Zero mode waveguides for single-molecule spectroscopy on lipid membranes.

Area of Science:

• Nanotechnology
• Biophysics
• Surface Science

Background:

• Zero mode waveguides (ZMWs) are subwavelength nanostructures used for studying ligand-receptor interactions.
• Lipid membranes can invaginate into ZMWs, confining optical excitation to subattoliter volumes.

Purpose of the Study:

• To investigate lipid membrane behavior within ZMWs.
• To characterize lipid diffusion and protein-membrane interactions using Fluorescence Correlation Spectroscopy (FCS).
• To determine the binding affinity of tetanus toxin C fragment to ganglioside-populated membranes.

Main Methods:

• Utilized ZMWs with dimensions of 50-200 nm.
• Employed FCS to analyze fluorescently tagged lipids (POPC and DSPC) in supported lipid bilayers.
• Measured the binding of labeled tetanus toxin C fragment to ganglioside-populated POPC membranes within ZMWs.

Main Results:

• Lipid membrane invagination into ZMWs was observed to be dependent on membrane rigidity (POPC vs. DSPC).
• Diffusion constants for POPC membranes within ZMWs were lower than expected, attributed to non-trivial membrane conformation.
• Determined the equilibrium binding constant for tetanus toxin C fragment at 500 nM, exceeding the capabilities of diffraction-limited FCS.

Conclusions:

• Supported lipid bilayers can be effectively studied within nanostructured devices like ZMWs.
• ZMWs facilitate single-molecule resolution studies of membrane-embedded receptors and proteins at physiological concentrations.
• Membrane conformation within ZMWs significantly influences diffusion characteristics.