David A. Christianson

Combinatorial materials research applied to the development of new surface coatings IV. A high-throughput bacterial biofilm retention and retraction assay for screening fouling-release performance of coatings

Abstract A high-throughput bacterial biofilm retention screening method has been augmented to facilitate the rapid analysis and down-selection of fouling-release coatings for identification of promising candidates. Coatings were cast in modified 24-well tissue culture plates and inoculated with the marine bacterium Cytophaga lytica for attachment and biofilm growth. Biofilms retained after rinsing with deionised water were dried at ambient laboratory conditions. During the drying process, retained biofilms retracted through a surface de-wetting phenomenon on the hydrophobic silicone surfaces. The retracted biofilms were stained with crystal violet, imaged, and analysed for percentage coverage. Two sets of experimental fouling-release coatings were analysed with the high-throughput biofilm retention and retraction assay (HTBRRA). The first set consisted of a series of model polysiloxane coatings that were systematically varied with respect to ratios of low and high MW silanol-terminated PDMS, level of cross-linker, and amount of silicone oil. The second set consisted of cross-linked PDMS-polyurethane coatings varied with respect to the MW of the PDMS and end group functionality. For the model polysiloxane coatings, HTBRRA results were compared to data obtained from field immersion testing at the Indian River Lagoon at the Florida Institute of Technology. The percentage coverage calculations of retracted biofilms correlated well to barnacle adhesion strength in the field (R2 = 0.82) and accurately identified the best and poorest performing coating compositions. For the cross-linked PDMS-polyurethane coatings, the HTBRRA results were compared to combinatorial pseudobarnacle pull-off adhesion data and good agreement in performance was observed. Details of the developed assay and its implications in the rapid discovery of new fouling-release coatings are discussed.

Combinatorial materials research applied to the development of new surface coatings III. Utilisation of a high-throughput multiwell plate screening method to rapidly assess bacterial biofilm retention on antifouling surfaces.

Abstract The authors recently reported on the development of a novel multiwell plate screening method for the high-throughput assessment of bacterial biofilm retention on surfaces. Two series of biocide containing coatings were prepared to assess the ability of the developed assay to adequately discern differences in antifouling performance: i) a commercially available poly(methyl methacrylate) (PMMA) and silicone elastomer (DC) physically blended with an organic antifouling biocide Sea-Nine 211 (SN211) (4,5-dichloro-2-n-octyl-3(2H)-isothiazolone), and ii) a silanol-terminated polydimethylsiloxane (PDMS-OH) reacted with an alkoxy silane-modified polyethylenimine containing bound ammonium salt groups (PEI-AmCl). Three marine bacteria were utilised to evaluate the SN211 blended coatings (Pseudoalteromonas atlantica ATCC 19262, Cobetia marina ATCC 25374, Halomonas pacifica ATCC 27122) and the marine bacterium Cytophaga lytica was utilised to evaluate the PEI-AmCl/PDMS-OH coatings. The SN211 blended coatings showed a general trend of decreasing biofilm retention as the concentration of SN211 increased in both PMMA and DC. HPLC analysis revealed that reduction in biofilm retention was positively correlated with the amount of SN211 released into the growth medium over the length of the bacterial incubation. When compared to PMMA, DC consistently showed an equal or greater percent reduction in biofilm retention as the level of SN211 loading increased, although at lower loading concentrations. Evaluations of the PEI-AmCl/PDMS-OH coatings with C. lytica showed that all PEI-AmCl loading concentrations significantly reduced biofilm retention (p < 0.0001) by a surface contact phenomenon. The high-throughput bacterial biofilm growth and retention assay has been shown to be useful as an effective primary screening tool for the rapid assessment of antifouling materials.

Barnacle reattachment: a tool for studying barnacle adhesion.

Standard approaches for measuring adhesion strength of fouling organisms use barnacles, tubeworms or oysters settled and grown in the field or laboratory, to a measurable size. These approaches suffer from the vagaries of larval supply, settlement behavior, predation, disturbance and environmental stress. Procedures for reattaching barnacles to experimental surfaces are reported. When procedures are followed, adhesion strength measurements on silicone substrata after 2 weeks are comparable to those obtained using standard methods. Hydrophilic surfaces require reattachment for 2–4 weeks. The adhesion strength of barnacles in reattachment assays was positively correlated to results obtained from field testing a series of experimental polysiloxane fouling-release coatings (r = 0.89). The reattachment method allows for precise barnacle orientation, enabling the use of small surfaces and the potential for automation. The method enables down-selection of coatings from combinatorial approaches to manageable levels for definitive field testing. Reattachment can be used with coatings that combine antifouling and fouling-release technologies.

Environmental and genetic factors that contribute to Escherichia coli K-12 biofilm formation.

Biofilms are communities of bacteria whose formation on surfaces requires a large portion of the bacteria’s transcriptional network. To identify environmental conditions and transcriptional regulators that contribute to sensing these conditions, we used a high-throughput approach to monitor biofilm biomass produced by an isogenic set of Escherichia coli K-12 strains grown under combinations of environmental conditions. Of the environmental combinations, growth in tryptic soy broth at 37°C supported the most biofilm production. To analyze the complex relationships between the diverse cell-surface organelles, transcriptional regulators, and metabolic enzymes represented by the tested mutant set, we used a novel vector-item pattern-mining algorithm. The algorithm related biofilm amounts to the functional annotations of each mutated protein. The pattern with the best statistical significance was the gene ontology ‘pyruvate catabolic process,’ which is associated with enzymes of acetate metabolism. Phenotype microarray experiments illustrated that carbon sources that are metabolized to acetyl-coenzyme A, acetyl phosphate, and acetate are particularly supportive of biofilm formation. Scanning electron microscopy revealed structural differences between mutants that lack acetate metabolism enzymes and their parent and confirmed the quantitative differences. We conclude that acetate metabolism functions as a metabolic sensor, transmitting changes in environmental conditions to biofilm biomass and structure.

Combinatorial materials research applied to the development of new surface coatings XIII: an investigation of polysiloxane antimicrobial coatings containing tethered quaternary ammonium salt groups.

High-throughput biological assays were used to develop structure - antimicrobial relationships for polysiloxane coatings containing chemically bound (tethered) quaternary ammonium salt (QAS) moieties. The QAS-functional polysiloxanes were derived from solution blends of a silanol-terminated polydimethylsiloxane, a trimethoxysilane-functional QAS (QAS-TMS), and methylacetoxysilane. Since the QAS moieties provide antimicrobial activity through interaction with the microorganism cell wall, most of the compositional variables that were investigated were associated with the chemical structure of the QAS-TMS. Twenty different QAS-TMS were synthesized for the study and the antimicrobial activity of sixty unique polysiloxane coatings derived from these QAS-TMS determined toward Escherichia coli , Staphylococcus aureus , and Candida albicans . The results of the study showed that essentially all of the compositional variables significantly influenced antimicrobial activity. Surface characterization of these moisture-cured coatings using atomic force microscopy as well as water contact angle and water contact angle hysteresis measurements indicated that the compositional variables significantly affected coating surface morphology and surface chemistry. Overall, compositional variables that produced heterogeneous surface morphologies provided the highest antimicrobial activity suggesting that the antimicrobial activity was primarily derived from the relationship between coating chemical composition and self-assembly of QAS moieties at the coating/air interface. Using data modeling software, a narrow region of the compositional space was identified that provided broad-spectrum antimicrobial activity.

Combinatorial materials research applied to the development of new surface coatings VII An automated system for adhesion testing

An automated, high-throughput adhesion workflow that enables pseudobarnacle adhesion and coating/substrate adhesion to be measured on coating patches arranged in an array format on 4x8 in.(2) panels was developed. The adhesion workflow consists of the following process steps: (1) application of an adhesive to the coating array; (2) insertion of panels into a clamping device; (3) insertion of aluminum studs into the clamping device and onto coating surfaces, aligned with the adhesive; (4) curing of the adhesive; and (5) automated removal of the aluminum studs. Validation experiments comparing data generated using the automated, high-throughput workflow to data obtained using conventional, manual methods showed that the automated system allows for accurate ranking of relative coating adhesion performance.

Combinatorial materials research applied to the development of new surface coatings XVI: fouling-release properties of amphiphilic polysiloxane coatings

High-throughput methods were used to prepare and characterize the fouling-release (FR) properties of an array of amphiphilic polysiloxane-based coatings possessing systematic variations in composition. The coatings were derived from a silanol-terminated polydimethylsiloxane, a silanol-terminated polytrifluorpropylmethylsiloxane (CF3-PDMS), 2-[methoxy(polyethyleneoxy)propyl]-trimethoxysilane (TMS-PEG), methyltriacetoxysilane and hexamethyldisilazane-treated fumed silica. The variables investigated were the concentration of TMS-PEG and the concentration of CF3-PDMS. In general, it was found that the TMS-PEG and the CF3-PDMS had a synergist effect on FR properties with these properties being enhanced by combining both compounds into the coating formulations. In addition, reattached adult barnacles removed from coatings possessing both TMS-PEG and relatively high levels of CF3-PDMS displayed atypical base-plate morphologies. The majority of the barnacles removed from these coatings exhibited a cupped or domed base-plate as compared to the flat base-plate observed for the control coating that did not contain TMS-PEG or CF3-PDMS. Coating surface analysis using water contact angle measurements indicated that the presence of CF3-PDMS facilitated migration of TMS-PEG to the coating/air interface during the film formation/curing process. In general, coatings containing both TMS-PEG and relatively high levels of CF3-PDMS possessed excellent FR properties.

High-Throughput Screening of Fouling-Release Properties: An Overview

Marine biofouling of ship hulls has significant cost, performance and environmental implications. Due to environmental concerns associated with traditional antifouling paints that mitigate fouling with the use of biocides, increasing research and development efforts have been made on fouling-release (FR) coatings. FR coatings do not actively deter settlement of marine organisms, but, instead, mitigate biofouling by minimizing the strength of adhesion. Ideally, an FR coating will allow the fouling community to be removed by simply running the vessel at relatively high speed. Traditional methods for characterizing FR properties involve immersion of relatively large samples in the ocean and waiting months for enough fouling to occur to enable reliable measurements to be made. To greatly enhance research and development relative to FR coatings, a combinatorial/high-throughput workflow was developed that includes a suite of FR laboratory assays involving marine bacteria, microalgae, and live, adult barnacles. The novel high-throughput FR measurement systems have been shown to allow for rapid screening of FR characteristics of miniaturized coating samples arranged in an array format.

Hybrid organic/inorganic coatings produced using a dual-cure mechanism

A coating precursor containing both acrylate functionality and trimethoxysilane functionality was produced by reacting bisphenol-A glycerolate diacrylate with 3-isocyanatopropyltimethoxysilane. With this precursor, two different crosslinked networks can be produced. A polyacrylate network can be produced using a radiation-cure mechanism while a polysiloxane network can be produced by hydrolysis and condensation reactions involving the trimethoxysilane groups. The objective of the study was to determine the utility of this dual-cure system for generating rapid-cure coatings for corrosion protection. Coating properties were determined as a function of cure conditions. The results of the study showed that the formation of siloxane crosslinks was significantly hindered by the crosslinked network induced by the UV-curing process. Even though the overall conversion of trimethoxysilane groups to siloxane crosslinks was relatively low, coating barrier properties were significantly enhanced and coating free volume reduced. At ambient conditions, additional crosslinking occurring through siloxane bond formation increased within the first 4 days after UV-curing. Beyond this period, siloxane bond formation remained unchanged as did coating properties.