Biomacromolecules 2008, 9, 2775–2783
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Poly(ethylene glycol)-Containing Hydrogel Surfaces for Antifouling Applications in Marine and Freshwater Environments Tobias Ekblad,† Gunnar Bergstro¨m,† Thomas Ederth,† Sheelagh L. Conlan,‡ Robert Mutton,‡ Anthony S. Clare,‡ Su Wang,§ Yunli Liu,§ Qi Zhao,§ Fraddry D’Souza,| Glen T. Donnelly,| Peter R. Willemsen,| Michala E. Pettitt,⊥ Maureen E. Callow,⊥ James A. Callow,⊥ and Bo Liedberg*,† Division of Molecular Physics, Department of Physics, Chemistry and Biology, Linko¨ping University, SE-581 83 Linko¨ping, Sweden, School of Marine Science and Technology, Newcastle University, Newcastle upon Tyne NE1 7RU, United Kingdom, Division of Mechanical Engineering, University of Dundee, Dundee DD1 4HN, United Kingdom, TNO Science and Industry, Bevesierweg MML (Harssens), Den Helder, The Netherlands, and School of Biosciences, University of Birmingham, Birmingham B15 2TT, United Kingdom Received May 20, 2008; Revised Manuscript Received July 9, 2008
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.
Introduction As the environmental and legislative demands on the antifouling surface coatings for man-made underwater structures increase,1 efforts are being made to replace current biocidal formulations with more environmentally benign compositions. In addition to the well-known fouling-release silicone elastomeric coatings that reduce the adhesion of macrofouling organisms,2,3 a number of other environmentally benign strategies are currently being investigated that either deter organisms from settling or reduce adhesion, for example, through topography,4-6 novel surface chemistries,7-9 enzymes,10,11 and pulsed electrical fields.12 Because a prerequisite for fouling is the secretion of adhesive molecules by the settling organism, surfaces that deter or reduce adhesive binding should also deter colonization. We have investigated this hypothesis by evaluating the settlement (attachment) and adhesion of a number of organisms to a protein-resistant, poly(ethylene glycol)-based hydrogel coating. Hydrogel-based materials have previously been evaluated for marine biofouling applications with varying results. For instance, poly(2-hydroxyethyl methacrylate) gels have been tested in field * To whom correspondence should be addressed. E-mail:
[email protected]. † Linko¨ping University. ‡ Newcastle University. § University of Dundee. | TNO Science and Industry. ⊥ University of Birmingham.
trials but failed to deter fouling except when loaded with biocides.13 In another study, a number of different hydrogel types were tested for barnacle settlement. The materials exhibited lower settlement than polystyrene references, but there were differences between the gels.14 These studies indicate that softness and hydrophilicity, two of the distinguishing features of hydrogels, are not enough to render a material resistant to biofouling. Instead, one must consider the interactions between the specific chemistry of the coating and the fouling organisms. One type of hydrogel chemistry that could be expected to perform quite well in this context, based on past experience within the biomedical field, is poly(ethylene glycol) (PEG). The protein resistance of this hydrophilic polymer and its oligomers is well-known and has been exploited in many applications. These properties have been attributed to strong interactions with water,15 in combination with charge neutrality and absence of hydrogen bond donors.16 For long-chained PEGs, steric repulsion effects may also play a role.17 In addition, the conformation of the ethylene glycol portion has also been discussed as a possible explanation to the favorable protein rejecting properties of oligo(ethylene glycol) self-assembled monolayers (SAMs).18,19 PEG coatings have successfully been used in biomaterial applications where low cell adhesion is desired. Examples of this are prevention of platelet adhesion in blood contact applications,20 limitation of the risk of bacterial infection on implanted biomaterials,21 or for selective patterning of cells.22-24 The performance of the coatings has generally been assumed to be related to the high protein resistance of PEG. This
10.1021/bm800547m CCC: $40.75 2008 American Chemical Society Published on Web 08/30/2008
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background, combined with the nontoxicity of the polymer, makes PEG an appealing candidate for novel antifouling coatings for specific end-uses such as oceanographic sensors. So far, in the aquatic biofouling context, the studies performed on PEGylated materials have focused on one or two fouling organisms, commonly two algae, namely, the green macroalga UlVa and the diatom NaVicula. The settlement (attachment) and removal behavior of these species has been studied for a small number of PEG-containing coating types with otherwise diverse chemical composition. The results have varied, but generally, settlement (attachment) and adhesion strength have been reduced.7,8,25-28 In a related application, the phase separation of hydrophilic PEG and hydrophobic fluoropolymer segments has been used to introduce chemical heterogeneity to further influence the settlement process.29 Together, these studies imply that inclusion of PEG in a coating can be an efficient way to reduce both settlement (attachment) of NaVicula cells and UlVa zoospores. The antifouling function of PEG-containing coatings toward many other key organisms, such as barnacles, has not been systematically examined to date. Therefore, we decided to undertake a study using a well-characterized PEG surface chemistry to investigate whether the reduction of settlement is also exhibited by other species and not limited to algae. The research presented here was aimed at producing thin, yet robust PEG-containing hydrogel coatings with low protein adsorption to provide model surfaces for the evaluation of biofouling resistance. In a previous work, we developed a hydrogel chemistry that could be coated in thin, optically transparent films onto a variety of substrates and exhibited little or no protein adsorption, even from such complex biofluids as blood plasma and serum.30 The hydrogel was prepared by UVinitiated free radical polymerization of the monomers, 2-hydroxyethyl methacrylate (HEMA) and PEG methacrylate, with an average PEG chain length of 10 units (PEG10MA). Combined with high reproducibility and relative ease of preparation, this coating appeared ideally suited for an extensive study within the field of biofouling. Additionally, the method of preparation allows for photolithographic patterning31 and manufacture of gradients of thickness and chemical composition,32 methods that may be used in future studies to further increase the understanding of settlement and adhesion of fouling organisms. For the current study, glass slides were homogeneously coated with the hydrogel and the surfaces were evaluated in laboratory assays using a range of common fouling organisms, specifically the marine algae, UlVa and NaVicula, two species of marine bacteria. Cobetia marina and Marinobacter hydrocarbonclasticus and the cypris larva (cyprid) of the barnacle, Balanus amphitrite. In addition, a freshwater bacterium, Pseudomonas fluorescens, was used. The physiochemical properties of the coatings were also investigated using contact angle measurements, infrared spectroscopy, and ellipsometry. This was specifically aimed at determining the stability of the hydrogel in both the short- and long-term. Several studies have shown that the long-term stability of PEG-based films can be quite low when exposed to demanding environments such as cell culture conditions. A maximum functional lifetime of a few months has been reported.22,33 Oxidative degradation and chain cleavage of PEG is the most common explanation for this poor performance. Additionally, in the case of methacrylate-based hydrogels, the ester bond with which the side chains are connected to the polymer backbone may be vulnerable to hydrolysis.34 Therefore, this critical issue was addressed by a series of measurements.
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Figure 1. Reactor chamber with the glass substrate hanging beneath the quartz disk by capillary forces. The reaction chamber was purged with a constant flow of nitrogen gas to remove oxygen. The monomer film, composed of an aqueous 1:1 solution of HEMA (denoted H in image) and PEG10MA (denoted P), was approximately 50 µm thick. UV light initiated the polymerization reaction directly, without any added initiator. The same free-radical formation mechanisms may also lead to branching (indicated in the figure) and a certain degree of cross-linking.32
Experimental Section Materials. HEMA, PEG10MA (PEG length, ca. 10 units) and xylene were purchased from Sigma-Aldrich Sweden AB. γ-Methacryloxypropyltrimethoxysilane (MPS, sold under the trade name PlusOne BindSilane) was purchased from GE Healthcare Life Sciences, Sweden. Glacial acetic acid was purchased from Merck KGaA, Germany. Polystyrene was cut from Petri dishes (VWR International, Sweden). Several different types of substrates were used in the different experiments. All biological evaluation experiments were carried out using glass microscope slides (Cleanroom-cleaned Nexterion B, 26 × 76 × 1 mm3, Schott AG, Germany) coated on one side with hydrogel. Silicon (100) wafers (Topsil Semiconductor Materials A/S, Denmark) with a 2 nm thick native oxide layer were used for the ellipsometry measurements. The substrates for the IR measurements were prepared by coating silicon with 2.5 nm titanium followed by 200 nm of gold in an electron beam evaporation system. Surface Preparation. Glass microscope slides were immersed in a 1:1 mixture of ethanol and Milli-Q water (Millipore, U.S.A.) containing 0.4% MPS and 0.05% glacial acetic acid. After 10 min, the slides were removed from the bath and dried with a stream of N2 gas. The silane layer was then cured by placing the slides in an oven at 115 °C for 10 min. To remove any silane multilayers, the slides were ultrasonicated in ethanol for 10 s, further rinsed with ethanol and dried. The hydrogel coating was prepared by polymerizing it directly onto the silanized substrates. The monomer solution consisted of 120 mM HEMA and 120 mM PEG10MA dissolved in Milli-Q water. No initiator was added and the monomers were used without purification. The polymerization process has been described elsewhere30-32 and the reactor setup is illustrated in Figure 1. In short, a sandwich of a UVtransparent quartz disk, the monomer solution, and the glass substrate was constructed by applying a 100 µL drop of monomer solution on the bottom face of the quartz disk and gently bringing the drop and the glass substrate in contact. The glass substrate thus hung underneath the quartz disk by capillary forces. The sandwich was then placed in a nitrogen-purged chamber equipped with a UV lamp with the main emission peak at 254 nm (Philips TUV PL-L 18 W). The glass slide and monomer solution was irradiated through the quartz disk for 10 min. The glass slide was then removed and ultrasonicated in ethanol and water, thoroughly rinsed with ethanol, and dried. After contact angle measurements, the coated slides were stored under nitrogen until used for physical characterization or biological evaluation. The same method as described above was used to prepare hydrogel coatings on silicon for ellipsometric thickness determination. The silicon wafer was cut into smaller pieces and washed in TL1 solution (1:1:5 proportions of 25% NH3, 30% H2O2, and Milli-Q water for 10 min at 85 °C) prior to silanization. For the long-term stability study an
Hydrogel Surfaces for Antifouling Applications additional layer of polystyrene (PS) was spin-coated on the silanized silicon. The gold substrate, which was used for the IR measurements, was also spin-coated with PS after being washed in TL1 solution. A solution of 0.25% PS in xylene was spin coated at 1000 r/min for 30 s using a WS-400B-6NPP/Lite spin coater (Laurell Technologies corp.). This gave a PS film thickness of approximately 16 nm. Characterization. Contact Angle Goniometry. The advancing and receding contact angles with Milli-Q water were measured using a CAM 200 Optical Contact Angle Meter (KSV Instruments Ltd., Finland) equipped with a manual liquid dispenser. Five advancing and five receding angles were measured in each point, and three points were measured on each surface. Contact angle measurements were routinely carried out on all slides to verify that the samples had consistent properties. Only slides that had mean advancing contact angles between 55 and 64° were accepted for the biological evaluation stages. Ellipsometry. Ellipsometry measurements were performed in air on dry samples. Silicon substrates were used. Measurements were carried out using a Rudolph Research AutoEL null ellipsometer equipped with a He-Ne laser (λ ) 632.8 nm) set at an angle of incidence of 70°. A total of 10 spots were measured on each sample. A refractive index of 1.5 was used in the model to calculate the thicknesses of the organic films. Infrared Spectroscopy. Infrared spectroscopy measurements were carried out using the same equipment and methods as described by us previously.30 In short, a Bruker IFS 66 FT-IR spectrometer equipped with a MCT detector was used with a grazing angle (85°) setup. The infrared spectra were recorded with a resolution of 2 cm-1. A clean gold substrate was used for collecting the background spectrum. The integration limits chosen for the quantitative estimation of the peak intensities were 1050-1215 cm-1 (CsO peak) and 1690-1760 cm-1 (CdO peak). Stability Studies. A short-term and a long-term stability study were carried out. The motivation for the shorter study was to confirm that the hydrogel, as prepared on glass slides, would not deteriorate during the course of the biological evaluation assays while the longer study served to investigate the long-term effect of dissolved oxygen and ions on the stability of the hydrogel. In both cases, samples were stored in artificial seawater (ASW, 0.45 µm filtered Instant Ocean, Aquarium Systems, France) in transparent 250 mL polypropylene containers with closed lids at normal room conditions. No attempts were made to remove dissolved oxygen from the ASW solution and the solutions were not stirred or agitated during storage. Samples were removed from the containers for characterization at set time intervals. This ranged from 24 h in the short term study to several weeks in the long term study. Prior to ellipsometry, IR, or contact angle measurements, the samples were rinsed in Milli-Q water. After analysis, they were immediately returned to the ASW solution. In the long-term study, the ASW was exchanged after each measurement series. The samples used for the short time-study were three glass slides and three silicon pieces, prepared as described above. The silicon substrates were included for thickness measurements by ellipsometry. Contact angles and thicknesses (of the silicon samples) were measured before immersion and after 24 and 48 h, the latter of which was the maximum duration time for any of the biological evaluation protocols. To further investigate the shortterm stability, an additional measurement series was performed after the samples had been immersed in ASW for 1 week. The long-term stability study was carried out over the course of 6 months. Note that a modification of the coating protocol was adopted for this study. We have previously shown that the hydrogel in question can be prepared on a number of alternative substrates, including plastics.32 Because silanes are known to be vulnerable to hydrolysis of the SisO bond,35 potentially diminishing the hydrogel adhesion to the substrate, the hydrogels used in this study were instead prepared on spin-coated PS to protect the silane from degradation and provide a stable basis for the coating. Gold substrates were included as these provided an ideal substrate for grazing angle IR measurements. The same coated sample was used for all IR measurements shown. Silicon
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substrates were used for the ellipsometry measurements. At the end of the stability study the resistance to protein adsorption, exemplified by human fibrinogen (Hyphen BioMed, 1 mg/mL in PBS pH 7.4 for 30 min), was assessed by ellipsometry. Biological Evaluation. The biological evaluation protocols were conducted in different ways depending on organism; the general goal (except for barnacles) was to quantify both the amount of settlement (attachment) and the adhesion strength of the settled organisms. To compare the settlement on the hydrogel with a common standard, acidwashed glass slides were included in all assays. Depending on the evaluation protocol, hydrogel-coated slides were equilibrated in either deionized water or ASW prior to the assay. Algae. Slides were equilibrated in ASW for 1 h prior to assay. UlVa zoospore settlement assays followed the principles outlined in Callow et al.36 In brief, each test surface (six replicates) was placed in a separate compartment of a Quadriperm dish (Greiner Bio-one Ltd.) to which 10 mL of a suspension containing 1.5 × 106 mL-1 zoospores were added. Zoospores were allowed to settle for 45 min, in the dark, before the slides were washed to remove unsettled (swimming) spores. Three replicate slides were fixed in 2.5% (v/v) glutaraldehyde, washed, and air-dried as described previously.36 The density of settled (adhered) zoospores was determined using a Zeiss Kontron 3000 image analysis system attached to a Zeiss epifluorescence microscope and video camera as described in Callow et al.37 A total of 30 fields of view were counted at 1 mm intervals along the length of each of three replicates slides. The attachment strength of zoospores was determined by exposing the remaining three replicate washed slides to a wall shear stress of 52 Pa in a calibrated flow channel.38 Zoospore removal data are expressed as a percentage of the initial settlement density. Diatom assays followed the methods described in Pettitt et al.39 In brief, cells of NaVicula perminuta were resuspended in ASW to a chlorophyll concentration of 0.3 µg mL-1. The surfaces (six replicates) were placed in Quadriperm dishes to which 10 mL of the diatom suspension was added. After 2 h, the slides were washed by submersion to remove any nonattached cells. Three replicate slides were then fixed and processed as described for UlVa zoospores. The remaining three slides were exposed to a wall shear stress of 51.5 Pa in a flow-channel,38 before identical fixation and processing. Cell density and percentage removal data were determined as described for UlVa zoospores. Barnacles. Cyprid settlement assays conformed in principle to standard methodology (see e.g. Hellio et al.40), but with minor modification for the glass slide format. Glass slides were housed within Quadriperm dishes as for the algal assays. Briefly, 20 3-day-old cyprids, obtained by larval culture from adult broodstock,40 were added in a 1 mL drop of ASW (Tropic Marin, Dr. Biener GmbH, Germany) to 8-10 replicate slides of the hydrogel coating and the glass control. Polystyrene was also assayed as a control by adding 10 3-day-old cyprids, in 2 mL of ASW, to each well of a 24-well plate (Iwaki). The cyprids were incubated for 48 h at 28 °C in the dark. Cyprid settlement was enumerated after 24 and 48 h and expressed as mean percent settlement. The data were analyzed statistically using the Kruskal-Wallis test with post hoc comparisons of treatment means made with the Dunn’s multiple comparison test. Marine Bacteria. The performance of the hydrogels for inhibition of attachment and detachment following exposure to hydrodynamic forces was investigated for two species of marine bacteria, Cobetia marina and Marinobacter hydrocarbonoclasticus. These two species were chosen because of their contrasting surface energies: C. marina being a hydrophilic species, whereas M. hydrocarbonoclasticus is a hydrophobic species.41 C. marina (formerly Halomonas marina) has also been previously used as a model species in studies on initial attachment of marine bacteria to surfaces.42 In brief, coated slides were conditioned in ASW for 2 h before the assay was started. The bacterial suspensions used for the testing were obtained after the cells were repetitively washed and centrifuged to remove excess extracellular polymeric substances (EPS) for optimal adhesion. For each bacterium, four replicate slides of the hydrogel and
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Table 1. Thicknesses and Advancing and Receding Contact Angles (CA) of Freshly Prepared Silane- and Hydrogel-Modified Glass and Silicon Substratesa sample silane on glass silane on silicon hydrogel on glass hydrogel on silicon a
thickness (nm) 2.0 ( 0.4 34.3 ( 1.9
adv. CA (degrees)
rec. CA (degrees)
75 ( 2 75 ( 3 60 ( 3 61 ( 3
61 ( 2 54 ( 4 18 ( 2 20 ( 2
N ) 3 in all cases.
glass reference, respectively, were immersed for 1 h in polystyrene Quadriperm dishes (Greiner Bio-One Ltd.) containing 8 mL of suspension of the bacteria with a standardized absorbance of 0.2 AU at 595 nm. The slides were rinsed to remove nonadhered cells and transferred back into Quadriperm dishes containing 8 mL of sterile filtered seawater with added growth medium (sea salt peptone) and incubated for 4 h at 30 °C. Two of the slides were immediately stained with the fluorochrome SYTO 13 (Molecular Probes, 1.5 µM) for quantification of attached bacteria in a Tecan plate reader (GENios model, equipped with Magellan software). A total of 27 measurement spots were distributed evenly over a 15 × 45 mm2 area located centrally on each slide. For the detachment of bacteria test, the two unstained slides were mounted on a rotating drum with a surface speed of 12 knots for 10 min in natural seawater. The remaining bacteria were then quantified using SYTO 13 stain as described above. The fluorescence intensity of the samples, proportional to the biomass, was then normalized by subtracting the intensity of a blank sample with the same coating, which had undergone all assay steps except immersion in the bacterial suspension. This was done to check for autofluorescence and contamination. Freshwater Bacteria. Six replicate slides were used for the freshwater bacteria assay. After equilibration in deionized water (1 h, 40 °C), the samples were immersed in a glass tank containing 500 mL of suspension of log-phase Pseudomonas fluorescens with a concentration of 106 cells/mL. The tank was transferred to a shaker-incubator at a low speed (20 rpm) at 28 °C for 1 h. The samples were removed and rinsed in sterile distilled water under controlled hydrodynamic conditions. A sample was moved down-up 20 times vertically in a glass tank A of sterile distilled water at 28 °C and a constant speed resulting in a shear stress of 0.014 N m-2 to remove adhered bacteria. The sample was then transferred to a second glass tank B containing sterile distilled water at 28 °C and sonicated in an ultrasonic bath to remove all the remaining attached bacteria. The number of bacteria in the tank A and the number of bacteria in the tank B were determined after a 24 h incubation on plate count agar medium at 28 °C, respectively, using a standard plating method for viable counts.43 The total number of bacteria, as colony-forming units (CFU) attached to the sample and the percentage removal were calculated.
Results Hydrogel Characterization. Table 1 shows the advancing and receding contact angles for modified glass and silicon substrates, combined with ellipsometric thicknesses of the silane and hydrogel films on silicon. It is clear from both these characterization methods that the two successive coating steps proceeded as intended with hydrogel-coated substrates as the end result. Clean silicon and glass have water contact angles below 10 degrees, but after the silanization procedure both substrates had advancing contact angles of 75°. Thus, the advancing contact angle after the silanization step appears to be independent of substrate, though the receding angle was lower on silicon. The thickness of MPS on silicon corresponds to slightly more than one monolayer44 and the higher receding contact angle on glass
Figure 2. Changes in advancing contact angle (squares) and thickness (triangles) for hydrogel-coated glass and silicon substrates immersed in ASW. Unfilled markers represent data from silicon samples, and filled markers represent glass samples. Note that the total thickness of the hydrogel was larger than 30 nm. Connecting solid and hatched lines are included as guides for the eye.
implies an even denser silane layer on that substrate. After the hydrogel coating process, the contact angles on the two substrates were very similar, implying that coherent hydrogel films had formed on both substrates. The ellipsometric thickness of the hydrogel on silicon was about 35 nm, and these measurements give no indications that the thickness was different for coatings prepared on glass. The substrate independence was also assessed by performing parallel biological assays (UlVa settlement) of hydrogels on both glass and silicon. A two-tailed Student’s t-test revealed no significant difference in the number of UlVa spores settled on the hydrogels on glass or silicon (p ) 0.05; data not shown). Short-Term Stability. As can be seen from the contact angle and ellipsometry measurements shown in Figure 2, the shortterm stability of the hydrogel (up to 7 days) was good. There were no statistically significant changes in the hydrogel thickness (on silicon) nor in contact angles (on glass and silicon) after 1 week in ASW. The fluctuations in thickness were on the scale of 0.5 nm and represented a change of about 1% of the total hydrogel thickness. Thus, from these experiments it can be concluded that the hydrogel coating should not be in risk of deteriorating during the biological evaluation assays performed. This is supported by the absence of any signs of degradation and/or delamination in the biological assays. Long-Term Stability. The chemical integrity of the hydrogel was studied using IR spectroscopy. Spectra taken before and after the six-month period are shown in figure 3A. As can be seen, the two spectra are almost identical. Note that the sixmonth sample was treated with HCl before the measurement (see below for further details). In the further analysis of the spectroscopic data, special attention was focused on two functional groups: the CdO bond of the ester in the methacrylate backbone and the CsO bond found mainly in the PEG side chains. These functionalities have characteristic IR-absorption peaks, as indicated by the notation in Figure 3A. In particular, note the relatively broad CsO peak in the region of 1150 cm-1, which indicates that the PEG conformation is disordered in the material.18 A quantitative assessment of the stability of the hydrogel was made by calculating the peak areas of these two absorption bands and plotting the development over time. The result is shown in Figure 3B. As there is one CdO group per monomer unit, regardless of type, the peak area of this absorption should give a quantitative measure of the total amount of hydrogel remaining on the surface. The integrated peak showed a slight decrease over time, with about 95% of the peak intensity remaining after 6 months. The CsO peak intensity decreased in a very similar
Hydrogel Surfaces for Antifouling Applications
Figure 3. (A) IR spectra of a HEMA/PEG10MA hydrogel on PS recorded in the 1850-1000 cm-1 regime before and after a six-month immersion in ASW. Before the measurement, the six-month sample was washed with 50 mM HCl for 5 min and then equilibrated in water. The integration limits used for (B) are marked with vertical lines. Inset: The 1520-1650 cm-1 portion of the spectra taken at 0 and 6 months, both after incubation in NaOH. The inset is magnified ×2 in adsorption direction for clarity. (B) Relative changes over time of the integrated areas of the IR absorption peaks of the carbonyl stretching vibration (1760-1690 cm-1) and the C-O stretching vibration (1050-1215 cm-1). (C) IR spectra of the hydrogel coating when freshly prepared and after 50 days in ASW (not washed with HCl). The calculated difference spectrum is plotted beneath the two spectra. (D) Ellipsometric thicknesses over time measured for samples washed in Milli-Q water (solid line). The hatched line illustrates a possibly more realistic end point, with a final value measured after washing away excess precipitated salts from the samples (see text).
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fashion, implying that a small amount of material was removed over time but that there were no significant changes in the chemistry of the hydrogel. The most obvious change that could be observed with IR spectroscopy was instead an increase in certain absorption peaks. This implies that an uptake of material from the surrounding ASW took place during the course of the study. A comparison between the freshly prepared hydrogel and the same sample after 50 days in ASW is shown in Figure 3C. Changes over time include two new peaks: one broad at 1300-1600 cm-1 and one sharper at approximately 850 cm-1. These absorption bands are typical for carbonate compounds.45 The new peaks diminished after washing the sample with 50 mM HCl for 5 min, implying dissolution of the salt. This treatment was performed before the six-month measurement shown in Figure 3A. The extent of hydrolysis of the ester bond, with potential sidechain cleavage and formation of carboxylic acids on the polymer backbone, was investigated by immersing the samples in 50 mM NaOH to ionize any carboxylic acids, shifting the IR absorption peak away from that of the dominating ester groups. This was only performed before and at the end of the stability study to avoid accelerated alkaline hydrolysis. As can be seen in the inset of Figure 3A, no increase was found for the carboxylate peak after 6 months, which implies that the extent of hydrolysis was very low. It should be noted that a small carboxylate absorption peak could be found even for the freshly prepared surface, most likely due to monomer impurities or formation during the UV irradiation. To complement the IR measurements, the development of the coating thickness over time was studied with ellipsometry. The results can be seen in Figure 3D, which shows the relative changes over time. The initial absolute thickness of hydrogels prepared on both PS-coated silicon and gold substrates was approximately 47 nm, which was somewhat thicker than hydrogels prepared directly on silanized silicon. Over the course of six months, there were no dramatic changes; actually, the thickness did not change significantly during the six-month period. However, it is possible that some of this apparent stability was the result of salt buildup occurring in parallel with slight hydrogel degradation. After the samples were washed with 50 mM HCl to remove the salts, the thickness decreased somewhat, implying that the actual thickness of the polymer layer had decreased to about 95% of the initial value during the period, in full agreement with the IR analysis. This harsher washing step was only performed at the end of the six-month period. To investigate whether the protein resistance of the hydrogel was affected by storage in ASW, the aged samples were incubated in fibrinogen solution for 30 min (1 mg/mL in PBS pH 7.4), a treatment that normally does not lead to any significant ellipsometric thickness increase for freshly prepared hydrogels.30 As can be seen in Figure 4, the aged samples behaved in the same way, with no detectable increase of the thickness after fibrinogen incubation. Biological Evaluation Assays. Green Algae and Diatoms. The UlVa zoospore assay was performed 3 times, and in all cases the number of spores that settled on the hydrogel was just 5-6% of the settlement density on the glass reference. Results of one assay are shown for the hydrogel and glass reference in Figure 5. While only a few cells settled on the hydrogel, those that did were not removed by exposure to a wall shear stress of 52 Pa (0.6% removed). It must be emphasized that this number should be treated with caution
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Figure 4. Thickness change after incubation in fibrinogen for freshly prepared (left) and aged (right) hydrogels. Three samples of each type were tested. Error bars denote standard deviations. The data for the freshly prepared surfaces have been published before along with data for serum and plasma.30
Figure 5. Settlement and removal results of an Ulva spore assay. The bars represent the initial density of settled spores and the remaining spores after exposure to 52 Pa shear stress in a flow channel. Error bars ) (2× standard error.
Figure 6. Density of Navicula cells on hydrogel and glass reference before and after exposure to 51.5 Pa wall shear stress. Error bars, as in Figure 5.
because removal is being expressed as a percentage of the low numbers of spores that settled on the hydrogel in the first place. However, it is possible to conclude in general terms that the hydrogel is unlikely to have any intrinsic foul-release properties for UlVa spores. Diatoms are unicellular algae that form biofilms (slimes) on surfaces. Unlike UlVa spores, diatom cells are not motile in the water column and reach a surface though transport in currents and gravity. Therefore, at the end of the 2 h assay period, the density of diatoms on a surface is similar on all surfaces. However, gentle washing removes those cells that have been unable to adhere to the substrate and only adhered cells are represented in the settlement density data. Figure 6 shows that the density of cells on the hydrogel after gentle washing was significantly lower than on the glass reference. This low density of settled cells thus reflects their poor ability to adhere to the hydrogel.
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Figure 7. Barnacle settlement on the hydrogel and two reference surfaces. Note that this graph, in contrast with the other evaluation results, does not show any removal data. Instead, it shows the settlement development at 24 and 48 h. Error bars represent double standard error, N ) 24 for polystyrene, 8 for glass, and 10 for the hydrogel.
The removal of settled (attached) diatoms from the hydrogel was lower than on the glass reference when exposed to a 51.5 Pa wall shear stress (Figure 6). However, because the removal data only reflect the percentage loss of adhered (settled) cells, fewer cells remained on the hydrogel than on glass after washing and exposure to flow. Barnacles. The barnacle cyprid larval settlement assay was performed three times. Settlement on the hydrogel never exceeded 21% of that on glass and, in one assay, no settlement occurred on the hydrogel, even after 48 h. The results of the assay with the highest settlement are shown in Figure 7. A comparison of treatment means using Dunn’s test revealed that, with the exception of the 48 h time point in this particular assay, settlement was significantly lower on the hydrogel surface than on glass (p < 0.05) for all measured time points in the three assays. Polystyrene was included in the assays as an additional control to assess the health and behavior of the different batches of cyprids based on extensive experience with this surface. In one of the assays, only 10% cyprid settlement occurred after 24 h, which is abnormally low for day 3 cyprids. Otherwise, cyprid behavior appeared normal. Settlement on glass was higher than on plastic, which is the normal trend for this species.46 Account must be taken, however, of the higher cyprid density on the glass slides, which likely leads to comparatively high settlement through enhanced intercyprid interactions.47,48 Nevertheless, the higher rate of settlement on glass compared to polystyrene allayed a concern that the comparatively shallow depth of the water droplet on the slides would constrain settlement behavior, leading to lower settlement. There was no difference in cypris larva mortality in the populations exposed to either the hydrogel samples or the references, which implies that the effect is not a result of coating toxicity. Marine Bacteria. The results of the marine bacterial assays, as shown in Figure 8, fit well with the data from the other organisms. Marinobacter showed both low settlement and high removal, while Cobetia marina had higher settlement. Freshwater Bacteria. As can be seen in Figure 9, the freshwater bacterium Pseudomonas fluorescens showed a settlement behavior similar to the marine bacteria. The behavior is particularly similar to that of Cobetia marina.
Discussion Hydrogel Stability. Both ellipsometry and IR measurements clearly show that the stability of the PEG hydrogel was excellent in our experiments, without signs of delamination or significant chemical degradation. The results of the two characterization
Hydrogel Surfaces for Antifouling Applications
Figure 8. Initial attachment and detachment (with removal percentage) of marine bacteria on the hydrogel and reference. The error bars represent (2× standard error.
Figure 9. Attachment of the freshwater bacterium Pseudomonas and the proportion of bacteria remaining after a flow test on the hydrogel and glass reference. Error bars represent ( standard error.
methods were very consistent, both indicating that approximately 95% of the hydrogel was left after 6 months. It is important to note that the function, in terms of protein resistance, was also preserved, as indicated by the results of the fibrinogen adsorption experiments (Figure 4). The stability of the coating could potentially be a most useful property of these coatings. However, the lack of degradation is a somewhat surprising result, considering the instability of PEG found in previous studies.22,33 It is well-known that PEG can undergo oxidative degradation when exposed to oxygen and elevated temperatures or light.49,50 However, when analyzing the IR data it can be noted that the appearance of the absorption peak of the characteristic ether stretching vibration (found between 1050 and 1215 cm-1) was highly consistent, with a slight intensity decrease as the only effect over time, just like the carbonyl peak of the hydrogel backbone seen at 1730 cm-1. This implies that the degradation that did occur was not specific to the PEG chains but affected the hydrogel uniformly. If the PEG side chains were significantly shortened or removed from the hydrogel backbone, it could be expected that the 1050-1215 cm-1 interval of the spectrum would not decrease homogeneously but become more like that of a polyHEMA homopoly-
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mer, with two separate peaks at 1075 and 1160 cm-1.30 The lack of PEG degradation in our case could possibly be due to depressed levels of oxygen in the sample containers during the six-month period. However, as no effort was made to reduce the oxygen exposure and the containers were opened with regular intervals, there should have been no immediate lack of oxygen in the containers. These results show that the structural, chemical, and functional stability of the hydrogel film was excellent under the conditions chosen for the stability study. It must be emphasized that these conditions represent a highly simplified simulation of the marine environment, lacking a multitude of dissolved organic species as well as living organisms. Addition of these factors could have dramatic effects on the stability of the hydrogel film. For instance, it is known that PEG can be oxidized enzymatically.51 Therefore, the main conclusion that can be drawn from the stability study is that the hydrogel can be stable for prolonged periods under the conditions mentioned above, which must be considered a prerequisite for any further, more challenging, stability evaluation. An explanation for the apparent stability may be the presence of cross-links, formed during the UV-irradiation process.32 The successive addition of salt into the hydrogel matrix could also be studied in detail using IR spectroscopy. We have not attempted to investigate the chemical mechanism causing this effect, but it is well-known that polyHEMA gels can imbibe calcium ions, followed by precipitation of salts.52 With this in mind, a likely candidate for the salt formed could be CaCO3, which agrees well with the spectroscopic data and the observation of a carbonate salt soluble at acidic pHs. It is as of now not known what effect this salt buildup would have on the resistance to biofouling, and this issue, and its possible prevention, must be investigated further. Biological Evaluation. UlVa spores swim until they locate a suitable surface on which to settle. The low density of settled spores indicates that the hydrogel is inhospitable for settlement and the results of the current study correlate well with results for other PEG-containing compositions.8,26,28 The low levels of removal are also consistent with the observation that UlVa spores generally adhere more strongly to hydrophilic than to hydrophobic surfaces.53 Recent work has highlighted the importance of the end group termination of oligoethylene glycol SAMs in terms of spore settlement.27 The apparent low settlement on hydroxy- and methoxy-terminated OEG6 SAMs was shown to not be due to an inhibition of settlement, per se, but rather to the inability of the adhesive secreted by settled spores to firmly attach to the surface. This contrasts with the results obtained here and for some other PEGylated surfaces28,54 that genuinely inhibit spore settlement, that is, the spores remain motile and do not settle and secrete adhesive. Diatoms exhibit a different type of settlement compared to UlVa spores because under the conditions of the laboratory assay they reach the surface by sinking, thus the same number of cells is in contact with all test surfaces at the end of the settlement period. The low number of cells retained on the hydrogel after gentle washing indicates that the diatoms were unable to adhere efficiently and this together with high removal of cells after exposure to flow suggests that the hydrogel prevents the cells from achieving good traction. Previous data27,53,55,56 have shown decreasing adhesion strength of diatoms with decreasing water contact angle, and the low number of cells retained on both the hydrogel and acid-washed glass (contact angle
