Article pubs.acs.org/Langmuir
Adhesion of Marine Fouling Organisms on Hydrophilic and Amphiphilic Polysaccharides Stella Bauer,*,†,‡ Maria Pilar Arpa-Sancet,†,‡ John A. Finlay,§ Maureen E. Callow,§ James A. Callow,§ and Axel Rosenhahn†,∥ †
Institute of Functional Interfaces, Karlsruhe Institute of Technology, P.O. Box 3640, 76021 Karlsruhe, Germany Applied Physical Chemistry, Ruprecht-Karls-University Heidelberg, Im Neuenheimer Feld 253, 69120 Heidelberg, Germany § School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, U.K. ∥ Analytical Chemistry - Biointerfaces, Ruhr-University Bochum, 44780 Bochum, Germany ‡
ABSTRACT: Polysaccharides are a promising material for nonfouling surfaces because their chemical composition makes them highly hydrophilic and able to form water-storing hydrogels. Here we investigated the nonfouling properties of hyaluronic acid (HA) and chondroitin sulfate (CS) against marine fouling organisms. Additionally, the free carboxyl groups of HA and CS were postmodified with the hydrophobic trifluoroethylamine (TFEA) to block free carboxyl groups and render the surfaces amphiphilic. All coatings were tested with respect to their protein resistance and against settlement and adhesion of different marine fouling species. Both the settlement and adhesion strength of a marine bacterium (Cobetia marina), zoospores of the seaweed Ulva linza, and cells of a diatom (Navicula incerta) were reduced compared to glass control surfaces. In most cases, TFEA capping increased or maintained the performance of the HA coatings, whereas for the very well performing CS coatings the antifouling performance was reduced after capping. the marine environment. On the basis of the egg-box model,25 which describes the situation for polysaccharides with guluronic acid building blocks, this loss of inert properties was related to the complexation of cations by free carboxyl groups. A number of recent studies showed that coatings with amphiphilic properties have a high potential for inert surface coatings,3,5 a property that can be established in the hydrophilic polysaccharide network via chemical modifications with hydrophobic molecules. In this Article, we focus on two very hydrophilic polysaccharides, hyaluronic acid and chondroitin sulfate (Figure 1). Both are glycosaminoglycans, belonging to the group of
1. INTRODUCTION Biofouling, the unwanted adhesion of macromolecules and organisms on surfaces, is an economic and ecological challenge for modern coating research.1,2 In the search for inert surfaces that prevent biofouling or facilitate fouling release, multiple surface properties including the chemistry, modulus, charge ,and morphology need to be considered.1,3−6 Hydrogels in particular have received increasing attention because they have the ability to store water, a property identified as important for resistance to fouling.7 Polymers based on ethylene glycol, a group of hydrogel-forming macromolecules, reveal significant antifouling properties.8−11 Despite these excellent resistant properties, all ethylene glycols suffer from rapid degradation.12−14 Nevertheless, ethylene glycol-containing model surfaces are very useful in biofouling research and recently allowed the importance of water binding for the inertness of surfaces to be identified.15−17 Alternatives to ethylene glycol with a similar ability to store water are polysaccharides.18,19 Because of their chemical composition, they are hydrophilic and can be used to produce inert coatings.20−23 Morra and Cassinelli showed that the highly hydrated polysaccharides hyaluronic acid and alginic acid are resistant to the adhesion of mammalian cells and bacteria.20 Cao et al.24 examined the interaction of different polysaccharide coatings with a range of marine organisms and hematopoietic cells in greater detail, observing that such coatings are unsuitable for application in © 2013 American Chemical Society
Figure 1. Polysaccharides used for the surface coatings: hyaluronic acid and chondroitin-6-sulfate. Received: September 21, 2012 Revised: February 7, 2013 Published: February 20, 2013 4039
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pressure 0.4 mbar, 3 min, 150 W, TePla, Wettenberg, Germany) to create sufficient free hydroxyl groups on the surface. A layer of 3aminopropyltrimethoxy silane (APTMS) was coupled onto the activated surfaces by immersion of the samples in a 5% APTMS solution in dry acetone for 30 min under a N2 atmosphere during ultrasonication. The polysaccharides used for coupling were the sodium salts of hyaluronic acid (HA) and chondroitin sulfate (CS). HA from Streptococcus equi had a molecular weight of (1.5−1.8) × 106 Da, and CS from shark cartilage had a molecular weight of (3.4 ± 0.3) × 104 Da, determined by SEC with a MALS detector (Wyatt, Dernbach, Germany) with a 6- to 4-sulfate ratio of 0.33:1. We used polysaccharides with a relatively high molecular weight and broad molecular distribution because they are the cheapest alternative for potential future upscaling. To couple the polysaccharides to the APTMS layers, EDC/NHS chemistry according to literature procedures has been used.48,49 Therefore, PS were dissolved in 4-(2hydroxyethyl)-piperazine-1-ethane sulfonic acid (HEPES) buffer solution (10 mM, pH 6−7, PS concentration 1 mg mL−1). After achieving a clear solution, N-hydroxysuccinimide (NHS, 0.01 M) and N-(3-dimethyl amino propyl)-3-ethyl carbodiimide hydrochloride (EDC, 0.05 M) were added. The APTMS-coated surfaces were then immersed in this solution of activated PS. After 18 h at room temperature on a shaker table (65 rpm), the reaction solution was diluted with an 8-fold volume of Milli-Q water. In order to remove physisorbed macromolecules this procedure was repeated twice on the subsequent days. After 3 days, the samples were rinsed with Milli-Q water and dried in a stream of N2. To modify the surface-bound polysaccharides, the coated samples were immersed in a solution of NHS (5 mM) and EDC (25 mM) in HEPES buffer in order to reactivate free-remaining carboxyl groups. After 15 min, 2,2,2-trifluoroethylamine (TFEA, 40 mM) was added to the activated surfaces. After 18 h, the solution was diluted with an 8fold volume of Milli-Q water, after 4 h the samples were rinsed with Milli-Q water and dried in a stream of N2. The samples were stored under Ar until they were characterized or under Milli-Q until they were evaluated in bioassays. 2.2. Surface Characterization. Sessile drop water (Milli-Q water) contact angles were measured at ambient conditions using a G1 goniometer (Krüss, Hamburg, Germany) without the tip being in contact with the droplet. To determine the film thickness of the prepared coatings, ellipsometric measurements were carried out on coated silicon wafers. A fixed angle M-44 spectral ellipsometer (J.A. Woollaam Co., Inc., Lincoln, USA) was used, operating in the wavelength range between 280 and 800 nm. The organic film was modeled as a single Cauchy layer, using the software WVASETM (J.A. Woollam Co.). The values shown are averages from measurements of three different spots on each sample from which the standard error was calculated. The films were further characterized by X-ray photoelectron spectroscopy (XPS), utilizing a Leybold-Heraeus MAX 200 X-ray photoelectron spectrometer with a polychromatic aluminum anode as X-ray source (Kα = 1486.4 eV). Peak fitting was performed with the software XPSPeak 4.1 (Prof. R.W.M. Kwok, Department of Chemistry, University of Hong Kong) using the background subtraction of Shirley.50 The thickness of the polysaccharide films was determined by its attenuation of the Si2p XPS signal from the previous APTMS layer, using Lambert−Beer’s law. A mean free path of the photoelectrons of λ = 39 Å was used.51 To calculate elemental ratios, cross sections calculated by Scofield were utilized.52 The surface roughness of the films was determined using atomic force microscopy in tapping mode (Dimension 3100 equipped with a Nanoscope IIIa controller) under ambient conditions. The shown rms values were calculated utilizing the Nanoscope software and display the mean roughness of 2 different spots (scanning area 500 × 500 nm) on each sample. The stability of the prepared surface coatings was tested in artificial seawater (ASW, Instant Ocean) over 7 d. The samples were immersed in the marine medium on a shaker table (50 rpm) and subsequently rinsed with water and blown dry in a stream of N2. The thickness of the coatings was measured by spectral ellipsometry prior and after immersion.
polysaccharides based on repeating disaccharide units composed of hexauronic acid and a hexosamine.26 Both sugars are negatively charged at physiological pH (and at the pH of seawater, ∼8.2)27 and are, for example, found in the extracellular matrix (ECM) of mammalian cells.28,29 They differ only in the sulfation of CS, which can be found in different positions. We used HA because it revealed superior inert properties compared to those of other polysaccharides such as alginic acid and pectic acid in recent studies.20,24 Resistance to the adsorption of proteins is a well-known characteristic of HA-coated substrates and does not depend on the applied grafting technique.30,31 The use of CS is inspired from its presence in fish mucus and its potential contribution to the protection of the skin of fish.32 Like HA, chondroitincontaining coatings are highly hydrophilic and the saccharide has been suggested to play a role in natural antifouling,33,34 but to the best of our knowledge, CS has never been tested for its antifouling properties. The CS used in this work was a natural polymer composed of disaccharides with sulfate groups at the 4 and/or 6 positions. Both acidic polysaccharides, HA and CS, carry carboxyl groups that can be utilized for surface binding and selective modifications.35 Because Morra et al. demonstrated that the grafting density does not dominate the resistance of HA coatings when the molecular weight of the polysaccharide is high,36 natural polymers with high MW were utilized. Surface grafting was achieved by carbodiimide coupling to silanized substrates.24 Besides the two pristine polysaccharide coatings, we capped the carboxylic acid groups with hydrophobic 2,2,2-trifluoroethylamine. Capping of the polysaccharides was attempted for three reasons: the blocking of free carboxyl groups should prevent the previously observed complexation of bivalent ions to preserve the resistance in seawater,24 it should shift the contact angle toward the minimum in the Baier curve to maximize inert properties,37−39 and amphiphilic properties introduced by the hydrophobic fluoro groups should enhance the resistance to fouling.3,5,40−43 The pristine and the capped polysaccharides produced a matrix of four surface coatings examined in this study (unmodified and amine-modified HA and CS). All coatings were tested for their protein resistance, adhesion of the marine bacterium Cobetia marina, and settlement and adhesion of zoospores of the green alga Ulva linza and cells of the diatom Navicula incerta.40 Resistance to protein adsorption is known to provide a general indication of the inert properties of coatings. Cobetia marina has been used as a model marine biofouling bacterium that readily forms biofilms.44 The attachment of cells of two algal species is a consequence of their different mechanisms to adhere to surfaces. Cells of N. incerta reach surfaces by gravity,45 and zoospores of U. linza are motile, with distinctive surface sensing prior to commitment to settlement (i.e., permanent attachment46,47).
2. MATERIALS AND METHODS 2.1. Preparation of the Polysaccharide Coatings. All chemicals were purchased from Sigma-Aldrich (Munich, Germany) and used without further purification. Deioinized water was purified with a Milli-Q Plus system (Millipore, Schwalbach, Germany). PBS buffer was used at pH 7.4 and a concentration of 0.01 M. NexterionB clean room cleaned glass slides were obtained from Schott (Jena, Germany). Samples (silicon wafers or glass slides) were cleaned by successive ultrasonication in solvents of increasing polarity (toluene, ethyl acetate, ethanol p.a., Milli-Q water) for 30 s in each solvent. After drying in a stream of N2, the samples were subjected to O2 plasma (O2 4040
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compartments of quadriperm dishes (Greiner-One). After 2 h at 20 °C on the laboratory bench, the slides were rinsed with ASW to remove cells that had not adhered to the surface. Three replicates were fixed with 2.5% glutaraldehyde in ASW, and the other three replicates were exposed to a shear stress of 32 Pa (320 dyn cm−2) in a calibrated water channel in order to determine the removability of the cells. The number of cells on the samples was counted, and percent removal was calculated as described for spores of U. linza.
2.3. Protein Adsorption Assay. The assays were carried out on silicon wafers coated with the corresponding polysaccharides. 1Dodecanethiol (DDT) self-assembled monolayers (SAMs) on goldcoated (30 nm) glass slides were used as a nonprotein resistant standard, as they are known to adsorb proteins readily.9 The protocol to assess protein resistance followed earlier work.53 Proteins were dissolved in phosphate-buffered saline (PBS, 0.01 M, pH 7.4) in concentrations of 2 mg mL−1 for pepsin, lysozyme, and fibrinogen and 160 units mL−1 for pyruvate kinase. The surfaces were immersed in 10 mL of PBS for 20 min, and then 10 mL of protein solution was added. After 30 min of incubation, the solutions were flooded with 1 L of deionized water. After being rinsed with water, the samples were dried under a stream of N2. The thickness of the adsorbed protein was determined by spectral ellipsometry. 2.4. Adhesion Strength of the Marine Bacterium Cobetia marina. Cobetia marina54 was obtained as a dried culture from DSMZ (Deutsche Sammlung von Mikroorganismen and Zellkulturen GmbH, German Collection of Microorganisms and Cell Cultures, Braunschweig, Germany) and stored frozen at −70 °C. Bacteria were cultured following established protocols.55 An overnight culture (∼14 h) of the bacteria in the stationary phase with an optical density, OD, of >1 (λ = 600 nm) was diluted with sterile growth medium (Marine Broth, 1:100). Then this diluted culture was grown at room temperature on a shaker table until OD = 0.1 was achieved and thus the log phase was reached (∼3 h). The medium was exchanged with ASW (Instant Ocean) by centrifugation at 10 000 rpm for 2 min and resuspension. The detachment assay was carried out in a microfluidic device as described earlier.55 Briefly, the microfluidic channel assembly consisted of the substrate of interest, a poly(dimethoxysiloxane) channel, and a sealing system. Four of these assemblies were installed on an inverted microscope and connected to a medium reservoir with ASW and a syringe pump. Prior to bacteria seeding, the microchannel systems were preconditioned with sterile ASW for 5 min. Then a suspension of the bacteria (107 cells mL−1) was injected into the channels and incubated for 2 h. After this time, the flow rate was increased stepwise by 26% every 5 s, and detachment was followed via video microscopy. The derived detachment curve yields two characteristic values. The adherent fraction FA is the number of adherent bacteria remaining on the surface after the application of a very weak flow divided by the number of initially visible bacteria. The critical shear stress τ50 is the shear stress value at which 50% of the initially adherent bacteria are detached. This measurement describes how strongly bacteria are able to stick to a specific surface. 2.5. Settlement and Attachment of Zoospores of the Green Alga Ulva linza. Zoospores were obtained from mature plants of U. linza by standard methods.56 The sample slides (six replicates of each chemistry) were stored in Milli-Q water until being tested. NexterionB glass slides were included in all assays as a comparative standard. One hour prior to the assay, they were immersed in 0.22 μm filtered ASW (Tropic Marin). A suspension of zoospores (10 mL: 1 × 106 spores mL−1) was added to individual compartments of quadriperm dishes (Greiner-One) each containing a slide. After 45 min in darkness at room temperature, the slides were washed in ASW to remove unsettled (motile) spores. Three replicates were fixed using 2.5% glutaraldehyde in seawater. The density of spores was counted on each of three replicate slides using an Axiovision image analysis system attached to a Zeiss Axioplan fluorescence microscope. Spores were visualized by the autofluorescence of chlorophyll. Counts were made for 30 fields of view (each 0.15 mm2) on each slide. The three remaining replicates were exposed to a shear stress of 52 Pa (520 dyn cm−2) in a calibrated water channel to determine the removability of the attached spores.57 The number of remaining spores was determined as described above. The percentage removal was calculated from the spore density before and after exposure to shear stress. 2.6. Settlement and Adhesion of Cells of the Diatom Navicula incerta. Samples were prepared as described for the zoospore experiments. Cells of N. incerta were cultured following a standard protocol.58 Cells in the log phase were washed with fresh medium three times, and the chlorophyll a concentration was adjusted to 0.25 μg mL−1. Ten milliliters of the culture were added to individual
3. RESULTS 3.1. Preparation and Characterization of the EndCapped Polysaccharide Coatings. For the preparation of the desired surface coatings, a strategy of substrate silanization and subsequent EDC/NHS coupling was chosen (Figure 2). In
Figure 2. Reaction scheme of the immobilization reaction and polysaccharide modification with 2,2,2-trifluoroethylamine.
the first step, substrates were coated with an amine-terminated silane. Polysaccharides with EDC/NHS chemistry-activated carboxyl groups were subsequently coupled to the aminecovered surfaces. Because not all carboxylic acid groups react in the linking step, free remaining carboxyl groups were reactivated with EDC/NHS for capping with hydrophobic TFEA. For the biological assays, coated glass slides were used because transparent substrates were required for light microscopy. Surface characterization and protein-resistance assays were carried out on silicon wafers coated with the different polysaccharides to provide the necessary conductance and reflectivity. The surface characterization results are summarized in Table 1. Static contact angles showed the expected transition from a highly hydrophilic hydroxyl-functionalized glass substrate to the more hydrophobic amine-terminated APTMS surface. The coupling of polysaccharides rendered the surface hydrophilic, and amide-functionalization with the fluorinated amine increased the contact angle again by ∼10°. The formation of Table 1. Static Water Contact Angles, Layer Thicknesses as Measured by Spectral Ellipsometry, and Attenuation of the Si 2p Photoemission Signal in XPS for the Different Coatings Representing the Average of Four Samples and rms Values as Determined by AFMa surface APTMS HA HA + TFEA CS CS + TFEA
contact angle (deg)
thickness (Å) from ellipsometry
thickness (Å) from XPS
rms (nm)
35 ± 5
