Vesna Svetličić

QUANTITATIVE NANOMECHANICAL MAPPING OF MARINE DIATOM IN SEAWATER USING PEAK FORCE TAPPING ATOMIC FORCE MICROSCOPY1

It is generally accepted that a diatom cell wall is characterized by a siliceous skeleton covered by an organic envelope essentially composed of polysaccharides and proteins. Understanding of how the organic component is associated with the silica structure provides an important insight into the biomineralization process and patterning on the cellular level. Using a novel atomic force microscopy (AFM) imaging technique (Peak Force Tapping), we characterized nanomechanical properties (elasticity and deformation) of a weakly silicified marine diatom Cylindrotheca closterium (Ehrenb.) Reimann et J. C. Lewin (strain CCNA1). The nanomechanical properties were measured over the entire cell surface in seawater at a resolution that was not achieved previously. The fibulae were the stiffest (200 MPa) and the least deformable (only 1 nm). Girdle band region appeared as a series of parallel stripes characterized by two sets of values of Young’s modulus and deformation: one for silica stripes (43.7 Mpa, 3.7 nm) and the other between the stripes (21.3 MPa, 13.4 nm). The valve region was complex with average values of Young’s modulus (29.8 MPa) and deformation (10.2 nm) with high standard deviations. After acid treatment, we identified 15 nm sized silica spheres in the valve region connecting raphe with the girdle bands. The silica spheres were neither fused together nor forming a nanopattern. A cell wall model is proposed with individual silica nanoparticles incorporated in an organic matrix. Such organization of girdle band and valve regions enables the high flexibility needed for movement and adaptation to different environments while maintaining the integrity of the cell.

Atomic force microscopy characterization of silver nanoparticles interactions with marine diatom cells and extracellular polymeric substance

This study highlights the capacity of atomic force microscopy (AFM) for investigating nanoparticle (NP) algal cell interaction with a subnanometer resolution. We designed a set of AFM experiments to characterize NP size, shape, and structure to visualize changes in the cell morphology induced by NPs and to characterize NP interaction with the extracellular polymeric substance (EPS). Samples for AFM imaging were prepared using the same protocol—drop deposition on mica and imaged in air. Here we address the interactions of Ag NPs with ubiquitous, lightly silicified marine diatoms Cylindrotheca fusiformis and Cylindrotheca closterium and their EPS. In natural seawater used throughout this study, the single Ag NPs adopted truncated tetrahedron morphology with particle heights of 10, 20, 30, and 40 nm. This size class Ag NPs penetrates the cell wall through the valve region built of silica NPs embedded in organic matrix. The Ag NPs cause a local damage inside the cell without disintegration of the cell wall. The EPS production has been shown to increase as a feedback response to Ag NP exposure and may contribute to detoxification mechanisms. Imaging EPS at high resolution revealed the incorporation of Ag NPs and their aggregates into the EPS–gel matrix, proving their detoxifying capacity. Copyright

A kinetic study of charge transfer and phase transitions of the methylene blue/leucomethylene blue couple adsorbed at the mercury/aqueous solution interface

The interfacial behaviour of a methylene blue (MB)/leucomethylene blue (LMB) redox couple has been studied using fast perturbation potentiostatic techniques at the dropping mercury electrode. The extent of adsorption and the structure of the adsorbed layer above the monolayer coverage are determined by the presence of anions at the interface, anion hydration playing the important role. In 1 M electrolytes (pH=7.9) the tendency of MB dimers towards a stacking interaction in the aqueous phase, as well as to an adsorption at the mercury/aqueous solution interface, increases in the sequence F− ≪NO3−<Cl− ≪ClO4−. Evidence is presented for a coadsorption of LMB and nitrate and well-defined phase transitions in the adsorbed film. There are three distinct potential-dependent phases of LMB at the interface: (1) flat molecules and/or smaller two-dimensional aggregates at low LMB coverages; (2) two-dimensional crystalline layers of flat molecules after a monolayer of LMB is generated by reduction at positive charges; (3) a compact LMB layer at the negatively charged electrode, after desorption of nitrate and a conformational change of LMB molecules. The characteristics of the charge-transfer process are best interpreted in terms of two MB/LMB redox couples: the prewave corresponds to redox reactions of adsorbed species at the mercury surface, while the process at more negative potentials corresponds to the MB/LMB couple at a chemically modified electrode-mercury, covered by a compact and conductive layer of solid LMB.

Electron transfer kinetics of an adsorbed redox couple by double potential-step chronocoulometry: Methylene blue/leucomethylene blue

Abstract The kinetics and mechanism of the surface reaction of the methylene blue/leucomethylene blue redox couple have been studied at the dropping mercury electrode/aqueous nitrate solution, pH = 7.9, using a fast double potential step chronocoulometric technique. Kinetic and thermodynamic parameters of the individual electron transfer reactions are determined and the mechanism of the charge transfer through the compact adsorbed monolayers is discussed.

Thionine self-assembly on polyoriented gold and sulphur-modified gold electrodes

Abstract A new type of self-assembled monolayer (SAM) on gold has been reported. Electroactive SAMs of thionine formed on polyoriented gold and sulphur-modified polyoriented gold from aqueous thionine solutions of pH 7.9 have been characterized by voltammetry. In thionine SAMs formed on gold modified by a monolayer of sulphur adatoms the attachment takes place through S-S bonding between the S adatom and S heteroatom of thionine with edge-on orientation. A fast 2c − reduction of thionine molecules in the SAM, at potentials 150 mV more negative than the solution couple, leads to a complete detachment of the reduced molecules leaving the sulphur adlayer intact. In the thionine SAM formed on a flame-treated surface of polyoriented gold, the surface concentration of 4.59 × 10 −10 mol cm −2 corresponds to the most efficient packing of thionine molecules in a monolayer (end-on orientation). On 2c reduction of thionine molecules, the initial thionine SAM transforms irreversibly to a new surface state with one-half of the initial surface concentration.

Heterocoalescence between dispersed organic microdroplets and a charged conductive interface

Abstract The Caselli-Lipollis-Gierst method of studying the heterocoagulation of colloids at a plane mercury electrode has been extended to the investigation of the initial attachment and spreading of liquid organic droplets dispersed in an aqueous environment. The first measurements of fast current-time transients due to displacement of the interfacial charge by spreading droplets were performed in aqueous dispersions of organic liquids of increasing polarity (pristane, hexadecane, 1-heptadecene, squalene and methyl oleate).

In vitro percutaneous penetration and characterization of silver from silver-containing textiles.

The objective of this study was to determine the in vitro percutaneous penetration of silver and characterize the silver species released from textiles in different layers of full thickness human skin. For this purpose, two different wound dressings and a garment soaked in artificial sweat were placed in the donor compartments of Franz cells for 24 hours. The concentration of silver in the donor phase and in the skin was determined by an electrothermal atomic absorption spectrometer (ET-AAS) and by inductively coupled plasma mass spectrometer (ICP-MS). The characterization of silver species in the textiles and in the skin layers was made by scanning electron microscopy with integrated energy dispersive X-ray spectroscopy (SEM-EDX). Additionally, the size distribution of silver nanoparticles in the textiles was performed by atomic force microscopy (AFM). On the surface of all investigated materials, silver nanoparticles of different size and morphology were found. Released silver concentrations in the soaking solutions (ie, exposure concentration) ranged from 0.7 to 4.7 μg/mL (0.6–4.0 μg/cm2), fitting the bactericidal range. Silver and silver chloride aggregates at sizes of up to 1 μm were identified both in the epidermis and dermis. The large size of these particles suggests that the aggregation occurred in the skin. The formation of these aggregates likely slowed down the systemic absorption of silver. Conversely, these aggregates may form a reservoir enabling prolonged release of silver ions, which might lead to local effects.

AFM imaging of extracellular polymer release by marine diatom Cylindrotheca closterium (Ehrenberg) Reiman & J.C. Lewin.

Extracellular polysaccharide production by marine diatoms is a significant route by which photosynthetically produced organic carbon enters the trophic web and may influence the physical environment in the sea. This study highlights the capacity of atomic force microscopy (AFM) for investigating diatom extracellular polysaccharides with a subnanometer resolution. Here we address a ubiquitous marine diatom Cylindrotheca closterium, isolated from the northern Adriatic Sea, and its extracellular polymeric substance (EPS) at a single cell level. We applied a simple procedure for AFM imaging of diatom cells on mica under ambient conditions (in air) to achieve visualization of their EPS with molecular resolution. The EPS represents a web of polysaccharide fibrils with two types of cross‐linking: fibrils association forming junction zones and fibril–globule interconnections with globules connecting two or more fibrils. The fibril heights were 0.4–2.6 nm while globules height was in the range of 3–12 nm. Polymer networks of native gel samples from the Northern Adriatic and the network formed by polysaccharides extracted from the C. closterium culture share the same features regarding the fibril heights, pore openings and the mode of fibril association, proving that the macroscopic gel phase in the Northern Adriatic can be formed directly by the self‐assembly of diatom released polysaccharide fibrils. Copyright

Seawater at the nanoscale: marine gel imaged by atomic force microscopy

The present study introduces atomic force microscopy (AFM) as a tool for characterization of marine gel network and marine biopolymers self‐assembly, not accessible by other techniques. AFM imaging of marine gel samples collected in summers 2003 and 2004 in the northern Adriatic Sea provided insight into molecular organization of gel network and associations between polysaccharide fibrils in the network. Initial stages of biopolymers self‐assembly were visualized by AFM in a phytoplankton bloom experiment performed in the same aquatorium. Based on AFM imaging and differential scanning calorimetry, the marine gel is characterized as a thermoreversible physical gel and the dominant mode of gelation as crosslinking of polysaccharide fibrils by hydrogen bonding which results in helical structures and their associations. Direct deposition of whole seawater on freshly cleaved mica followed by rinsing was the procedure that caused the least impact on the original structures of biopolymer assemblies in seawater. Copyright

Organic monolayer formation at a sulphur modified gold electrode. The methylene blue/leucomethylene blue redox couple

Abstract The electrochemical formation of a condensed methylene blue (MB+)/leucomethylene blue (LMB) monolayer at the sulphur covered gold electrode (1 M aqueous electrolyte solution, pH = 7.9) is reported. The strong interaction between MB+ and adsorbed sulphur in the monolayer is ascribed to a disulphide linkage between a sulphur adatom and the sulphur heteroatom of the perpendicularly oriented organic molecule. This binding also results in a change in the electrical properties of the sulphur monolayer, which increases the reversibility of the electron transfer in the first adsorbed MB monolayer. The stability of the reduced monolayer at the Au-S/aqueous electrolyte interface is affected strongly by the type of anion (F− > Cl− ⪢ NO−3).