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

Abstract

Zero-mode waveguides (ZMW) have the potential to be powerful confinement tools for studying electron transfer dynamics at single molecule occupancy conditions. Flavin mononucleotide contains an isoalloxazine chromophore, which is fluorescent in the oxidized state (FMN) while the reduced state (FMNH2) exhibits dramatically lower light emission, i.e. a dark-state. This allows fluorescence emission to report the redox state of single FMN molecules, an observation that has been used previously to study single electron transfer events in surface-immobilized flavins and flavoenzymes, e.g. sarcosine oxidase, by direct wide-field imaging of ZMW arrays. Single molecule electron transfer dynamics have now been extended to the study of freely diffusing molecules using fluorescence measurements of Au ZMWs under single occupancy conditions. The Au in the ZMW serves both as an optical cladding layer and as the working electrode for potential control, thereby accessing single molecule electron transfer dynamics at μM concentrations. Consistent with expectations, the probability of observing single reduced molecules increases as the potential is scanned negative, E(appl) < E(eq), and the probability of observing emitting oxidized molecules increases at E(appl) > E(eq). Different single molecules exhibit different electron transfer properties as reflected in the position of E(eq) and the distribution of E(eq) among a population of FMN molecules. Two types of actively-controlled electroluminescence experiments were used: chronofluorometry experiments, in which the potential is alternately stepped between oxidizing and reducing potentials, and cyclic potential sweep fluorescence experiments, analogous to cyclic voltammetry, these latter experiments exhibiting a dramatic scan rate dependence with the slowest scan rates showing distinct intermediate states that are stable over a range of potentials. These states are assigned to flavosemiquinone species that are stabilized in the special environment of the ZMW nanopore.

SERS speciation of the electrochemical oxidation–reduction of riboflavin

“…Flavin is brightly fluorescent in the oxidized state and dim in the reduced state, while the semi-quinone is somewhere in between. 20, 28 Chronofluorometry experiments of FMN showed the ability to cycle between the bright (oxidized) and dark (reduced) states of the molecule. However, cyclic potential sweep fluorescence of the same species showed a scan rate dependence wherein at fast scan rates the emission spectra is similar to the bulk solution measurement, while at slow scan rates distinct intermediates states are observed between the oxidized and reduced species.…”

“…However, cyclic potential sweep fluorescence of the same species showed a scan rate dependence wherein at fast scan rates the emission spectra is similar to the bulk solution measurement, while at slow scan rates distinct intermediates states are observed between the oxidized and reduced species. 28 It was hypothesized that this appearance was a result of the stabilization of the semi-quinone in the zero-mode waveguide nanopore. The speciation observed in our experiments agrees with this hypothesis.…”

Single‐Molecule Sensors: Challenges and Opportunities for Quantitative Analysis

“…[38] There are many variants of this type of experiment for the monitoring of enzyme kinetics,a shasb een recently reviewed. [41] FMN is aredox enzyme cofactor,and closely related electrochemically and optically to the ubiquitous enzyme cofactor flavin adenine dinucleotide.I mportantly, when excited, the oxidized form is fluorescent but the reduced form is less fluorescent. [40] A particularly exciting development in this field is the development by Bohn and co-workers of arrays of zero-mode waveguides for spectroelectrochemical investigation of as ingle flavin mononucleotide (FMN) imaged in aw idefield format.…”

Micro‐ and Nanoscopic Imaging of Enzymatic Electrodes: A Review

“…Moreover, reduction and oxidation half‐reactions were shown to be independent . The second case concerns immobilized FAD and flavin mononucleotide freely diffusing in the nanopores . A similar setup allowed to study the spectroelectrochemical behavior, and especially fluorescence arising from single‐electron transfer events of these two cofactors.…”