Synthesis of Novel Sulfobetaine Polymers with Differing Dipole Orientations in Their Side Chains, and Their Effects on the Antifouling Properties.

The impact of the orientation of zwitterionic groups, with respect to the polymer backbone, on the antifouling performance of thin hydrogel films made of polyzwitterions is explored. In an extension of the recent discussion about differences in the behavior of polymeric phosphatidylcholines and choline phosphates, a quasi-isomeric set of three poly(sulfobetaine methacrylate)s is designed for this purpose. The design is based on the established monomer 3-[N-2-(methacryloyloxy)ethyl-N,N-dimethyl]ammonio-propane-1-sulfonate and two novel sulfobetaine methacrylates, in which the positions of the cationic and the ionic groups relative to the polymerizable group, and thus also to the polymer backbone, are altered. The effect of the varied segmental dipole orientation on their water solubility, wetting behavior by water, and fouling resistance is compared. As model systems, the adsorption of the model proteins bovine serum albumin (BSA), fibrinogen, and lysozyme onto films of the various polyzwitterion surfaces is studied, as well as the settlement of a diatom (Navicula perminuta) and barnacle cyprids (Balanus improvisus) as representatives of typical marine fouling communities. The results demonstrate the important role of the zwitterionic group's orientation on the polymer behavior and fouling resistance.

Antifouling Properties of Dendritic Polyglycerols against Marine Macrofouling Organisms.

Dendritic polyglycerols (PGs) were synthesized and postmodified by grafting of poly(ethylene glycol) (PEG) and polypropylene glycol (PPG) diglycidyl ether groups, and their antifouling and fouling-release properties were tested. Coating characterization by spectroscopic ellipsometry, contact angle goniometry, attenuated total internal reflection-Fourier transform infrared spectroscopy (ATR-FTIR), and atomic force microscopy showed brushlike morphologies with a high degree of microscale roughness and the ability to absorb large amounts of water within seconds. PGs with three different thicknesses were tested in laboratory assays against settlement of larvae of the barnacle Balanus improvisus and against the settlement and removal of zoospores of the alga Ulva linza. Very low coating thicknesses, e.g., 11 nm, reduced the settlement of barnacles, under static conditions, to 2% compared with 55% for an octadecyltrichlorosilane reference surface. In contrast, zoospores of U. linza settled readily but the vast majority were removed by exposure to a shear force of 52 Pa. Both PEG and PPG modification increased the antifouling properties of the PG films, providing a direct comparison of the ultralow fouling properties of all three polymers. Both, the modified and the nonmodified PGs are promising components for incorporation into amphiphilic fouling-resistant coatings.

Effects of ocean warming and acidification, combined with nutrient enrichment, on chemical composition and functional properties of Ulva rigida.

Ulva is increasingly viewed as a food source in the world. Here, Ulva rigida was cultured at two levels of temperature (14, 18 °C), pH (7.95, 7.55, corresponding to low and high pCO2), and nitrate conditions (6 µmol L-1, 150 µmol L-1), to investigate the effects of ocean warming, acidification, and eutrophication on food quality of Ulva species. High temperature increased the content of each amino acid. High nitrate increased the content of all amino acids except aspartic acid and cysteine. High temperature, pCO2, and nitrate also increased the content of most fatty acids. The combination of high temperature, pCO2, and nitrate increased the swelling capacity, water holding capacity, and oil holding capacity by 15.60%, 7.88%, and 16.32% respectively, compared to the control. It seems that the future ocean environment would enhance the production of amino acid and fatty acid as well as the functional properties of Ulva species.

Marine antifouling performance of polymer coatings incorporating zwitterions.

Zwitterionic materials display antifouling promise, but their potential in marine anti-biofouling is still largely unexplored. This study evaluates the effectiveness of incorporating small quantities (0-20% on a molar basis) of zwitterions as sulfobetaine methacrylate (SBMA) or carboxybetaine methacrylate (CBMA) into lauryl methacrylate-based coatings whose relatively hydrophobic nature encourages adhesion of the diatom Navicula incerta, a common microfouling organism responsible for the formation of 'slime'. This approach allows potential enhancements in antifouling afforded by zwitterion incorporation to be easily quantified. The results suggest that the incorporation of CBMA does provide a relatively minor enhancement in fouling-release performance, in contrast to SBMA which does not display any enhancement. Studies with coatings incorporating mixtures of varying ratios of the cationic monomer [2-(methacryloyloxy)ethyl]trimethylammonium chloride and the anionic monomer (3-sulfopropyl)methacrylate, which offer a potentially lower cost approach to the incorporation of anionic and cationic charge, suggest these monomers impart little significant effect on biofouling.

Fouling-Release Performance of Silicone Oil-Modified Siloxane-Polyurethane Coatings.

The effect of incorporation of silicone oils into a siloxane-polyurethane fouling-release coatings system was explored. Incorporation of phenylmethyl silicone oil has been shown to improve the fouling-release performance of silicone-based fouling-release coatings through increased interfacial slippage. The extent of improvement is highly dependent upon the type and composition of silicone oil used. The siloxane-polyurethane (SiPU) coating system is a tough fouling-release solution, which combines the mechanical durability of polyurethane while maintaining comparable fouling-release performance with regard to commercial standards. To further improve the fouling-release performance of the siloxane-PU coating system, the use of phenylmethyl silicones oils was studied. Coatings formulations were prepared incorporating phenylmethyl silicone oils having a range of compositions and viscosities. Contact angle and surface energy measurements were conducted to evaluate the surface wettability of the coatings. X-ray photoelectron spectroscopy (XPS) depth profiling experiments demonstrated self-stratification of silicone oil along with siloxane to the coating-air interface. Several coating formulations displayed improved or comparable fouling-release performance to commercial standards during laboratory biological assay tests for microalgae (Navicula incerta), macroalgae (Ulva linza), adult barnacles (Balanus amphitrite syn. Amphibalanus amphitrite), and mussels (Geukensia demissa). Selected silicone-oil-modified siloxane-PU coatings also demonstrated comparable fouling-release performance in field immersion trials. In general, modifying the siloxane-PU fouling-release coatings with a small amount (1-5 wt % basis) of phenylmethyl silicone oil resulted in improved performance in several laboratory biological assays and in long-term field immersion assessments.

In vivo and in situ synchrotron radiation-based µ-XRF reveals elemental distributions during the early attachment phase of barnacle larvae and juvenile barnacles.

Barnacles are able to establish stable surface contacts and adhere underwater. While the composition of adult barnacle cement has been intensively studied, far less is known about the composition of the cement of the settlement-stage cypris larva. The main challenge in studying the adhesives used by these larvae is the small quantity of material available for analysis, being on the order of nanograms. In this work, we applied, for the first time, synchrotron radiation-based µ-X-ray fluorescence analysis (SR-µ-XRF) for in vivo and in situ analysis of young barnacles and barnacle cyprids. To obtain biologically relevant information relating to the body tissues, adhesives, and shell of the organisms, an in situ sample environment was developed to allow direct microprobe investigation of hydrated specimens without pretreatment of the samples. In 8-day-old juvenile barnacles (Balanus improvisus), the junctions between the six plates forming the shell wall showed elevated concentrations of calcium, potassium, bromine, strontium, and manganese. Confocal measurements allowed elemental characterization of the adhesive interface of recently attached cyprids (Balanus amphitrite), and substantiated the accumulation of bromine both at the point of initial attachment as well as within the cyprid carapace. In situ measurements of the cyprid cement established the presence of bromine, chlorine, iodine, sulfur, copper, iron, zinc, selenium, and nickel for both species. The previously unrecognized presence of bromine, iron, and selenium in the cyprid permanent adhesive will hopefully inspire further biochemical investigations of the function of these substances.

Slippery liquid-infused porous surfaces showing marine antibiofouling properties.

Marine biofouling is a longstanding problem because of the constant challenges placed by various fouling species and increasingly restricted environmental regulations for antifouling coatings. Novel nonbiocidal strategies to control biofouling will necessitate a multifunctional approach to coating design. Here we show that slippery liquid-infused porous surfaces (SLIPSs) provide another possible strategy to obtaining promising antifouling coatings. Microporous butyl methacrylate-ethylene dimethacrylate (BMA-EDMA) surfaces are prepared via UV-initiated free-radical polymerization. Subsequent infusion of fluorocarbon lubricants (Krytox103, Krytox100, and Fluorinert FC-70) into the porous microtexture results in liquid-repellent slippery surfaces. To study the interaction with marine fouling organisms, settlement of zoospores of the alga Ulva linza and cypris larvae of the barnacle Balanus amphitrite is tested in laboratory assays. BMA-EDMA surfaces infused with Krytox103 and Krytox100 exhibit remarkable inhibition of settlement (attachment) of both spores and cyprids to a level comparable to that of a poly(ethylene glycol) (PEG)-terminated self-assembled monolayer. In addition, the adhesion strength of sporelings (young plants) of U. linza is reduced for BMA-EDMA surfaces infused with Krytox103 and Krytox100 compared to pristine (noninfused) BMA-EDMA and BMA-EDMA infused with Fluorinert FC-70. Immersion tests suggest a correlation between the stability of slippery coatings in artificial seawater and fouling resistance efficacy. The results indicate great potential for the application of this concept in fouling-resistant marine coatings.

Probing biofouling resistant polymer brush surfaces by atomic force microscopy based force spectroscopy.

The protein repellency and biofouling resistance of zwitterionic poly(sulfobetaine methacrylate)(pSBMA) brushes grafted via surface initiated polymerization (SIP) from silicon and glass substrata was assessed using atomic force microscopy (AFM) adherence experiments. Laboratory settlement assays were conducted with cypris larvae of the barnacle Balanus amphitrite. AFM adherence includes the determination of contact rupture forces when AFM probe tips are withdrawn from the substratum. When the surface of the AFM tip is modified, adherence can be assessed with chemical specifity using a method known as chemical force microscopy (CFM). In this study, AFM tips were chemically functionalized with (a) fibronectin- here used as model for a nonspecifically adhering protein - and (b) arginine-glycine-aspartic acid (RGD) peptide motifs covalently attached to poly(methacrylic acid) (PMAA) brushes as biomimics of cellular adhesion receptors. Fibronectin functionalized tips showed significantly reduced nonspecific adhesion to pSBMA-modified substrata compared to bare gold (2.3±0.75 nN) and octadecanethiol (ODT) self-assembled monolayers (1.3±0.75 nN). PMAA and PMAA-RGD modified probes showed no significant adhesion to pSBMA modified silicon substrata. The results gathered through AFM protein adherence studies were complemented by laboratory fouling studies, which showed no adhesion of cypris larvae of Balanus amphitrite on pSBMA. With regard to its unusually high non-specific adsorption to a wide variety of materials the behavior of fibronectin is analogous to the barnacle cyprid temporary adhesive that also binds well to surfaces differing in polarity, charge and free energy. The antifouling efficacy of pSBMA may, therefore, be directly related to the ability of this surface to resist nonspecific protein adsorption.

Poly(dimethyl siloxane) (PDMS) network blends of amphiphilic acrylic copolymers with poly(ethylene glycol)-fluoroalkyl side chains for fouling-release coatings. II. Laboratory assays and field immersion trials.

Amphiphilic copolymers containing different amounts of poly(ethylene glycol)-fluoroalkyl acrylate and polysiloxane methacrylate units were blended with a poly(dimethyl siloxane) (PDMS) matrix in different proportions to investigate the effect of both copolymer composition and loading on the biological performance of the coatings. Laboratory bioassays revealed optimal compositions for the release of sporelings of Ulva linza, and the settlement of cypris larvae of Balanus amphitrite. The best-performing coatings were subjected to field immersion tests. Experimental coatings containing copolymer showed significantly reduced levels of hard fouling compared to the control coatings (PDMS without copolymer), their performance being equivalent to a coating based on Intersleek 700™. XPS analysis showed that only small amounts of fluorine at the coating surface were sufficient for good antifouling/fouling-release properties. AFM analyses of coatings under immersion showed that the presence of a regular surface structure with nanosized domains correlated with biological performance.

Modulation of barnacle (Balanus amphitrite Darwin) cyprid settlement behavior by sulfobetaine and carboxybetaine methacrylate polymer coatings.

Zwitterionic polymers such as poly(sulfobetaine methacrylate) (polySBMA) and poly(carboxybetaine methacrylate) (polyCBMA) have demonstrated impressive fouling-resistance against proteins and mammalian cells. In this paper, the effects of these surface chemistries on the settlement and behavior of an ubiquitous fouling organism, the cypris larva of the barnacle Balanus amphitrite (=Amphibalanus amphitrite), were studied in the laboratory. Conventional settlement assays and behavioral analysis of cyprids using Noldus Ethovision 3.1 demonstrated significant differences in settlement and behavior on different surfaces. Cyprids did not settle on the polySBMA or polyCBMA surfaces over the course of the assay, whereas settlement on glass occurred within expected limits. Individual components of cyprid behavior were shown to differ significantly between glass, polySBMA and polyCBMA. Cyprids also responded differently to the two zwitterionic surfaces. On polySBMA, cyprids were unwilling or unable to settle, whereas on polyCBMA cyprids did not attempt exploration and left the surface quickly. In neither case was toxicity observed. It is concluded that a zwitterionic approach to fouling-resistant surface development has considerable potential in marine applications.

Resistance of polysaccharide coatings to proteins, hematopoietic cells, and marine organisms.

The interaction of covalently coupled hyaluronic acid, alginic acid, and pectic acid with proteins, cells (hematopoietic KG1a and Jurkat cells), and marine organisms (algal zoospores and barnacle cypris larvae) is compared. In contrast to cells and proteins for which such polysaccharide coatings are known for their antiadhesive properties, marine algal spores and barnacle cyprids were able to colonize the surfaces. Of the three polysaccharides, hyaluronic acid showed the lowest settlement of both Ulva zoopores and barnacles. Photoelectron spectroscopy reveals that the polysaccharide coatings tend to bind bivalent ions, such as calcium, from salt water. Such pretreatment with a high salinity medium significantly changes the protein and hematopoietic cell resistance of the surfaces. Complexation of bivalent ions is therefore considered as one reason for the decreased resistance of polysaccharide coatings when applied in the marine environment.

Consensus multivariate methods in gas chromatography mass spectrometry and denaturing gradient gel electrophoresis: MHC-congenic and other strains of mice can be classified according to the profiles of volatiles and microflora in their scent-marks.

House mice (Mus domesticus) communicate using scent-marks, and the chemical and microbial composition of these 'extended phenotypes' are both influenced by genetics. This study examined how the genes of the major histocompatibility complex (MHC) and background genes influence the volatile compounds (analysed with Gas Chromatography Mass Spectrometry or GC/MS) and microbial communities (analysed using Denaturating Gradient Gel Electrophoresis or DGGE) in scent-marks produced by congenic strains of mice. The use of Consensus Principal Components Analysis is described and shows relationships between the two types of fingerprints (GC/MS and DGGE profiles). Classification methods including Support Vector Machines and Discriminant Partial Least Squares suggest that mice can be classified according to both background strain and MHC-haplotype. As expected, the differences among the mice were much greater between strains that vary at both MHC and background loci than the congenics, which differ only at the MHC. These results indicate that the volatiles in scent-marks provide information about genetic similarity of the mice, and support the idea that the production of these genetically determined volatiles is influenced by commensal microflora. This paper describes the application of consensus methods to relate two blocks of analytical data.

Poly(ethylene glycol)-containing hydrogel surfaces for antifouling applications in marine and freshwater environments.

This work describes the fabrication, characterization, and biological evaluation of a thin protein-resistant poly(ethylene glycol) (PEG)-based hydrogel coating for antifouling applications. The coating was fabricated by free-radical polymerization on silanized glass and silicon and on polystyrene-covered silicon and gold. The physicochemical properties of the coating were characterized by infrared spectroscopy, ellipsometry, and contact angle measurements. In particular, the chemical stability of the coating in artificial seawater was evaluated over a six-month period. These measurements indicated that the degradation process was slow under the test conditions chosen, with the coating thickness and composition changing only marginally over the period. The settlement behavior of a broad and diverse group of marine and freshwater fouling organisms was evaluated. The tested organisms were barnacle larvae (Balanus amphitrite), algal zoospores (Ulva linza), diatoms (Navicula perminuta), and three bacteria species (Cobetia marina, Marinobacter hydrocarbonoclasticus, and Pseudomonas fluorescens). The biological results showed that the hydrogel coating exhibited excellent antifouling properties with respect to settlement and removal.

Larval Development and Vitellin-like Protein Expression in Palaemon elegans Larvae Following Xeno-oestrogen Exposure.

Certain anthropogenic chemicals, most notably xeno-oestrogens, are known to have the potential to disrupt vertebrate endocrine systems. For example, induction of the female-specific protein, vitellogenin, in male fish is a well-known effect of exposure to xeno-oestrogens and serves as a biomarker of such exposure. There have been few comparable studies of putative biomarkers of endocrine disruption in invertebrates. An exception is the upregulation of vitellin-like larval storage protein (LSP) expression in the barnacle cypris larva following exposure to oestrogenic chemicals. The current study aimed to establish whether larvae of the glass prawn, Palaemon elegans, are likewise susceptible to xeno-oestrogen exposure. Using a polyclonal antiserum to P. elegans apolipovitellin, an 86 kDa polypeptide was detected by western blotting in the larval and early postlarval stages of this species. An indirect ELISA applied to the soluble protein fraction of larval homogenates determined that the titre of this putative LSP ranged, depending on larval stage, from 0.48-0.67 ng µg(-1). Exposure of P. elegans larvae to the xeno-oestrogen 4-n-nonylphenol (4-NP), at 0.2-20 µg L(-1), resulted in a significant, concentration-independent increase in putative LSP levels of 5-18%. Conversely, exposure to the natural oestrogen, 17ß-oestradiol (E(2)), at 0.2 and 20 µg L(-1), led to a significant concentration-independent decline (up to 11%) in LSP levels. Whether the effect of 4-NP results from endocrine disruption is not known, however, an oestrogen receptor-mediated effect is unlikely. Other than a slight increase in larval mortality when exposed to 4-NP at 2 µg L(-1), neither 4-NP nor E(2) significantly affected development, growth or survival of P. elegans larvae.