Single occupancy spectroelectrochemistry of freely diffusing flavin mononucleotide in zero-dimensional nanophotonic...

Area of Science:

• Single-molecule biophysics
• Electrochemistry
• Nanophotonics

Background:

• Zero-mode waveguides (ZMWs) enable single-molecule studies.
• Flavin mononucleotide (FMN) fluorescence reports its redox state (oxidized FMN vs. reduced FMNH2).
• Previous studies focused on surface-immobilized flavins.

Purpose of the Study:

• To extend single molecule electron transfer dynamics studies to freely diffusing molecules.
• To utilize gold ZMWs for optical confinement and electrochemical control.
• To investigate FMN redox state dynamics at micromolar concentrations.

Main Methods:

• Single molecule fluorescence measurements in gold ZMWs.
• Controlled electroluminescence experiments: chronofluorometry and cyclic potential sweep fluorescence.
• Utilizing ZMWs as optical cladding and working electrodes for potential control.

Main Results:

• Observed redox state changes of single diffusing FMN molecules.
• Correlated molecule's redox state with applied potential (E(appl) vs. E(eq)).
• Identified distinct intermediate states, assigned to flavin semiquinone species, in cyclic potential sweep experiments.

Conclusions:

• Gold ZMWs facilitate single molecule electron transfer studies of freely diffusing molecules.
• Electroluminescence experiments reveal population heterogeneity in electron transfer properties.
• ZMW nanopore environment stabilizes intermediate flavin semiquinone species.