Variations in the formal electrochemical potential (E0) and electron‐transfer rates (k0) of the blue copper protein azurin have been directly observed. A new method, fluorescent cyclic voltammetry (FCV), was used to resolve the properties of 100–1000 proteins. On this scale, the presence of large variations in the values of both E0 and k0 could be established and several forms of heterogeneity were differentiated.

Impedance derived electroanalytical assays are inherently spectroscopic (frequency resolved) and potentially exceedingly sensitive indicators of interfacial change (such as target binding at an appropriate receptor). We introduce here the use of a portfolio of mathematically derived immittance functions and related components, capable, from the same raw data sets, of enabling increased assay sensitivity and markedly shorter assay times in comparison to traditional impedance analyses. The methodology, applied herein to faradaic (redox probe amplified) and non-faradaic assays, requires no equivalent circuit analysis or prior assumption of response. Its focus is to optimize analytical potency and to enable the user to select and apply the most frequency-optimized reporter of interfacial change and to, thereafter, run rapid (optimized) analyses at single frequencies.

With the view of enhancing the functionality of label-free single molecule nanopore-based detection, we have designed and developed a highly robust, mechanically stable, integrated nanopipette-microfluidic device which combines the recognized advantages of microfluidic systems and the unique properties/advantages of nanopipettes. Unlike more typical planar solid-state nanopores, which have inherent geometrical constraints, nanopipettes can be easily positioned at any point within a microfluidic channel. This is highly advantageous, especially when taking into account fluid flow properties. We show that we are able to detect and discriminate between DNA molecules of varying lengths when motivated through a microfluidic channel, upon the application of appropriate voltage bias across the nanopipette. The effects of applied voltage and volumetric flow rates have been studied to ascertain translocation event frequency and capture rate. Additionally, by exploiting the advantages associated with microfluidic systems (such as flow control and concomitant control over analyte concentration/presence), we show that the technology offers a new opportunity for single molecule detection and recognition in microfluidic devices.

To stabilize nanotube-modified atomic force microscopy tips and extend their functionality, a method for conformal polymer coating of the tips and removal of the polymer from just the probing nanotube end is described. Expressions quantifying stabilization of the tips against buckling and bending due to stresses encountered while imaging are developed. Electrical conductivity of the probes is demonstrated by their use in scanned conductance microscopy, where a substantial sensitivity advantage over standard tips is realized and explained. Further advantages of these electrically insulated (save for the tip) probes are discussed, including their proposed application in bioelectrochemical research.

Among the numerous label free electronic biomarker assay methodologies now available, impedance based electrochemical capacitance spectroscopy (ECS), based upon mapping the perturbations in interfacial charging of redox elements incorporated into a biologically receptive interface, has recently been shown to be a convenient and highly sensitive mode of transduction and one which, additionally, requires no predoping of analytical solution. We present, herein, a data acquisition and analysis methodology based on frequency resolved immittance function analysis. Ultimately, this enables both a maximization of assay sensitivity and a reduction in assay acquisition time by an order of magnitude.

Bacteriorhodopsin (BR) is a robust light-driven proton pump embedded in the purple membrane of the extremophilic archae Halobacterium salinarium. Its photoactivity remains in the dry state, making BR of significant interest for nanotechnological use. Here, in a novel configuration, BR was depleted from most of its endogenous lipids and covalently and asymmetrically anchored onto a gold electrode through a strategically located and highly responsive cysteine mutation; BR has no indigenous cysteines. Chemisorption on gold was characterized by surface plasmon resonance, reductive striping voltammetry, ellipsometry, and atomic force microscopy (AFM). For the first time, the conductance of isolated protein trimers, intimately probed by conducting AFM, was reproducibly and reversibly switched under wavelength-specific conditions (mean resistance of 39 ± 12 MΩ under illumination, 137 ± 18 MΩ in the dark), demonstrating a surface stability that is relevant to potential nanodevice applications.

Schwankungen im formalen elektrochemischen Potential (E0) und in der Elektronentransfergeschwindigkeit (k0) des blauen Kupferproteins Azurin wurden direkt beobachtet. Die Fluoreszenz‐Cyclovoltammetrie wurde als ein neues Verfahren angewendet, um die Eigenschaften von 100–1000 Proteinen zu charakterisieren. Dabei wurden starke Schwankungen für E0 und k0 bestimmt, und man konnte unterschiedliche Arten von Heterogenitäten unterscheiden.

In coupling the redox state of an adsorbed molecule to its spectral characteristics redox profiles can be directly imaged by means of far-field fluorescence. At suitable levels of dilution, on optically transparent electrode surfaces, reversible interfacial electron transfer processes can be followed pixel by pixel down to scales which approach the molecular. In mapping out switching potentials across a surface population, thermodynamic dispersion, related to variance in the orientation, electronic coupling, protein fold, electric field drop, and general surface order, can be quantified. The self-assembled monolayer buffering the protein from the underlying metallic electrode surface not only acts to tune electronic coupling between the two but also potentially provides a variable more easily segmented from other contributions to molecular dispersion. We have, specifically, considered the possibility that the supporting monolayer crystallinity is a significant contributor to the subsequently observed spread in half-wave potentials. We report here that this is indeed the case and that this spread diminishes from 17 to 12 mV for the blue copper protein azurin as the supporting alkanethiol layer crystallinity increases. The work herein, then, presents not only a direct determination of submonolayer scale variance in redox character but also a means of tuning this through gross surface and entirely standard chemical means.