Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

Area of Science:

• Biophysics
• Surface Science
• Electrochemistry

Background:

• Proteins interacting with surfaces is crucial for biosensors and biomaterials.
• Understanding these interactions at the single-molecule level provides detailed insights.
• Azurin is a model redox protein with well-characterized electrochemical properties.

Purpose of the Study:

• To characterize the interaction between fluorescently labeled azurin and gold thin films.
• To investigate the effects of proximity and surface morphology on azurin's fluorescence.
• To demonstrate fluorescence-detected redox activity of single azurin molecules near a gold surface.

Main Methods:

• Single-molecule fluorescence intensity and lifetime measurements.
• Utilizing fluorescently labeled azurin.
• Characterizing interactions with gold thin films of varying roughness.

Main Results:

• Fluorescence quenching observed below 2.3 nm from the gold surface.
• Quantum yield decreased up to fourfold for direct protein attachment to bare gold.
• Up to fivefold fluorescence enhancement observed with increasing gold layer roughness.
• Demonstrated fluorescence-detected redox activity of individual azurin molecules near gold (lifetime switching ratio of 0.4).

Conclusions:

• Azurin's fluorescence is significantly modulated by its proximity to and interaction with gold surfaces.
• Surface roughness plays a key role in modulating fluorescence intensity.
• Single-molecule fluorescence techniques can reveal redox activity of proteins near surfaces.