Andrew S. Mount

Noradrenaline-functionalized hyperbranched fluoropolymer-poly(ethylene glycol) cross-linked networks as dual-mode, anti-biofouling coatings.

The strategy of decorating antibiofouling hyperbranched fluoropolymer-poly(ethylene glycol) (HBFP-PEG) networks with a settlement sensory deterrent, noradrenaline (NA), and the results of biofouling assays are presented. This example of a dual-mode surface, which combines both passive and active modes of antibiofouling, works in synergy to improve the overall antibiofouling efficiency against barnacle cyprids. The HBFP-PEG polymer surface, prior to modification with NA, was analyzed by atomic force microscopy, and a significant distribution of topographical features was observed, with a nanoscopic roughness measurement of 110 ± 8 nm. NA attachment to the surface was probed by secondary ion mass spectrometry to quantify the extent of polymer chain-end substitution with NA, where a 3- to 4-fold increase in intensity for a fragment ion associated with NA was observed and 39% of the available sites for attachment were substituted. Cytoskeletal assays confirmed the activity of tethered NA on adhering oyster hemocytes. Settlement assays showed deterrence toward barnacle cyprid settlement, while not compromising the passive biofouling resistance of the surface. This robust strategy demonstrates a methodology for the incorporation of actively antibiofouling moieties onto a passively antibiofouling network.

Synergistic roles for lipids and proteins in the permanent adhesive of barnacle larvae

Thoracian barnacles rely heavily upon their ability to adhere to surfaces and are environmentally and economically important as biofouling pests. Their adhesives have unique attributes that define them as targets for bio-inspired adhesive development. With the aid of multi-photon and broadband coherent anti-Stokes Raman scattering microscopies, we report that the larval adhesive of barnacle cyprids is a bi-phasic system containing lipids and phosphoproteins, working synergistically to maximize adhesion to diverse surfaces under hostile conditions. Lipids, secreted first, possibly displace water from the surface interface creating a conducive environment for introduction of phosphoproteins while simultaneously modulating the spreading of the protein phase and protecting the nascent adhesive plaque from bacterial biodegradation. The two distinct phases are contained within two different granules in the cyprid cement glands, implying far greater complexity than previously recognized. Knowledge of the lipidic contribution will hopefully inspire development of novel synthetic bioadhesives and environmentally benign antifouling coatings.

Detection of phospholipid-carbon nanotube translocation using fluorescence energy transfer

Single-walled carbon nanotubes (SWNTs) and lysophospholipids readily assemble into supramolecular complexes in aqueous solutions. Upon light excitation the fluorescence of rhodamine-labeled lysophospholipids was redshifted and quenched due to the optical absorption of the SWNTs. Utilizing fluorescence energy transfer, the authors detected the translocation and disassembly of SWNT complexes in MCF breast cancer cells. These lipid-coated SWNT complexes enable drugs to be delivered at an effective dose and their subsequent release to be monitored in real time.

Confocal microscopy-based goniometry of barnacle cyprid permanent adhesive

SUMMARY Biological adhesives are materials of particular interest in the fields of bio-inspired technology and antifouling research. The adhesive of adult barnacles has received much attention over the years; however, the permanent adhesive of the cyprid – the colonisation stage of barnacles – is a material about which very little is presently known. We applied confocal laser-scanning microscopy to the measurement of contact angles between the permanent adhesive of barnacle cyprid larvae and self-assembled monolayers of OH- and CH3-terminated thiols. Measurement of contact angles between actual bioadhesives and surfaces has never previously been achieved and the data may provide insight into the physicochemical properties and mechanism of action of these functional materials. The adhesive is a dual-phase system post-secretion, with the behaviour of the components governed separately by the surface chemistry. The findings imply that the cyprid permanent adhesion process is more complex than previously thought, necessitating broad re-evaluation of the system. Improved understanding will have significant implications for the production of barnacle-resistant coatings as well as development of bio-inspired glues for niche applications.

Toxicity of aqueous C70-gallic acid suspension in Daphnia magna.

The present study assessed the toxic effects of stable aqueous colloidal suspensions of gallic-acid-stabilized C(70) fullerene on Daphnia magna. The suspensions were stabilized through noncovalent surface modification with gallic acid. In addition to whole-organism responses, changes in antioxidative processes in D. magna were quantified. Acute toxicity was observed with 96LC50 for C(70) -gallic acid of 0.4 ± 0.1 mg/L C(70) . Daphnia magna fecundity was significantly reduced in 21-d bioassays at C(70) -gallic aqcid concentrations below quantifiable limits. Antioxidant enzyme activities of glutathione peroxidase and superoxide dismutase as well as lipid peroxidation suggested that exposed organisms experienced oxidative stress. Microscopic techniques used to determine cellular toxicity via apoptosis proved unsuccessful.

Barnacle biology before, during and after settlement and metamorphosis: a study of the interface

ABSTRACT Mobile barnacle cypris larvae settle and metamorphose, transitioning to sessile juveniles with morphology and growth similar to that of adults. Because biofilms exist on immersed surfaces on which they attach, barnacles must interact with bacteria during initial attachment and subsequent growth. The objective of this study was to characterize the developing interface of the barnacle and substratum during this key developmental transition to inform potential mechanisms that promote attachment. The interface was characterized using confocal microscopy and fluorescent dyes to identify morphological and chemical changes to the interface and the status of bacteria present as a function of barnacle developmental stage. Staining revealed patchy material containing proteins and nucleic acids, reactive oxygen species amidst developing cuticle, and changes in bacteria viability at the developing interface. We found that as barnacles metamorphose from the cyprid to juvenile stage, proteinaceous materials with the appearance of coagulated liquid were released into and remained at the interface. It stained positive for proteins, including phosphoprotein, as well as nucleic acids. Regions of the developing cuticle and the patchy material itself stained for reactive oxygen species. Bacteria were absent until the cyprid was firmly attached, but populations died as barnacle development progressed. The oxidative environment may contribute to the cytotoxicity observed for bacteria and has the potential for oxidative crosslinking of cuticle and proteinaceous materials at the interface. Summary: Barnacles have complex interactions with bacteria during settlement and metamorphosis. Barnacle attachment uses multiple proteinaceous glues, which attract bacteria to the interface; bacteria at this interface are eventually killed.

Direct Deposition of Crystalline Aragonite in the Controlled Biomineralization of the Calcareous Tubeworm

Although space delineation is a well-accepted requirement for biologically controlled biomineralization, the actual location of the mineralizing compartment within marine invertebrates has only recently been determined. We observed that the biomineralization was compartmented within the collar region of the metamorphosing larvae of Hydrodies elegans at its earliest possible time, i.e. at the post-metamorphic stage. We have also found that these highly regulated compartments contained aragonite crystals, as detected by EBSD and confirmed by electron diffraction TEM. Within these compartments, the metamorphosed larvae maintained a pH 9, at the pKa for CaCO3 formation. This model describes how biomineralization is a space delineation event in which calcium carbonate formation is an intracellular phenomenon.

Electrochemical characterization of a bioceramic material: The shell of the Eastern oyster Crassostrea virginica.

The shell of the Eastern oyster (Crassostrea virginica) is composed of multiple incongruent mineralized layers. This bioceramic composite material was investigated to determine the effects of shell thickness, orientation and layer composition on its electrochemical behavior using electrochemical impedance spectroscopy, potentiodynamic polarization and scanning electron microscopy-energy dispersive spectroscopy. SEM-EDS analysis of the oyster shell revealed that the multilayered biocomposite material is composed of calcium carbonate (CaCO(3)). EIS measurements in 3.5wt.% NaCl indicated that the impedance of the whole oyster shell in the low frequency region exhibited high impedance values which exhibited a decreasing trend with increasing immersion time. In terms of overall shell thickness, limiting currents measured by potentiodynamic techniques through the shell were observed to increase when the outer layers of the shell were sequentially removed by grinding, thus decreasing the shell thickness. These limiting current values remained relatively constant when the inner layers of the shell were removed. The impedance values of the oyster shell material as measured by EIS were shown to decrease with decreasing shell thickness. These findings suggest that the prismatic (outermost) shell layer in combination with the soluble organic matrix between all shell layers may influence the ionic conductivity through the oyster shell.

Chitin Facilitated Mineralization in the Eastern Oyster

Chitin is often reported in molluscan shell, where it likely contributes to the mechanical strength of the biomineral. However, the role of this polysaccharide in relation to the process of shell formation is not well understood. We investigated the deposition of chitin during shell repair in the Eastern oysters, Crassostrea virginica, by inserting stainless steel and glass implants in a region of shell damage. This work documents the time course of deposition of both chitin fibrils and calcium carbonate layers. Chitin is detected by confocal laser scanning microscopy (CLSM) using a chitin-specific fluorescent probe that was produced from clones of a chitin-binding domain. The presence of fibrils was confirmed using electron microscopy of implants. The fibrils’ dimensions were reduced after treatment with both acid and bleach, suggesting that chitin interacts with inorganic minerals and other organic components such as proteins and lipid as early as in 5 hours after shell damage. With CLSM, it was shown that chitin co-localized with membrane, suggesting the importance of cells located on the implants in the process of fibril formation. Using observations from this study as well as those from the literature on chitin synthase production in molluscs and fungi, we propose a cellular mechanism of chitin deposition related to shell formation.

Characterization of Calcification Events Using Live Optical and Electron Microscopy Techniques in a Marine Tubeworm

Characterizing the first event of biological production of calcium carbonate requires a combination of microscopy approaches. First, intracellular pH distribution and calcium ions can be observed using live microscopy over time. This allows identification of the life stage and the tissue with the feature of interest for further electron microscopy studies. Life stage and tissues of interest are typically higher in pH and Ca signals. Here, using H. elegans, we present a protocol to characterize the presence of calcium carbonate structures in a biological specimen on the scanning electron microscope (SEM), using energy-dispersive X-ray spectroscopy (EDS) to visualize elemental composition, using electron backscatter diffraction (EBSD) to determine the presence of crystalline structures, and using transmission electron microscopy (TEM) to analyze the composition and structure of the material. In this protocol, a focused ion beam (FIB) is used to isolate samples with dimension suitable for TEM analysis. As FIB is a site specific technique, we demonstrate how information from the previous techniques can be used to identify the region of interest, where Ca signals are highest.