Jens Enevold Thaulov Andersen

In situ STM and AFM of the copper protein Pseudomonas aeruginosa azurin

Abstract Scanning tunnel (STM) and atomic force microscopy (AFM) in the in situ mode under potentiostatic control have opened new perspectives for mapping the two-dimensional organization of surface adsorbates in aqueous solution. In situ STM and AFM, however, also raise recognized problems. In the context of biological macromolecules, sample immobilization and the mechanism of the imaging process are, for example, outstanding issues. We have shown that the blue single-copper redox protein azurin is well suited for gentle surface immobilization and mapping. Azurin has a surface disulphide group which adsorbs to gold and facile electron tunnel routes between this group and the copper atom. Azurin adsorbed on Au(111) can be imaged to molecular resolution by in situ STM and shows regular arrays of individual structures corresponding well to the known molecular size of azurin. The current falls off approximately exponentially with increasing distance with a decay constant of 0.4–0.5 A −1 . In comparison in situ AFM shows structures laterally convoluted with the tip while the vertical extension is in the same range as the structural size of azurin. The results are of interest in relation to electron tunnel mechanisms of redox metalloproteins and in technological contexts such as electrochemical biosensors, microbial corrosion and broadly for protein adsorption from biological liquids.

Electrochemistry of Self-Assembled Monolayers of the Blue Copper Protein Pseudomonas Aeruginosa Azurin on Au(111)

Abstract We report the self-assembly and electrochemical behaviour of the blue copper protein Pseudomonas aeruginosa azurin on Au(111) electrodes in aqueous acetate buffer (pH=4.6). The formation of monolayers of this protein is substantiated by electrochemical measurements. Capacitance results indicate qualitatively that the protein is strongly adsorbed at sub-μM concentrations in a broad potential range (about 700 mV). This is further supported by the attenuation of a characteristic cyclic voltammetric peak of Au(111) in acetate solution with increasing azurin concentration. Reductive desorption is clearly disclosed in NaOH solution (pH=13), strongly suggesting that azurin is adsorbed via its disulphide group. An anodic peak and a cathodic peak associated with the copper centre of azurin are finally observed in the differential pulse voltammograms. These peaks are, interestingly, indicative of long-range electrochemical electron transfer such as paralleled by intramolecular electron transfer between the disulphide anion radical and the copper atom in homogeneous solution, and anticipated by theoretical frames. Together with reported in situ scanning tunnelling microscopy (STM) results they constitute the first case for electrochemistry of self-assembled monolayers of azurin, even redox proteins. This integrated investigation provides a new approach to both structure and function of adsorbed redox metalloproteins at the molecular level.

A novel method for the filterless preconcentration of iron

A novel method of analysis of iron by filterless preconcentration is presented. This is the first example of efficient preconcentration of a refractory transition metal where coprecipitation and columns were omitted. The method applies a manifold of flow injection analysis (FIA) to iron species that are preconcentrated on the inner walls of a tubular reactor. It was found that the adsorption of iron species to the walls was particularly pronounced in reactors of nylon material and enrichment factors of 30-35 could be attained, as dependent on the length of the reactor and on the time of preconcentration. In the preconcentration step of the FIA accessory, the optimum efficacy was obtained when the acidity of the samples was adjusted by HCl to pH = 2.5 whereas the ammonia preconcentration buffer should be kept alkaline at pH = 9.8. After being preconcentrated on the tubular reactor, the iron species were eluted by hydrochloric acid and analysed by flame atomic absorption spectrometry (FAAS). An unprecedented low limit of detection (LOD, 3sigma) of 1.8 microg L(-1) was thus obtained for the analysis of iron by FAAS, and the calibration line was linear up to 100 microg L(-1). A high sampling frequency of 40 per hour was obtained and the protocol allowed analysis of small amounts of iron in drinking water, in digested samples and in saline water. The major influence of interferences originated from ligands that are known to complex strongly to iron species. The method thus developed was uncomplicated in operation and it exhibited an excellent long-term stability and a high selectivity. Further, it was environmentally safe owing to production of non-toxic waste disposals. Equally high enrichment factors (EF) were obtained for Fe(ii) and Fe(iii), which is explained by the very low solubility product of both species.

Cytochrome c dynamics at gold and glassy carbon surfaces monitored by in situ scanning tunnel microscopy

Abstract We have investigated the absorption of cytochrome c on gold and glassy carbon substrates by in situ scanning tunnel microscopy under potentiostatic control of both substrate and tip. Low ionic strength and potential ranges where no Faradaic current flows were used. Cyt c aggregates into flat composite structures of about 50 nm lateral extension at gold surfaces. The aggregates evolve in time, and structures resembling individual cyt c molecules can be distinguished in the space between the 50 nm structures. Cyt c aggregates also form at glassy carbon but have a different, unbroken character where cyt c both sticks well to the surface and exhibits notable mobility. The observations suggest that characteristic surface specific, internally mobile protein aggregates are formed at both surfaces and that in situ molecular resolution of the STM pictures may have been achieved.

The influence of intermediate particles on the nucleation of copper on polycrystalline platinum

Abstract The influences of the Cu adatoms and Cu + ions on the initial stages of copper electrodeposition on polycrystalline Pt in acidified copper sulphate solution have been studied by cyclic voltammetry, potentiostatic current transients, rotating ring-disk electrode ( rrde ), and in situ scanning tunnelling microscopy (STM). It has been established that during the long-time polarization of the platinum electrode, at potentials close to the copper equilibrium potential, the concentration of Cu + ions in the vicinity of the electrode increases and a Cu microdeposit if formed. The dissolution of the microdeposit occurs very slowly and takes several minutes of potential cycling in the range of 0.27–1.25 V. While the microdeposit is present on the platinum, the electrode surface is more active with respect to upd and bulk copper deposition. The combination of electrochemical and in situ STM results indicates that formation of surface alloy occurs during electrodeposition of copper on polycrystalline platinum.

Impurity‐induced 900 °C (2×2) surface reconstruction of SrTiO3(100)

A p(2×2)surface reconstruction is observed over the 800–900 °C range for SrTiO3 (100), the temperature range which is used in the oxygen anneal of Y, Ba, Cu layers on SrTiO3 (100) substrates in the synthesis of high Tc superconducting thin films. As observed by Auger electron spectroscopy, high‐temperature annealing causes calcium to segregate to the surface. Initially, at a Ca content of 3% upon a SrO‐terminated SrTiO3 (100) surface, the Ca atom is incorporated in a superstructure, which is attributed to multiple scattering across the two topmost interfaces. When the surface Ca content is below 1% the p(2×2) surface reconstruction is induced. The p(2×2) surface reconstruction disappears when the calcium content is removed.

PTH (1–34), but not strontium ranelate counteract loss of trabecular thickness and bone strength in disuse osteopenic rats

PTH and strontium ranelate (SrR) have both been shown to reduce bone loss induced by immobilization. PTH is a potent bone anabolic agent, whereas SrR has been suggested to be an antiresorptive as well as a bone anabolic agent. The aim of the study was to investigate whether PTH, SrR, and PTH and SrR in combination could counteract immobilization-induced bone loss in a rat model. Immobilization was induced by injecting 4IU Botox (BTX) into the muscles of the right hind limb. Seventy-two female Wistar rats, 3-months-old, were divided into the following groups: Baseline, Controls, BTX, BTX+PTH, BTX+SrR, and BTX+PTH+SrR (n=12 in each group). PTH was given as injections (SC) at a dosage of 60μg/kg/d, and SrR as 900mg/kg/d in the diet. The experiment lasted for 4weeks. BTX resulted in lower trabecular bone formation rate (-68%) and periosteal bone formation rate (-91%), and a higher fraction of osteoclast-covered surfaces (+53%) compared with controls. This was accompanied by significantly lower trabecular bone volume fraction (-24%), trabecular thickness (-16%), and bone strength (-14% to -32% depending on site). PTH alone counteracted immobilization-induced losses in trabecular (4-fold increase vs. BTX) and periosteal (5-fold increase vs. BTX) bone formation rate, trabecular thickness (+25% vs. BTX) and femoral neck strength (+24% vs. BTX). In contrast, SrR did not influence BTX-induced loss of bone formation rate, trabecular bone volume fraction, trabecular thickness, or bone strength. Finally, no additive effect was found when PTH and SrR treatments were combined. In conclusion, PTH counteracted loss in bone architecture and bone strength in immobilized rats, whereas as no effect of SrR was found. Moreover, no additional effect was found by combining PTH with SrR.

Metalloprotein adsorption on Au(111) and polycrystalline platinum investigated by in situ scanning tunneling microscopy with molecular and submolecular resolution

Redox metalloproteins exhibit interesting features such as long-range electron transfer (ET), cooperative effects etc. of importance in relation to fundamental ET theory, and mapped in considerable detail. Adsorption and interfacial electrochemical ET of metalloproteins at metallic surfaces is also broadly important in a range of contexts, and has been addressed by spectroscopic, voltammetric, and thermodynamic methods. In situ scanning tunneling (STM) and atomic force microscopy (AFM) have opened new perspectives for addressing adsorbed metalloproteins in their natural functional aqueous medium at the molecular level. In addition to broadly recognized problems of in situ STM AFM imaging, sample preparation, mobility, and adsorbate stability are, however, particular problems. We illustrate here the perspectives by recent in situ STM imaging of covalently bound horse heart cytochrome c on polycrystalline platinum, and of chemisorbed Pseudomonas aeruginosa azurin on Au(111). Molecular resolution is achieved, but azurin gives by far the best images which show, moreover, an interesting submolecular feature. This is likely to be associated with the disulphide group as a natural unit for gentle linking, facile ET routes through the protein, and tunnel enhancement by the low-lying redox level of the copper atom. The particular electronic-vibrational three-level configuration in in situ STM of metalloproteins. finally, offers a new way of distinction between superexchange, coherent, and sequential ET modes in the long-range ET patterns of metalloproteins.

Electron tunnelling in electrochemical processes and in situ scanning tunnel microscopy of structurally organized systems

Abstract The electronic tunnel factor in homogeneous long-range electron transfer (ET) systems has been characterized experimentally and theoretically in great detail. Outstanding questions, however, remain, for example interference in multiple ET routes and environmentally induced barrier fluctuations. Well characterized electrochemical long-range ET systems have also become available where the tunnel factor can be accurately deconvoluted from the total current. Intriguing new effects here are associated with the electrode charge or overpotential effects on the tunnel factor and with the feasibility of observation of critical current behaviour near phase transitions in thin films across which electron tunnelling occurs. In situ scanning tunnel microscopy (STM) offers a conceptual and technical new frame for mapping of molecular ET routes through large adsorbate molecules with low-lying local fluctuating levels. Such a configuration would be representative of large transition metal complexes or redox metalloproteins. High-resolution images and new theoretical approaches to STM mapping of large adsorbates are, moreover reported. The independent in situ electrode potential control provides new, non-monotonous features in the current-voltage relations which differ from electrochemical ET behaviour at a single interface.

Bulk copper electrodeposition on gold imaged by in situ STM : morphology and influence of tip potential

Electrochemical measurements were carried out simultaneously with acquisition of in situ STM images of copper electrodeposition at low cathodic overpotentials and subsequent dissolution from the underlying polycrystalline gold surfaces. The morphologies of the copper deposits were examined for correlation with features of the current-voltage diagram. Copper growth is by nucleation and formation of 3D islands. During the initial stages of bulk copper growth the potentials were fixed at selected values and a balance observed between formation of polycrystalline copper nuclei and of copper crystals. After the first cycle of copper deposition and dissolution the morphology of the polycrystalline gold surface had apparently changed into a recrystallized phase of a copper-gold alloy. At a given stage of the cycle the potential of the electrode was found to depend linearly on the tip potential. In a wide range of tip potentials the onset of copper deposition and end of dissolution showed a potential separation of 59 ± 5 mV indicating a single electron process.