Marine microbial natural products in antifouling coatings

Ecological problems associated with current antifouling technologies have increased interest in the natural strategies that marine organisms use to keep their surfaces clean and free from fouling. Bacteria isolated from living surfaces in the marine environment have been shown to produce chemicals that are potential antifoulants. Active compounds from the cells and culture supernatant of two bacterial strains, FS‐55 and NudMB50–11, isolated from surface of the seaweed, Fucus serratus, and the nudibranch, Archidoris pseudoargus, respectively, were extracted using solid phase extraction. The extracts were combined with acrylic base paint resin and assayed for antifouling activity by measuring their ability to inhibit the growth of fouling bacteria. These formulations were found to be active against fouling bacteria isolated from marine surfaces. The formulation of antifouling paints that incorporate marine microbial natural products is reported here for the first time. This is a significant advance towards the production of an environmentally friendly antifouling paint that utilises a sustainable supply of natural biodegradable compounds.     

The characterization, replication and testing of dermal denticles of Scyliorhinus canicula for physical mechanisms of biofouling prevention

There is a current need to develop novel non-toxic antifouling materials. The mechanisms utilized by marine organisms to prevent fouling of external surfaces are of interest in this regard. Biomimicry of these mechanisms and the ability to transfer the antifouling characteristics of these surfaces to artificial surfaces are a highly attractive prospect to those developing antifouling technologies. In order to achieve this, the mechanisms responsible for any antifouling ability must be elucidated from the study of the natural organism and the critical surface parameters responsible for fouling reduction. Dermal denticles of members of the shark family have been speculated to possess some natural, as yet unidentified antifouling mechanism related to the physical presence of denticles. In this study, the dermal denticles of one particular member of the slow-swimming sharks, Scyliorhinus canicula were characterized and it was found that a significant natural variation in denticle dimensions exists in this species. The degree of denticle surface contamination was quantified on denticles at various locations and it was determined that the degree of contamination of the dorsal surface of denticles varies with the position on the shark body. In addition, we successfully produced synthetic sharkskin samples using the real skin as a template. Testing of the produced synthetic skin in field conditions resulted in significant differences in material attachment on surfaces exhibiting denticles of different dimensions.     

Poly(vinylpyrrolidone) for bioconjugation and surface ligand immobilization

Poly(vinylpyrrolidone) (PVP), a nonionic and nontoxic polymer with antifouling properties, has been synthesized via RAFT polymerization to obtain thiol-terminated PVP. We demonstrate that when the polymer is adsorbed onto the surface of colloidal silica particles, the terminal thiol groups of PVP remain accessible for chemical modification and lend themselves to the immobilization of ligands. We show that ligand attachment onto the surface via conjugation to PVP is reversible, as the polymer can be desorbed from the surface for conjugate and surface recovery. We present the conjugation of a model peptide and an oligonucleotide to PVP via the polymer terminal thiol and demonstrate that conjugates remain functional in molecular recognition assay. The developed technique offers a novel method to functionalize low-fouling surfaces for a variety of biomedical applications and presents opportunities to use PVP as a macromolecular drug carrier.     

Tannin and tannate from the quebracho tree: an eco-friendly alternative for controlling marine biofouling

Current antifouling coatings are based on toxic compounds that can be harmful to the natural environment. A promising alternative to these compounds is the use of natural products that are non-toxic, but have antifouling properties. Tannins are natural, water-soluble, complex polyphenolic substances, which precipitate proteins and have anticorrosive and antimicrobial properties. In this study, the effect of quebracho tannin as a probable antifouling pigment in both laboratory and field trials is evaluated. As tannins have high solubility in aqueous media and consequently would leach rapidly, they were precipitated as aluminium tannate, which has an adequate solubility for use as a component in marine paints. In vitro exposure of Balanus amphitrite and Polydora ligni larvae to low concentrations of both quebracho tannin and saturated aluminium tannate solutions produced complete appendage immobilisation. In 28-d field trials of test gels, a significant decrease in micro- and macrofouling density and diversity in relation to the control gel was detected (p < 0.05). This study suggests that natural tannins could be employed as bioactive pigment for new antifouling technologies.     

Surface PEGylation via native chemical ligation

Native chemical ligation (NCL) is an emerging chemoselective chemistry that forms an amide bond by trans-thioesterification followed by intramolecular nucleophilic rearrangement between thioester and cysteine. The reaction is simple, occurs in a mild aqueous solution, and gives near-quantitative yields of a desired product. Since the first report in 1994, most studies involving the use of NCL have focused on the total synthesis of proteins to address fundamental questions pertaining to many aspects of protein science, such as folding, mirror images, and site-specific labeling of proteins, but applications of the NCL reaction for other areas remain largely unexplored. Herein, we present a facile strategy for surface immobilization of poly(ethylene glycol) (PEG) utilizing the NCL reaction. Surface immobilization of PEG (i.e., PEGylation) plays a key role in preventing nonspecific protein adsorption on surfaces, which is crucial in a wide variety of medical devices. Using cysteine-PEG and thioester-containing phosphonic acid conjugates, we achieved efficient surface PEGylation on titanium surfaces. Ellipsometry, goniometry, and X-ray photoelectron spectroscopy (XPS) unambiguously confirmed the presence of PEGs, which provided nonfouling effects of surfaces. This study indicates that the NCL reaction will be a useful toolkit for surface bioconjugation and functionalization.     

Controlled antibody immobilization onto immunoanalytical platforms by synthetic peptide

Antibody immobilization on a solid surface is inevitable in the preparation of immunochips/sensors. Antibody-binding proteins such as proteins A and G have been extensively employed to capture antibodies on sensor surfaces with right orientations, maintaining their full functionality. Because of their synthetic versatility and stability, in general, small molecules have more advantages than proteins. Nevertheless, no small molecule has been used for oriented and specific antibody immobilization. Here is described a novel strategy to immobilize an antibody on various sensor surfaces by using a small antibody-binding peptide. The peptide binds specifically to the Fc domain of immunoglobulin G (IgG) and, therefore, affords a properly oriented antibody surface. Surface plasmon resonance analysis indicated that a peptide linked to a gold chip surface through a hydrophilic linker efficiently captured human and rabbit IgGs. Moreover, antibodies captured by the peptide exhibited higher antigen binding capacity compared with randomly immobilized antibodies. Peptide-mediated antibody immobilization was successfully applied on the surfaces of biosensor substrates such as magnetic particles and glass slides. The antibody-binding peptide conjugate introduced in this work is the first small molecule linker that offers a highly stable and specific surface platform for antibody immobilization in immunoassays.     

Patterning of proteins and cells on functionalized surfaces prepared by polyelectrolyte multilayers and micromolding in capillaries

A method for protein and cell patterning on polyelectrolyte-coated surfaces using simple micromolding in capillaries (MIMIC) is described. MIMIC produced two distinctive regions. One contained polyethylene glycol (PEG) microstructures fabricated using photopolymerization that provided physical, chemical, and biological barriers to the nonspecific binding of proteins, bacteria, and fibroblast cells. The second region was the polyelectrolyte (PEL) coated surface that promoted protein and cell immobilization.

The difference in surface functionality between the PEL region and background PEG microstructures resulted in simple patterning of biomolecules. Fluorescein isothiocyanate-tagged bovine serum albumin, E. coli expressing green fluorescence protein (GFP), and fibroblast cells were successfully bound to the exposed PEL surfaces at micron scale. Compared with the simple adsorption of protein, fluorescence intensity was dramatically improved (by about six-fold) on the PEL-modified surfaces. Although animal cell patterning is prerequisite for adhesive protein layer to survive on desired area, the PEL surface without adhesive proteins provides affordable microenvironment for cells.

The simple preparation of functionalized surface but universal platform can be applied to various biomolecules such as proteins, bacteria, and cells.     

Functional glass surface displaying a glutamyl donor substrate for transglutaminase-mediated protein immobilization

A chemically modified glass surface displaying a glutamyl donor substrate peptide (Z-QG) was developed for microbial transglutaminase (MTG)-mediated immobilization of recombinant proteins tagged with an MTG-reactive lysine-containing substrate peptide (K-tag). To evaluate the surface modification conditions affecting the enzymatic protein immobilization, we employed an amino-modified 96-well glass plate as a base and prepared three types of glass surfaces displaying Z-QG. Validation of the Z-QG modified glass surfaces with recombinant enhanced green fluorescent proteins revealed that the insertion of a di(ethylene glycol) linker between the terminal Z-QG moiety and the base not only enhances enzymatic protein immobilization efficiency but also decreases nonselective protein adsorption. A bacterial alkaline phosphatase fused with a K-tag at the N terminus was also successfully immobilized to the designed glass surface, suggesting that the chemically modified glass surface displaying a spatially controlled glutamyl donor substrate is a potential platform for MTG-mediated fabrication of protein-based solid biomaterials.     

Ultrathin functional star PEG coatings for DNA microarrays

In this study, star PEG coatings on glass substrates have been used as support material for oligonucleotide microarrays. These coatings are prepared from solutions of six armed star shaped prepolymers that carry reactive isocyanate endgroups. As described earlier, such films prevent the adsorption of proteins and the adhesion of cells but can easily be functionalized for specific biological recognition. Here we used the high functionality of these coatings for the covalent immobilization of amino terminated 20mer oligonucleotides, both by microcontact printing and spotting techniques. The permanent immobilization of fluorescently labeled DNA as well as hybridization of 20mer oligonucleotides have been monitored by fluorescence microscopy. The hybridization efficiency as determined by fluorescence intensity varied from 30% to 80% depending on the way of layer preparation. The direct spotting without additional activation and blocking steps of the surface demonstrates the potential of star PEG coatings as ultrathin surface modification for microarrays.     

Chemical defensive substances of soft corals and gorgonians

Despite lack of efficient physical protection in the highly competitive and hostile environment, the marine invertebrates including soft corals and gorgonians can survive, mainly relying on their chemical defensive system by a series of secondary metabolites accumulating in their bodies or releasing to their surroundings. The chemical defensive functions of these secondary metabolites were found to serve as antipredatory, antimicrobial, allelopathy and antifouling agents. Study on chemical defensive substances from corals and gorgonians is one of the most important topics in marine chemical ecology. The research results could help us to understand the chemical ecological relationships between corals and their surrounding organisms. The research strategy and methodology played an enlightening role in the discovery of bioactive natural products and the generation of new drug lead compounds from marine sources. The chemical defensive substances from soft corals and gorgonians were reviewed. This review focused on the structures of these secondary metabolites as well as their functions including antipredatory, allelopathy and antifouling activities.     

Parasitism of Iron-siderophore Receptors of Escherichia Coli by the Siderophore-peptide Microcin E492m and its Unmodified Counterpart

Microcin E492 (MccE492) is an antibacterial peptide naturally secreted by Klebsiella pneumoniae RYC492. Initially described as an 84-residue unmodified peptide, it was also recently isolated in a posttranslationally modified form, MccE492m. The production of MccE492m is dependent on the synthesis of enterobactin and the mceABCDEFGHIJ gene cluster. The posttranslational modification was characterized as a trimer of N-(2,3-dihydroxybenzoyl)-l-serine (DHBS) linked to the Ser84-carboxylate via a β-d-glucose moiety. MccE492m was shown to bind ferric ions through the trimer of DHBS. This is the first example of a novel type of antibacterial peptide termed siderophore-peptide. Recognition of MccE492m, but also of the unmodified MccE492, was shown to be mediated by the catecholate siderophore receptors FepA, Cir and Fiu at the outer membrane of E. coli. The siderophore-type modification was shown to be responsible for a significant enhancement of the microcin antibacterial activity. Therefore, we propose that MccE492 and MccE492m use iron-siderophore receptors for uptake into the target bacteria and that improvement of MccE492 antimicrobial activity upon modification results from an increase in the microcin/receptor affinity.     

The Influence of Natural Surface Microtopographies on Fouling

Multiple antifouling strategies of marine organisms may consist of combinations of physical, chemical and mechanical mechanisms. In this study, the role of surface microtopography (?<?500?μm) of different marine organisms, such as Cancer pagurus, Mytilus edulis, Ophiura texturata and the eggcase of Scyliorhinus canicula, has been investigated as a possible component of their defence systems. High resolution resin replicates of these natural surface structures were exposed to natural fouling in field experiments. Abundances of recruits were determined and compared to those on untextured, but otherwise identical, control surfaces to quantify the influence of the different microtopographies on fouling rates. Antifouling effects of microtopographies varied with type of microtopography and coloniser species. The surface microtopography of C. pagurus significantly rejected macrofoulers. The surface structures of the eggcase and O. texturata had repellent effects on microfoulers. Barnacle settlement was temporarily reduced on surface microtopographies of M. edulis and the eggcase. These results emphasise the promising non-toxic antifouling properties of microtextured surfaces.     

The influence of low surface energy materials on bioadhesion — a review

Investigations on the adhesion of a diverse range of biological systems including proteins, tissues, microbes, algae and invertebrates all indicate that minimal long-term adhesion is associated with surfaces having initial surface tensions between 20 and 30 dynes/cm (mN/m), i.e. low energy surfaces. However, all surfaces rapidly become modified on immersion in natural waters through the adsorption of ‘conditioning films’, which may influence subsequent adhesive events associated with the permanent attachment of organisms. In this review the various methods which have been used to measure the strength of attachment of both micro- and macrofouling to surfaces will be outlined and results presented for substrata with a range of surface energies. Data will be presented which show that surface energy can elicit different responses in different organisms. For most organisms, minimal adhesion is associated with low surface energy. Silicone elastomers and fluoropolymers have received most attention regarding their potential use as foul release coatings. Results on the antifouling performance of these classes of materials will be discussed.     

软珊瑚(Sinularia flexibilis)和柳珊瑚(Plexaura homomalla)化学防御物质研究进展

软珊瑚(Sinularia flexibilis)和柳珊瑚(Plexaura homomalla)属于海洋低等无脊椎动物,虽然这些动物自身缺乏有效的物理防御手段,却能在竞争激烈的海洋环境中生存与繁衍,这主要是依靠其次级代谢产物的化学防御作用.这些次级代谢产物聚积在体内或释放到环境中,作用主要体现在抵御捕食者、抗病原微生物、克生与防附着等方面.珊瑚化学防御物质的研究有助于探讨珊瑚与其环境中其它生物的化学生态关系,属于海洋化学生态学研究的重要内容之一,其研究方法和思路对海洋活性天然产物乃至海洋新药先导化合物的发现,具有重要的启迪作用.综述了软珊瑚和柳珊瑚化学防御物质的研究进展,并阐释了软珊瑚和柳珊瑚中具有拒捕食、克生、防生物附着等生物活性的次级代谢产物的结构及其化学防御作用.     

Molecular templates for bio-specific recognition by low-energy electron beam lithography

Protein patterning has become an important topic as advances are made in biologically integrated devices and protein chip technology. Versatile and effective patterning requires substrates that can be quantified, with active presentation of proteins and control over protein density and orientation. Herein we describe a model system and the use of low-energy electron beam lithography to pattern molecular templates for immobilization of antibodies through ligand recognition. The templates were patterned over a background of poly(ethylene glycol) (PEG) modified silicon oxide (SiO _x ). These substrates were exposed to a low-voltage (2 keV) electron beam to remove PEG selectively from exposed regions. These regions were then functionalized with a dinitrophenyl (DNP) ligand and tested for specific binding of fluorescently labeled anti-DNP antibodies. The PEG modified regions in conjunction with ligand-presenting regions in the patterned arrays substantially reduces non-specific adsorption of proteins, yielding a specific/nonspecific ratio of approx 10. The surface coverage of the biologically active DNP groups on SiO _x and the amount of immobilized antibody on DNP were measured with a fluorescence-based, enzyme-linked immunosorbent assay. The specificity of the interaction between DNP ligand and fluorescently labeled anti-DNP antibodies was evaluated with fluorescence microscopy. This approach to patterning of molecular templates and assays for quantification are generally applicable to immobilization of any ligand-receptor pair on a wide range of substrates.     

Chemical defensive substances of soft corals and gorgonians

Despite lack of efficient physical protection in the highly competitive and hostile environment, the marine invertebrates including soft corals and gorgonians can survive, mainly relying on their chemical defensive system by a series of secondary metabolites accumulating in their bodies or releasing to their surroundings. The chemical defensive functions of these secondary metabolites were found to serve as antipredatory, antimicrobial, allelopathy and antifouling agents. Study on chemical defensive substances from corals and gorgonians is one of the most important topics in marine chemical ecology. The research results could help us to understand the chemical ecological relationships between corals and their surrounding organisms. The research strategy and methodology played an enlightening role in the discovery of bioactive natural products and the generation of new drug lead compounds from marine sources. The chemical defensive substances from soft corals and gorgonians were reviewed. This review focused on the structures of these secondary metabolites as well as their functions including antipredatory, allelopathy and antifouling activities.     

Carbohydrate and protein immobilization onto solid surfaces by sequential Diels-Alder and azide-alkyne cycloadditions

We demonstrate the applicability of sequential Diels-Alder and azide-alkyne [3 + 2] cycloaddition reactions (click chemistry) for the immobilization of carbohydrates and proteins onto a solid surface. An alpha,omega-poly(ethylene glycol) (PEG) linker carrying alkyne and cyclodiene terminal groups was synthesized and immobilized onto an N-(epsilon-maleimidocaproyl) (EMC)-functionalized glass slide via an aqueous Diels-Alder reaction. In the process, an alkyne-terminated PEGylated surface was provided for the conjugation of azide-containing biomolecules via click chemistry, which proceeded to completion at low temperature and in aqueous solvent. As anticipated, alkyne, azide, cyclodiene, and EMC are independently stable and do not react with common organic reagents nor functional groups in biomolecules. Given an appropriate PEG linker, sequential Diels-Alder and azide-alkyne [3 + 2] cycloaddition reactions provide an effective strategy for the immobilization of a wide range of functionally complex substances onto solid surfaces.     

Rapidly-Deposited Polydopamine Coating via High Temperature and Vigorous Stirring: Formation,Characterization and Biofunctional Evaluation

Polydopamine (PDA) coating provides a promising approach for immobilization of biomolecules onto almost all kinds of solid substrates. However, the deposition kinetics of PDA coating as a function of temperature and reaction method is not well elucidated. Since dopamine self-polymerization usually takes a long time, therefore, rapid-formation of PDA film becomes imperative for surface modification of biomaterials and medical devices. In the present study, a practical method for preparation of rapidly-deposited PDA coating was developed using a uniquely designed device, and the kinetics of dopamine self-polymerization was investigated by QCM sensor system. It was found that high temperature and vigorous stirring could dramatically speed up the formation of PDA film on QCM chip surface. Surface characterization, BSA binding study, cell viability assay and antibacterial test demonstrates that the polydopamine coating after polymerization for 30 min by our approach exhibits similar properties to those of 24 h counterpart. The method has a great potential for rapid-deposition of polydopamine films to modify biomaterial surfaces.     

Immobilization of oligonucleotides on poly(ethylene glycol) brush-coated Si surfaces

High-density poly(ethylene glycol) (PEG) molecules are grafted onto Si surfaces in a brush-like configuration. We demonstrate that this surface is an excellent substrate for oligonucleotide immobilization. p-Maleimidophenyl isocyanate is used as a heterobifunctional cross-linker to tether thiol-modified oligonucleotides to terminal OH groups on the PEG brush. This approach gives excellent immobilization specificity and low background. The immobilized oligonucleotides show high sensitivity for the detection of complementary targets.     

Amphiphilic triblock copolymers with PEGylated hydrocarbon structures as environmentally friendly marine antifouling and fouling-release coatings

The ideal marine antifouling (AF)/fouling-release (FR) coating should be non-toxic, while effectively either resisting the attachment of marine organisms (AF) or significantly reducing their strength of attachment (FR). Many recent studies have shown that amphiphilic polymeric materials provide a promising solution to producing such coatings due to their surface dual functionality. In this work, poly(ethylene glycol) (PEG) of different molecular weights (M_w?=?350, 550) was coupled to a saturated difunctional alkyl alcohol to generate amphiphilic surfactants (PEG-hydrocarbon-OH). The resulting macromolecules were then used as side chains to covalently modify a pre-synthesized PS₈?_K-b-P(E/B)₂₅?_K-b-PI₁₀?_K (SEBI or K3) triblock copolymer, and the final polymers were applied to glass substrata through an established multilayer surface coating technique to prepare fouling resistant coatings. The coated surfaces were characterized with AFM, XPS and NEXAFS, and evaluated in laboratory assays with two important fouling algae, Ulva linza (a green macroalga) and Navicula incerta, a biofilm-forming diatom. The results suggest that these polymer-coated surfaces undergo surface reconstruction upon changing the contact medium (polymer/air vs polymer/water), due to the preferential interfacial aggregation of the PEG segment on the surface in water. The amphiphilic polymer-coated surfaces showed promising results as both AF and FR coatings. The sample with longer PEG chain lengths (M_w?=?550?g?mol^?1) exhibited excellent properties against both algae, highlighting the importance of the chemical structures on ultimate biological performance. Besides reporting synthesis and characterization of this new type of amphiphilic surface material, this work also provides insight into the nature of PEG/hydrocarbon amphiphilic coatings, and this understanding may help in the design of future generations of fluorine-free, environmentally friendly AF/FR polymeric coatings.