Probing the location of displayed cytochrome b562 on amyloid by scanning tunnelling microscopy.

Amyloid fibres displaying cytochrome b562 were probed using scanning tunnelling microscopy (STM) in vacuo. The cytochromes are electron transfer proteins containing a haem cofactor and could, in principle, mediate electron transfer between the tip and the gold substrate. If the core fibres were insulating and electron transfer within the 3D haem network was detected, then the electron transport properties of the fibre could be controlled by genetic engineering. Three kinds of STM images were obtained. At a low bias (<1.5 V) the fibres appeared as regions of low conductivity with no evidence of cytochrome mediated electron transfer. At a high bias, stable peaks in tunnelling current were observed for all three fibre species containing haem and one species of fibre that did not contain haem. In images of this kind, some of the current peaks were collinear and spaced around 10 nm apart over ranges longer than 100 nm, but background monomers complicate interpretation. Images of the third kind were rare (1 in 150 fibres); in these, fully conducting structures with the approximate dimensions of fibres were observed, suggesting the possibility of an intermittent conduction mechanism, for which a precedent exists in DNA. To test the conductivity, some fibres were immobilized with sputtered gold, and no evidence of conduction between the grains of gold was seen. In control experiments, a variation of monomeric cytochrome b562 was not detected by STM, which was attributed to low adhesion, whereas a monomeric multi-haem protein, GSU1996, was readily imaged. We conclude that the fibre superstructure may be intermittently conducting, that the cytochromes have been seen within the fibres and that they are too far apart for detectable current flow between sites to occur. We predict that GSU1996, being 10 nm long, is more likely to mediate successful electron transfer along the fibre as well as being more readily detectable when displayed from amyloid.

Self-assembled gold nanoparticles on functionalized gold (111) studied by scanning tunneling microscopy

Nanogold colloidal solutions are prepared by the reduction of HAuClO4 with sodium citrate and sodium borohydride. 4-Aminothiophenol (ATP) self-assembled monolayers (SAMs) are formed on gold(lll) surface, on which gold nanoparticles are immobilized and a sub-monolayer of the particles appears. This sub-monolayer of gold nanoparticles is characterized with scanning tunneling microscopy (STM), and a dual energy barrier tunneling model is proposed to explain the imageability of the gold nanoparticles by STM. This model can also be used to construct multiple energy barrier structure on solid/liquid interface and to evaluate the electron transport ability of some organic monolayers with the aid of electrochemical method.

Nanostructure and mechanical measurement of highly oriented lamellae of melt-drawn HDPE by scanning probe microscopy

Scanning probe microscopy was used to simultaneously determine the molecular chain structure and intrinsic mechanical properties, including anisotropic elastic modulus and friction, for lamellae of highly oriented high-density polyethylene (HDPE) obtained by the melt-drawn method. The molecular-scale image of the highly oriented lamellae by friction force microscopy (FFM) clearly shows that the molecular chains are aligned parallel to the drawing direction, and the periodicities along and perpendicular to the drawing direction are 0.26 and 0.50 nm, respectively. The results indicate that the exposed planes of the lamellae resulting from the melt-drawn method are (200), which is consistent with results of transmission electron microscopy and electron diffraction. Because of the high degree of anisotropy in the sample, coming from alignment of the molecular chains along the drawing direction, the measured friction force, F, determined by FFM is strongly dependent on the angle, theta, between the scanning direction and the chain axis. The force increases as theta is increased from 0 degrees (i.e., parallel to the chain axis) to 90 degrees (i.e., perpendicular to the chain axis). The structural anisotropy was also found to strongly influence the measurements of the transverse chain modulus of the polymer by the nanoindentation technique. The measured value of 13.8 GPa with transverse modulus was larger than the value 4.3 GPa determined by wide-angle X-ray diffraction, which we attributed to anisotropic deformation of the lamellae during nanoindentation measurements that was not accounted for by the elastic treatment we adopted from Oliver and Pharr. The present approach using scanning probe microscopy has the advantage that direct correlations between the nanostructure, nanotribology, and nanomechanical properties of oriented samples can be determined simultaneously and simply.

Recent advances in scanning electrochemical microscopy

The basic principle, instrumentation and recent advances of scanning electrochemical microscopy are briefly reviewed with 55 references.

In situ electrochemical scanning tunneling microscopy investigation of renewing graphite surface accompanied by electrochemical reaction

In situ electrochemical scanning tunneling microscopy (ECSTM) has been employed to follow the renewal process of a graphite electrode accompanied by flavin adenine dinucleotide (FAD) electrochemical reaction which involves adsorption of the reduced form (FADH(2)) and desorption of the oxidized form (FAD). The renewal process initiates from steps or kinks on the electrode surface, which provide high active sites for adsorption. This renewal depends on the working electrode potential, especially in the range near the FAD redox potential. Our experiment suggests that delamination of the graphite surface is caused by interaction between the substrate and adsorbed molecules. A simple model is proposed to explain this phenomenon.

In situ electrochemical scanning tunnelling microscopy investigation of structure for horseradish peroxidase and its electrocatalytic property

Immobilization of protein molecules is a fundamental problem for scanning tunnelling microscopy (STM) measurements with high resolution. In this paper, an electrochemical method has been proved to be an effective way to fix native horseradish peroxidase (HRP) as well as inactivated HRP from electrolyte onto a highly oriented pyrolytic graphite (HOPG) surface. This preparation is suitable for both ex situ and in situ electrochemical STM (ECSTM) measurements. In situ STM has been successfully employed to observe totally different structures of HRP in three typical cases: (1) in situ ECSTM reveals an oval-shaped pattern for a single molecule in neutral buffer solution, which is in good agreement with the dimension determined as 6.2 x 4.3 x 1.2. nm(3) by ex situ STM for native HRP; (2) in situ ECSTM shows that the adsorbed HRP molecules on HOPG in a denatured environment exhibit swelling globes at the beginning and then change into a V-shaped pattern after 30 min; (3) in situ ECSTM reveals a black hole in every ellipsoidal sphere for inactivated HRP in strong alkali solution. The cyclic voltammetry results indicate that the adsorbed native HRP can directly catalyse the reduction of hydrogen peroxide, demonstrating that a direct electron transfer reduction occurred between the enzyme and HOPG electrode, whereas the corresponding cyclic voltammograms for denatured HRP and inactivated HRP adsorbed on HOPG electrodes indicate a lack of ability to catalyse H2O2 reduction, which confirms that the HRP molecules lost their biological activity. Obviously, electrochemical results powerfully support in situ STM observations.

EFFECTS OF ANODIC-OXIDATION ON THE SURFACE-STRUCTURE OF HIGHLY ORIENTED PYROLYTIC-GRAPHITE REVEALED BY IN-SITU ELECTROCHEMICAL SCANNING-TUNNELING-MICROSCOPY IN H2SO4 SOLUTION

Effects of the potential of anodic oxidation and of potential cycling on the surface structure of a highly oriented pyrolytic graphite (HOPG) electrode were observed by in situ electrochemical scanning tunnelling microscopy (ECSTM) in dilute H2SO4 solution with atomic resolution. With potential cycling between -0.1 V and 1.8 V vs. Ag/AgCl (sat. KCI), some atoms on the top layer of HOPG protrude out of the base plane, and the graphite lattice of these protrusions is still intact but is strained and expanded. With further potential cycling, some protrusions coalesced and some grew larger, and an anomalous superperiodic feature was observed (spacing 90 Angstrom with a rotation 30 degrees relative to atomic corrugations) which superimposed on the atomic corrugation of HOPG. On the topmost of these protrusions, some atoms form oxides and others are still resolved by the ECSTM image. With potential cycling between -0.1 V and + 2.0 V vs. Ag/AgCl (sat. KCl), damage to freshly cleaved HOPG surface is more serious and fast, some ridges are observed, the atomic structure of the HOPG surface is partially and then completely damaged due to the formation of oxide. We also found that anodic oxidation occurred nonuniformly on the surface of HOPG near defects during potential cycling.