Fluorescence-enabled electrochemical microscopy with dihydroresorufin as a fluorogenic indicator.

Recently, we introduced a new electrochemical imaging technique called fluorescence-enabled electrochemical microscopy (FFEM). The central idea of FEEM is that a closed bipolar electrode is utilized to electrically couple a redox reaction of interest to a complementary fluorogenic reaction converting an electrochemical signal into a fluorescent signal. This simple strategy enables one to use fluorescence microscopy to observe conventional electrochemical processes on very large electrochemical arrays. The initial demonstration of FEEM focused on the use of a specific fluorogenic indicator, resazurin, which is reduced to generate highly fluorescent resorufin. The use of resazurin has enabled the study of analyte oxidation reactions, such as the oxidation of dopamine and H2O2. In this report, we extend the capability of FEEM to the study of cathodic reactions using a new fluorogenic indicator, dihydroresorufin. Dihydroresorufin is a nonfluorescent molecule, which can be electrochemically oxidized to generate resorufin. The use of dihydroresorufin has enabled us to study a series of reducible analyte species including Fe(CN)6(3-) and Ru(NH3)6(3+). Here we demonstrate the correlation between the simultaneously recorded fluorescence intensity of resorufin and electrochemical oxidation current during potential sweep experiments. FEEM is used to quantitatively detect the reduction of ferricyanide down to a concentration of approximately 100 µM on a 25 µm ultramicroelectrode. We also demonstrate that dihydroresorufin, as a fluorogenic indicator, gives an improved temporal response and significantly decreases diffusional broadening of the signal in FEEM as compared to resazurin.

Fluorescence coupling for direct imaging of electrocatalytic heterogeneity.

Here we report the use of fluorescence microscopy and closed bipolar electrodes to reveal electrochemical and electrocatalytic activity on large electrochemical arrays. We demonstrate fluorescence-enabled electrochemical microscopy (FEEM) as a new electrochemical approach for imaging transient and heterogeneous electrochemical processes. This method uses a bipolar electrode mechanism to directly couple a conventional oxidation reaction, e.g., the oxidation of ferrocene, to a special fluorogenic reduction reaction. The generation of the fluorescent product on the cathodic pole enables one to directly monitor an electrochemical process with optical microscopy. We demonstrate the use of this method on a large electrochemical array containing thousands or more parallel bipolar microelectrodes to enable spatially and temporally resolved electrochemical imaging. We first image molecular transport of a redox analyte in solution using an array containing roughly 1000 carbon fiber ultramicroelectrodes. We then carry out a simple electrocatalysis experiment to show how FEEM can be used for electrocatalyst screening. This new method could prove useful for imaging transient electrochemical events, such as fast exocytosis events on single and networks of neurons, and for parallel, high-throughput screening of new electrocatalysts.

Steady-state voltammetry of a microelectrode in a closed bipolar cell.

Here we report the theory and experimental study of the steady-state voltammetric behavior of a microelectrode used as a limiting pole in a closed bipolar electrochemical cell. We show that the steady-state voltammetric response of a microelectrode used in a closed bipolar cell can be quantitatively understood by considering the responses of both poles in their respective conventional two-electrode setups. In comparison to a conventional electrochemical cell, the voltammetric response of the bipolar cell has a similar sigmoidal shape and limiting current; however, the response is often slower than that of the typical two-electrode setup. This leads to a broader voltammogram and a decreased wave slope, which can be somewhat misleading, causing the appearance that the process being studied is irreversible when it instead can be a result of the coupling of two reversible processes. We show that a large limiting current on the excess pole would facilitate the observation of a faster voltammetric response and that both redox concentration and electrode area of the excess pole affect the wave shape. Both factors should be maximized in electroanalytical experiments in order to obtain fast voltammetric responses on the main electrode of interest and to detect quick changes in analyte concentrations.

Coupled electrochemical reactions at bipolar microelectrodes and nanoelectrodes.

Here we report the voltammetric study of coupled electrochemical reactions on microelectrodes and nanoelectrodes in a closed bipolar cell. We use steady-state cyclic voltammetry to discuss the overall voltammetric response of closed bipolar electrodes (BPEs) and understand its dependence on the concentration of redox species and electrode size. Much of the previous work in bipolar electroanalytical chemistry has focused on the use of an "open" cell with the BPE located in an open microchannel. A closed BPE, on the other hand, has two poles placed in separate compartments and has remained relatively unexplored in this field. In this work, we demonstrated that carbon-fiber microelectrodes when backfilled with an electrolyte to establish conductivity are closed BPEs. The coupling between the oxidation reaction, e.g., dopamine oxidation, on the carbon disk/cylinder and the reduction of oxygen on the interior fiber is likely to be responsible for the conductivity. We also demonstrated the ability to quantitatively measure voltammetric properties of both the cathodic and anodic poles in a closed bipolar cell from a single cyclic voltammetry (CV) scan. It was found that "secondary" reactions such as oxygen reduction play an important role in this process. We also described the fabrication and use of Pt bipolar nanoelectrodes which may serve as a useful platform for future advances in nanoscale bipolar electrochemistry.

Voltammetric behavior of gold nanotrench electrodes.

We report the fabrication and electrochemical response of a gold nanoband electrode located at the bottom of a glass/epoxy nanotrench, hereafter referred to as a gold nanotrench electrode. Gold nanotrench electrodes of 12.5 and 40 nm in width with various depths from a few tens of nanometers to approximately 4 µm are fabricated and further characterized by cyclic voltammetry. The fabrication of a Au nanotrench electrode follows a simple electrochemical etching process in which a small AC signal is applied to an inlaid Au nanoband electrode submersed in a NaCl solution. The voltammetric behavior of a Au nanotrench electrode is characterized by a quasi-steady-state response at lower scan rates (e.g., <1 V/s for a 12.5-nm-wide electrode). We present an analytical expression for the quasi-steady-state diffusion-limited current of the nanotrench electrode based upon the analysis of the mass-transport resistance. Finite-element simulation of steady-state and transient voltammetric responses of the nanotrench electrodes provides additional insights for the analytical model. Peak-shaped transient voltammetric responses were observed at scan rates as low as 5 V/s for both inlaid and nanotrench electrodes. This result may suggest that the exposed area of the nanoband electrode is much greater than that expected from the fabrication of the inlaid bands. However, the extent to which this is seen is greatly decreased in the nanotrench electrode by a smoothing effect during etching. Our results confirm previous reports of excess overhanging metal and delamination crack contributing significantly to the shape and magnitude of the voltammetric response.

Scan-rate-dependent current rectification of cone-shaped silica nanopores in quartz nanopipettes.

Here we report the voltammetric behavior of cone-shaped silica nanopores in quartz nanopipettes in aqueous solutions as a function of the scan rate, v. Current rectification behavior for silica nanopores with diameters in the range 4-25 nm was studied. The rectification behavior was found to be strongly dependent on the scan rate. At low scan rates (e.g., v < 1 V/s), the rectification ratio was found to be at its maximum and relatively independent of v. At high scan rates (e.g., v > 200 V/s), a nearly linear current-voltage response was obtained. In addition, the initial voltage was shown to play a critical role in the current-voltage response of cone-shaped nanopores at high scan rates. We explain this v-dependent current-voltage response by ionic redistribution in the vicinity of the nanopore mouth.

Substrate specificity and scope of MvdD, a GRASP-like ligase from the microviridin biosynthetic gene cluster.

The cyanobacterial protease inhibitor microviridin K is ribosomally biosynthesized as a prepeptide (MvdE) and subsequently modified posttranslationally by double lactonization followed by lactamization. Two proteins belonging to the GRASP superfamily of ligases catalyze these ring closures. We here show that one of these ligases (MvdD) forms the lactones in a specific order, the larger ring being formed first, and that the ring size requirement for both lactonizations is stringent. However, for the first cyclization MvdD accepts alanine substitution in all C-terminal positions of the microviridin prepeptide that are not directly involved in the cross-linking, whereas the second lactonization is dependent on the presence of specific residues in MvdE. This suggests that MvdD possesses some, albeit limited, substrate tolerance that might be useful for the modification of peptides and proteins not belonging to the microviridin group of metabolites.