Precise Electrochemical Sizing of Individual Electro-Inactive Particles.

Nanoimpact electrochemistry enables the time-resolved in situ characterization (e.g., size, catalytic activity) of single nanomaterial units, providing a means of elucidating heterogeneities that would be masked in ensemble studies. To implement this technique with redox inactive particles, a solution-phase redox reaction is used to produce a steady-state background current on a disk ultramicroelectrode. When a particle adsorbs onto the electrode, it produces a stepwise reduction in the exposed electrode area, which produces, in turn, a stepwise decrease in the current commensurate with the size of the adsorbing species. Historically, however, nanoimpact electrochemistry has suffered from edge effects, in which the radial diffusion layer formed at the circumference of the ultramicroelectrodes renders the step size dependent not only on the size of the particle but also on where it lands on the electrode. The introduction of electrocatalytic current generation, however, mitigates the heterogeneity caused by edge effects, thus improving the measurement precision. In this approach, termed electrocatalytic interruption, a substrate that regenerates the redox probe at the diffusion layer is introduced. This shifts the rate-limiting step of the current generation from diffusion to the homogeneous reaction rate constant, thus reducing flux heterogeneity and increasing the precision of particle sizing by an order of magnitude. The protocol described here explains the set-up and data collection employed in nanoimpact experiments implementing this effect for improved precision in the sizing of redox in-active materials

Temperature and force dependence of nanoscale electron transport via the Cu protein Azurin

The mechanisms of solid-state electron transport (ETp) via a monolayer of immobilized Azurin (Az) was examined by conducting probe atomic force microscopy (CP-AFM), both as function of temperature (248 - 373K) and of applied tip force (6-12 nN). By varying both temperature and force in CP-AFM, we find that the ETp mechanism can alter with a change in the force applied via the tip to the proteins. As the applied force increases, ETp via Az changes from temperature-independent to thermally activated at high temperatures. This is in contrast to the Cu-depleted form of Az (apo-Az), where increasing the applied force causes only small quantitative effects, that fit with a decrease in electrode spacing. At low force ETp via holo-Az is temperature-independent and thermally activated via apo-Az. This observation agrees with macroscopic-scale measurements, thus confirming that the difference in ETp dependence on temperature between holo- and apo-Az is an inherent one that may reflect a difference in rigidity between the two forms. An important implication of these results, which depend on CP-AFM measurements over a significant temperature range, is that for ETp measurements on floppy systems, such as proteins, the stress applied to the sample should be kept constant or, at least controlled during measurement.Comment: 24 pages, 6 figures, plus Supporting Information with 4 pages and 2 figure

On the Disinfection of Electrochemical Aptamer-Based Sensors.

Electrochemical aptamer-based (EAB) sensors encompass the only biosensor approach yet reported that is simultaneously: (1) independent of the chemical or enzymatic reactivity of its target, rendering it general; (2) continuous and real-time; and (3) selective enough to deploy in situ in the living body. Consistent with this, in vivo EAB sensors supporting the seconds-resolved, real-time measurement of multiple drugs and metabolites have been reported, suggesting the approach may prove of value in biomedical research and the diagnosis, treatment, and monitoring of disease. However, to apply these devices in long-duration animal models, much less in human patients, requires that they be free of any significant pathogen load. Thus motivated, here we have characterized the compatibility of EAB sensors with standard sterilization and high-level disinfection techniques. Doing so, we find that, while many lead to significant sensor degradation, treatment with CIDEX OPA (0.55% ortho-phthalaldehyde) leads to effective disinfection without causing any detectable loss in sensor performance

Direct Electricity Production from <i>Nematostella</i> and <i>Arthemia</i>’s Eggs in a Bio-Electrochemical Cell

In recent years, extensive efforts have been made to develop clean energy technologies to replace fossil fuels to assist the struggle against climate change. One approach is to exploit the ability of bacteria and photosynthetic organisms to conduct external electron transport for electricity production in bio-electrochemical cells. In this work, we first show that the sea anemones Nematostella vectensis and eggs of Artemia (brine shrimp) secrete redox-active molecules that can reduce the electron acceptor Cytochrome C. We applied 2D fluorescence spectroscopy and identified NADH or NADPH as secreted species. Finally, we broaden the scope of living organisms that can be integrated with a bio-electrochemical cell to the sea anemones group, showing for the first time that Nematostella and eggs of Artemia can produce electrical current when integrated into a bio-electrochemical cell

Understanding single enzyme activity via the nano-impact technique.

To evaluate the possible detection of single enzyme activity via electrochemical methods, a combined finite difference and random walk simulation is used to model individual enzyme-electrode collisions where such events are monitored amperometrically via the measurement of products formed by the enzyme in solution. It is found that the observed signal is highly sensitive to both the enzyme turnover number, the size of the electrode and the bandwidth of the electronics. Taking single catalase impacts as an example, simulation results are compared with experimental data. Our work shows the requirement for the detection of electrochemically active product formed by individual enzymes and gives guidance for the design of experiments

Calibration-Free, Seconds-Resolved In Vivo Molecular Measurements using Fourier-Transform Impedance Spectroscopy Interrogation of Electrochemical Aptamer Sensors.

Electrochemical aptamer-based (EAB) sensors are capable of measuring the concentrations of specific molecules in vivo, in real time, and with a few-second time resolution. For their signal transduction mechanism, these sensors utilize a binding-induced conformational change in their target-recognizing, redox-reporter-modified aptamer to alter the rate of electron transfer between the reporter and the supporting electrode. While a variety of voltammetric techniques have been used to monitor this change in kinetics, they suffer from various drawbacks, including time resolution limited to several seconds and sensor-to-sensor variation that requires calibration to remove. Here, however, we show that the use of fast Fourier transform electrochemical impedance spectroscopy (FFT-EIS) to interrogate EAB sensors leads to improved (here better than 2 s) time resolution and calibration-free operation, even when such sensors are deployed in vivo. To showcase these benefits, we demonstrate the approachs ability to perform real-time molecular measurements in the veins of living rats

Electrochemistry of Single Enzymes: Fluctuations of Catalase Activities

Dynamic fluctuations of the catalytic ability of single catalase enzymes toward hydrogen peroxide decomposition are observed via the nanoimpact technique. The electrochemical signals of single enzymes show that the catalytic ability of single enzymes can temporarily be much higher than expected from the classical, time-averaged Michaelis–Menten description. By combination of experimental data with a new theoretical model, we interpret the unusual enhancement of the single catalase signal and find that single catalases show large fluctuations of the catalytic ability