated from single-molecule data.
• Mobility-based adsorption isotherms were successfully constructed, providing insights into RPE surface interactions.

Conclusions:

• The study provides a quantitative understanding of RPE adsorption on fused-silica surfaces.
• These findings are valuable for optimizing RPE-based applications in capillary electrophoresis and other analytical methods.
• The combined use of CE and single-molecule imaging offers a powerful approach for characterizing protein-surface interactions.