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.
Single molecules exhibit a set of behaviors that are characteristic and distinct from larger ensembles. Blinking is one such behavior that involves episodic transitions between luminescent and dark states. In addition to the common blinking mechanisms, flavin adenine dinucleotide (FAD), a cofactor in many common redox enzymes, exhibits blinking by cycling between a highly fluorescent oxidized state and a dark reduced state. In contrast to its behavior in flavoenzymes, where the transitions are coupled to chemical redox events, here we study single FAD molecules that are chemically immobilized to the Au region of a zero-mode waveguide (ZMW) array through a pyrroloquinoline quinone (PQQ) linker. In this structure, the Au functions both to confine the optical field in the ZMW and as the working electrode in a potentiostatically controlled 3-elecrode system, thus allowing potential-dependent blinking to be studied in single FAD molecules. The subset of ZMW nanopores housing a single molecule were identified statistically, and these were subjected to detailed study. Using equilibrium potential, E(eq), values determined from macroscopic planar Au electrodes, single molecule blinking behavior was characterized at potentials E < E(eq), E - E(eq), and E > E(eq). The probability of observing a reduced (oxidized) state is observed to increase (decrease) as the potential is scanned cathodic of E(eq). This is understood to reflect the potential-dependent probability of electron transfer for single FAD molecules. Furthermore, the observed transition rate reaches a maximum near E(eq) and decreases to either anodic or cathodic values, as expected, since the rate is dependent on having significant probabilities for both redox states, a condition that is obtained only near E(eq).
The ability of zero-mode waveguides (ZMW) to guide light into subwavelength-diameter nanoapertures has been exploited for studying electron transfer dynamics in zeptoliter-volume nanopores under single-molecule occupancy conditions.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.