Anna L. Whitworth

Scanning electrochemical microscopy: principles and applications to biophysical systems

This review highlights numerous and wide ranging biophysical and biochemical applications of scanning electrochemical microscopy (SECM). SECM instrumentation and theoretical modelling, necessary for experimental interpretation, are outlined, followed by a detailed discussion of the diverse applications of this technique. These include the measurement of flow through membranes, the determination of kinetic parameters of reactions, the investigation of the permeability of small molecules in tissues and monitoring biological processes, such as the production of oxygen or nitric oxide by cells. The significant impact of micro-electrochemical techniques on our understanding of basic physicochemical processes at biologically relevant interfaces is also considered. Studies reviewed include transport across and within bilayers and monolayers. Recent advances in SECM include the combination of SECM with other techniques, such as atomic force microscopy and optical microscopy. These developments are highlighted, along with prospects for the future.

Scanning Ion Conductance Microscopy: a Model for Experimentally Realistic Conditions and Image Interpretation

Scanning ion conductance microscopy (SICM) is a scanned probe microscopy technique in which the probe is a fine glass pipet filled with a contact (reference) electrode and an electrolyte solution. The current flow between the reference electrode and a second reference electrode positioned in bulk solution when the two electrodes are biased externally can be used as a feedback signal to maintain a constant separation between the tip and a surface during imaging. In usual practice the tip position is modulated over a small amplitude perpendicular to the surface, and the resulting alternating current (AC) is used as the feedback signal, although the direct current can also be used. A comprehensive model for the current response is reported. Laplaces equation has been solved for the electrolyte solution for a range of tip geometries, enabling the factors controlling the tip current to be identified. The approach developed is shown to represent an improvement over earlier semiempirical treatments. To explore the influence of surface topography on the (AC) current response, various surfaces have been considered, including a tip moved toward a planar surface (in the normal direction) and tips scanned over a pit and a step in the surface. The results have allowed a critical assessment of the SICM response as a means of probing surface topography. Features identified through simulation have been found in experiments through studies of two model substrates for which imaging results are reported. In typical experimental practice, the response of the SICM tip to surface features occurs over much greater lateral distances than the size of the pipet aperture.

Theory of scanning electrochemical microscopy (SECM) as a probe of surface conductivity

Scanning electrochemical microscopy (SECM) can be used in the feedback mode as a ‘contactless’ method for probing the conductivity of ultra-thin films. With this new technique a sample is bathed with one form of a reversible redox (Red/Ox) couple, e.g. Red, and an ultramicroelectrode (UME), located close to the film surface is used to locally convert Red to Ox by diffusion-limited oxidation. Ox diffuses from the UME to the film where it may undergo conversion back to Red. This process is driven by a potential gradient, which depends on the surface conductivity. A model is developed for the SECM feedback process from which the conductivity of the film can be extracted. Two routes arising from the model are considered: an approximate method which allows an order of magnitude estimate of the film conductivity and a more involved approach, which can be used with greater accuracy. The merits of the two approaches are assessed by analysing experimental data for the effect of surface pressure on the conductivity of a polyaniline monolayer at a water/air interface.

Quantitative Analysis and Application of Tip Position Modulation-Scanning Electrochemical Microscopy

Tip position modulation (TPM) involves moving the ultramicroelectrode (UME) tip of a scanning electrochemical microscope (SECM) perpendicular to the substrate in a sinusoidal fashion with a small amplitude compared to the tip/sample separation. The UME, which serves as the working electrode in a conventional voltammetric setup, is held at a potential to detect a species in solution at a transport-limited rate and the resulting current (ac and dc) is measured. This paper shows that tip-induced convection is an important factor in TPM. A model has been developed that describes the TPM response for the most challenging case of an inert substrate, where tip-induced convective effects compared to diffusion are greatest. The model provides an improved description of the ac response compared to existing treatment, as evidenced by the analysis of TPM-SECM approach curves (current-distance characteristics). The extension of the model to SECM-induced transfer is considered and it is shown that one can extract highly precise information on the permeability of a sample from such measurements, for which experiments and theory are compared. The prospects for using the technique more widely are highlighted and routes to improving the theoretical analysis further are briefly discussed.

Polyaniline Langmuir-Blodgett films: formation and properties

The deposition and characterisation of Langmuir-Blodgett (LB) layers of polyaniline (PAN) on solid supports is described. Langmuir films were spread as a mixture of PAN and dodecylbenzenesulfonic acid (DBSA) at the water/air interface and deposited on either glass or indium tin oxide (ITO). Mono- and multi-layer films of PAN/DBSA were characterized by X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM), absorption spectroscopy and cyclic voltammetry (CV). The ultrathin films produced were found to be highly uniform and very stable. Further characterisation of the films was accomplished by scanning electrochemical microscopy (SECM) in the feedback mode. It was found that the conductivity depended strongly on the pH of the solution and the number of layers deposited. Values for the pH-dependent lateral conductivity of PAN LB films are reported.

Discovery of cyanophage genomes which contain mitochondrial DNA polymerase

DNA polymerase γ is a family A DNA polymerase responsible for the replication of mitochondrial DNA in eukaryotes. The origins of DNA polymerase γ have remained elusive because it is not present in any known bacterium, though it has been hypothesized that mitochondria may have inherited the enzyme by phage-mediated nonorthologous displacement. Here, we present an analysis of two full-length homologues of this gene, which were found in the genomes of two bacteriophages, which infect the chlorophyll-d containing cyanobacterium Acaryochloris marina. Phylogenetic analyses of these phage DNA polymerase γ proteins show that they branch deeply within the DNA polymerase γ clade and therefore share a common origin with their eukaryotic homologues. We also found homologues of these phage polymerases in the environmental Community Cyberinfrastructure for Advanced Microbial Ecology Research and Analysis (CAMERA) database, which fell in the same clade. An analysis of the CAMERA assemblies containing the environmental homologues together with the filter fraction metadata indicated some of these assemblies may be of bacterial origin. We also show that the phage-encoded DNA polymerase γ is highly transcribed as the phage genomes are replicated. These findings provide data that may assist in reconstructing the evolution of mitochondria.

MICROELECTROCHEMICAL TECHNIQUES FOR PROBING KINETICS AT LIQUID/LIQUID INTERFACES

We provide an overview of recent advances in microelectrochemical approaches to investigate the kinetics of various physicochemical processes that occur at the interface between two immiscible electrolyte solutions (ITIES). To place the advances in context, background material on the structure of the ITIES, derived from both experimental studies and computer simulation, is also provided. The main focus of the article is micro-ITIES techniques, single droplet measurements, microelectrochemical measurements at expanding droplets (MEMED) and scanning electrochemical microscopy (SECM). Recent developments in a combined SECM-Langmuir trough technique for probing diffusion processes across Langmuir monolayers at the water/air (W/A) interface are also highlighted, by considering an organic monolayer at a water surface as a special case of a liquid/liquid interface.

Genomic and proteomic characterization of two novel siphovirus infecting the sedentary facultative epibiont cyanobacterium Acaryochloris marina

Acaryochloris marina is a symbiotic species of cyanobacteria that is capable of utilizing far-red light. We report the characterization of the phages A-HIS1 and A-HIS2, capable of infecting Acaryochloris. Morphological characterization of these phages places them in the family Siphoviridae. However, molecular characterization reveals that they do not show genetic similarity with any known siphoviruses. While the phages do show synteny between each other, the nucleotide identity between the phages is low at 45-67%, suggesting they diverged from each other some time ago. The greatest number of genes shared with another phage (a myovirus infecting marine Synechococcus) was four. Unlike most other cyanophages and in common with the Siphoviridae infecting Synechococcus, no photosynthesis-related genes were found in the genome. CRISPR (clustered regularly interspaced short palindromic repeats) spacers from the host Acaryochloris had partial matches to sequences found within the phages, which is the first time CRISPRs have been reported in a cyanobacterial/cyanophage system. The phages also encode a homologue of the proteobacterial RNase T. The potential function of RNase T in the mark-up or digestion of crRNA hints at a novel mechanism for evading the host CRISPR system.