Microchannel Voltammetry in the Presence of Large External Voltages and Electric Fields.

The ability to perform electrochemistry in the presence of large voltages and electric field magnitudes without concern for the local potential has many possible applications in micro/nanofluidic assays and in capillary electrophoresis. Traditionally, electrochemistry in the presence of significant external electric fields has been dominated by end-channel detection for capillary and microchip electrophoresis detection. We describe novel instrumentation for potentiostatically controlled voltammetry that can be applied in the presence of high external voltages and electric fields. Cyclic voltammetry is demonstrated without significant shifts in the half-wave potential at working electrodes at local potentials of up to ∼1500 V and field strengths of up to 3000 V/cm, using a standard Ag/AgCl reference electrode.

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

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.

Potential-dependent single molecule blinking dynamics for flavin adenine dinucleotide covalently immobilized in zero-mode waveguide array of working electrodes.

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).