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The Quartz Crystal Microbalance: a New Tool for the Investigation of the Bioadhesion of Diatoms to Surfaces of Differing Surface Energies Paul J. Molino,† Oliver M. Hodson,† John F. Quinn,‡ and Richard Wetherbee*,† School of Botany, The UniVersity of Melbourne, ParkVille, Victoria 3010, Australia, and Centre for Nanoscience and Nanotechnology, Department of Chemical and Biomolecular Engineering, The UniVersity of Melbourne, ParkVille, Victoria 3010, Australia ReceiVed March 3, 2008 Diatoms are a major component of the biofoul layer found on modern low-surface-energy, ‘foul release’ coatings. While diatoms adhere more strongly to hydrophobic, as opposed to hydrophilic, surfaces, surprisingly little is known of the chemical composition of their adhesives. Even less is known about the underlying processes that characterize the interaction between the adhesive and a given surface, including those of differing wettability. Using the quartz crystal microbalance with dissipation monitoring (QCM-D), we examined differences in the viscoelastic properties of the extracellular adhesives produced by the marine diatoms Amphora coffeaeformis Cleve and Craspedostauros australis Cox interacting with surfaces of differing wettability; 11-mercaptoundecanoic acid (MUA) that is hydrophilic and 1-undecanethiol (UDT) that is hydrophobic. While the overall ∆f/∆D ratios were slightly different, the trends were the same for both diatom species, with the layer secreted upon UDT to be more viscoelastic and far more consistent over several experiments, compared to that on MUA which was less viscoelastic and demonstrated far more variability between experiments. While the nature of the parameter shifts for C. australis were the same for both surfaces, A. coffeaeformis cells settling upon UDT illustrated significant positive f and D shifts during the initial stages of cell settlement and adhesion to the surface. Further experiments revealed the parameter shifts to occur only during the initial adhesion of cells upon the pristine virgin UDT surface. The mechanism behind these parameter responses was isolated to the actin-myosin/adhesion complex (AC), using the myosin inhibitor 2,3-butanedione 2-monoxime (BDM) to remove the cells ability to ‘pull’ on adhesive strands emanating from the cell raphe. The observations made herein have revealed that adhesives secreted by fouling diatoms differ significantly in their interaction with surfaces depending on their wettability, as well as illustrating the unique mechanics behind the adhesion of A. coffeaeformis upon hydrophobic surfaces, a mechanism that may contribute significantly to the cells success in colonizing hydrophobic surfaces.
Introduction The biofouling of artificial surfaces in the marine environment has long been a challenging problem of major economic and ecological importance. The effect fouling has on the form and function of these surfaces is most obvious in the fouling of oceangoing vessels. The fouling by marine organisms increases the skin friction of ship hulls, therefore reducing their efficiency and increasing fuel costs by up to 30% (for reviews, see Robinson et al.,1 Hoagland et al.,2 and Brady3). Attempts to limit fouling on marine surfaces have commonly included the use of toxic antifoul paints. Largely containing copper and tin, these coatings function as biocides, continually expelling toxins into the environment in order to dissuade fouling organisms. Recently, the heavy metals from these paints have been found to bioaccumulate in the marine food chain, causing major environmental impacts on a diverse range of species.4 Consequently, they have been slowly outlawed over time.3 These biocidal coatings have been replaced by a new generation of paints, termed ‘foul-release’ paints. Based on a low-energy (hydrophobic) * To whom correspondence should be addressed. Email: richardw@ unimelb.edu.au. † School of Botany, The University of Melbourne. ‡ Centre for Nanoscience and Nanotechnology, Department of Chemical and Biological Engineering, The University of Melbourne. (1) Robinson, M. G.; Hall, B.; Voltolina, D. J. Coatings Technol. 1985, 57(725), 35–41. (2) Hoagland, K. D.; Rosowsk, J. R.; Gretz, M. R. J. Phycol. 1993, 29, 537– 566. (3) Brady, R. F. J. Coatings Technol. 2000, 72(900), 45–56. (4) Terlizzi, A.; Frashetti, S.; Gianguzza, P.; Faimali, M.; Boero, F. Aquat. ConserV. 2001, 11, 311–317.
surface, foul-release paints act by preventing organisms from forming a strong adhesive bond to the paint surface, thus promoting their release from the hull under the shear stress caused as the ship moves through the water.3 This strategy has been extremely effective for most fouling organisms but is ineffective for diatoms that actually adhere more strongly to hydrophobic surfaces.5 Diatoms (Bacillariophyceae) are a diverse group of algae that are ubiquitous in aquatic habitats and are a major component of the biofoul layer found on artificial surfaces placed in the marine environment. Diatom morphology is characterized by a highly ornamented, siliceous cell wall plus associated organic layers and coverings that define the diatom frustule. Diatoms exhibit both planktonic and benthic life strategies in the marine environment, assisted by the various specialized structures and cellular components derived within the diatom frustule. Pennate diatom morphology allows for an elongate slit termed a ‘raphe’ to exist within the cell silica wall, providing a structure from which adhesive mucilage is secreted and therefore for the cell to attach to a given substratum. Most pennate species are defined by their ability to adhere and then ‘glide’ over a surface, a process that results in the deposition of an adhesive mucilaginous trail on the substrate surface (for review, see Edger and Pickett-Heaps,6 Hoagland et al.,7 and Wetherbee et al.8). The secreted trail material (5) Callow, M. E.; Callow, J. A. Biologist 2002, 49(1), 1–5. (6) Edgar, L. A.; Pickett-Heaps, J. D. Prog. Phycol. Res. 1984, 3, 47–84. (7) Hoagland, K. D.; Rosowsk, J. R.; Gretz, M. R. J. Phycol. 1993, 29, 537– 566. (8) Wetherbee, R.; Lind, J. L.; Burke, J.; Quatrano, R. S. J. Phycol. 1998, 34, 9–15.
10.1021/la800672h CCC: $40.75 2008 American Chemical Society Published on Web 05/29/2008
The Quartz Crystal Microbalance
accumulates to form a layer, which can comprise a major component of the slime layer found on both natural and manmade structures in the marine environment. Diatom gliding is generated by an adhesion complex (AC) that consists of an actin/ myosin motor that interacts through a continuum of connector molecules to an adhesive, extracellular strand that interacts with the substratum to provide the traction for gliding.8 Researchers have employed a range of techniques in order to probe the interactions between diatoms and a variety of surfaces. Shultz et al.9 utilized a turbulent channel flow apparatus to measure the adhesion strength of microfouling organisms to test surfaces. They were able to apply various wall sheer stress forces on the diatom Amphora in order to test its adhesive strength to acidwashed glass. Subsequent work using Amphora and the zoospores of the biofouling green alga Enteromorpha investigated the influence of surface wettability on cell adhesion strength via the adsorption of self assembled monolayers (SAMs) onto a gold substrate surface, where depending on the terminal functional group of the alkanethiol, we were able to provide either a highly hydrophobic or hydrophilic surface.10 Atomic force microscopy (AFM) has been used to characterize the tensile properties of the adhesive mucilage that mediates diatom adhesion from a number of species.11–16 Recently, this technique was used to describe the nanomechanical properties of single adhesive nanofibers emanating from the adhesive mucilage of the known biofouling diatom Toxarium undulatum,14 with further research revealing the fibers to be comprised of supramolecular assemblies characteristic of singular modular proteins.15 Techniques employed to investigate the antifouling ability of various surfaces have generally encompassed the counting of irreversibly adhered cells to the substrate surface after a given period of time, or following treatments aimed at removing weakly adhered cells. These techniques fail to give any measure of changes in the specific interaction between the adhesive mucilage and surfaces of differing physical and/or chemical properties, instead giving macroscopic observations. The quartz crystal microbalance (QCM) has previously been used to investigate (9) Schultz, M. P.; Finlay, J. A.; Callow, M. E.; Callow, J. A. Biofouling 2000, 243–251. (10) Finlay, J. A.; Callow, M. E.; Ista, L. I.; Lopez, G. P.; Callow, J. A. Integr. Comp. Biol. 2002, 42, 1116–1122. (11) Higgins, M. J.; Crawford, S. A.; Mulvaney, P.; Wetherbee, R. Protist 2002, 153, 25–38. (12) Higgins, M. J.; Sader, J. E.; Mulvaney, P.; Wetherbee, R. J. Phycol. 2003, 39, 722–734. (13) Higgins, M. J.; Molino, P.; Mulvaney, P.; Wetherbee, R. J. Phycol. 2003, 39, 1181–1193. (14) Dugdale, T. M.; Dagastine, R.; Chiovitti, A.; Mulvaney, P.; Wetherbee, R. Biophys. J. 2005, 89, 4252–4260. (15) Dugdale, T. M.; Dagastine, R.; Chiovitti, A.; Wetherbee, R. Biophys. J. 2006, 90, 2987–2993. (16) Dugdale, T. M.; Willis, A.; Wetherbee, R. Biophys. J. 2006, 90, L58–60L (17) Nivens, D. E.; Chambers, J. Q.; Anderson, T. R.; White, D. C. Anal. Chem. 1993, 65, 65–69. (18) Gryte, D. M.; Ward, M. D.; Hu, W. Biotechnol. Prog. 1993, 9, 105–108, 1993. (19) Redepenning, J.; Schlesinger, T. K.; Mechalke, E. J.; Puleo, D. A.; Bizios, R. Anal. Chem. 1993, 65, 3378–3381. (20) Wegener, J.; Janshoff, A.; Galla, H. Eur. Biophys. J. 1998, 28, 26–37. (21) Fredriksson, C.; Kihlman, S.; Rodahl, M.; Kasemo, B. Langmuir 1998, 14, 248–251. (22) Zhou, T.; Marx, K. A.; Warren, M.; Schulze, H.; Braunhut, S. J. Biotechnol. Prog. 2000, 16, 268–277. (23) Marx, K. A.; Zhou, T.; Montrone, A.; Schulze, H.; Braunhut, S. J. Biosens. Bioelectron. 2001, 16, 773–782. (24) Marxer, C. G.; Coen, M. C.; Bissig, H.; Greber, U. F.; Schlapbach, L. Anal. Bioanal. Chem. 2003, 377, 570–577. (25) Marxer, C. G.; Coen, M. C.; Greber, T.; Greber, U. F.; Schlapbach, L. Anal. Bioanal. Chem. 2003, 377, 578–586. (26) Marx, K. A.; Zhou, T.; Warren, M.; Braunhut, S. J. Biotechnol. Prog. 2003, 19, 987–999. (27) Lu¨thgens, E.; Herrig, A.; Kastl, K.; Steinem, C.; Reiss, B.; Wegener, J.; Pignataro, B.; Janshoff, A. Meas. Sci. Technol. 2003, 14, 1865–1875.
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Figure 1. Transmission-mode SLCM images of C. australis (a) and A. coffeaeformis (b). Scale bars ) 13 (a) and 11 µm (b).
the adhesion of cells to the sensor surface in liquid phase17–29 (for general reviews, see O’Sullivan and Guilbault,30 and Marx31). The adhesion and propagation of cells on the crystal surface can be followed by measuring the mass deposited on the crystal surface through changes in the oscillating frequency of the sensor crystal.17–20 Later, by combining measurement of the oscillating frequency of the crystal (f) with measurement of the dissipation factor (D),21 researchers were able to additionally examine the viscoelastic properties of the adhered layer.22–32 Previously, we have identified the QCM-D as an excellent technique with which to probe the interactions between the adhesive mucilage secreted by marine diatoms and the substrate surface.33 Here we aim to investigate the interactions between these mucilaginous adhesives and surfaces of differing surface energy, by functionalizing the QCM-D sensor surface using SAMs prior to introduction of the fouling organisms.
Materials and Methods Algal Cell Culture. Craspedostauros australis Cox was collected from Western Port Bay, Victoria, Australia (Figure 1a). Amphora coffeaeformis Cleve was collected from the Defense Science and Technology Organisation (DSTO) Maritime Testing Facility at Williamstown, Victoria, Australia (Figure 1b). A. coffeaeformis was chosen because it is known as a notorious ubiquitous biofouler, while C. australis was chosen for examination as it is regarded as a relatively weak biofouling diatom species. Cells were isolated and maintained in 200 mL Pyrex conical flasks with sterile K-medium containing silicates34 at 16 °C under Sylvania 58 W Luxline Plus and Gro-Lux fluorescent lamps with a daily 14:10 h light/dark cycle. In order to inhibit any potential bacterial contamination, cells for QCM analysis were substituted into 50 mL Cellstar (Greiner Bioone) tissue culture flasks containing K + Si medium with 0.1 mg mL-1 streptomycin sulfate and 100 units mL-1 sodium penicillin G for a duration of 24 h. Thereafter, the cells were rinsed with sterile K + Si medium 24 h prior to use. SAM Adsorption onto the Gold QCM Sensor Surface. The QCM sensors employed here were A-T cut 5 MHz quartz crystals with a 10 mm diameter gold electrode (QSX301, Standard Gold) from Q-Sense AB, Va¨stra Fro¨lunda, Sweden. Prior to each experiment, the gold sensor surface on the quartz crystal was cleaned with Piranha solution for 3 min, 40% w/v sodium hydroxide solution (28) Jenkins, M. S.; Wong, K. C. Y.; Chhit, O.; Bertram, J. F.; Young, R. J.; Subaschandar, N. Biotechnol. Bioeng. 2004, 88(3), 392–398. (29) Li, J.; Thielemann, C.; Reuning, U.; Johannsmann, D. Biosens. Bioelectron. 2005, 20, 1333–1340. (30) O’Sullivan, C. K.; Guilbault, G. G. Biosens. Bioelectron. 1999, 14, 663– 670. (31) Marx, K. A. Biomacromolecules 2003, 4(5), 1099–1120. (32) Ho¨o¨k, F.; Kasemo, B. Anal. Chem. 2001, 73, 5796–5804. (33) Molino, P. J.; Hodson, O. M.; Quinn, J. F.; Wetherbee, R. Biomacromolecules 2006, 7, 3276–3282. (34) Anderson, R. A.; Jacobsen, D. M.; Sexton, J. P. Provasoli Guillard center for culture of marine phytoplankton-catalogue of strains. West Boothbay Harbour, Maine, 1991; 98.
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Figure 2. (a) Schematic of Q-Sense axial flow chamber (QAFC301). Diameter of fluid injection port is 0.1 mm, sitting directly above the QCM sensor. The sensor rests upon an O-ring at ∼0.2-0.4 mm from the injection point. Chamber volume is ∼80 µL. (b) Image paths used to sample cell concentration upon the sensor surface after QCM analysis (area sampled ∼23% of total electrode area).
for 3 min, and subsequently twice more with Piranha solution for 3 min. After each cleaning step, the crystals were rinsed twice with deionized water and dried with nitrogen gas. Immediately following the final Piranha cleaning step, the QCM sensor was transferred to 1 mM ethanolic solutions of either 11mercaptoundecanoic acid (MUA, Sigma-Aldrich 450561) (hydrophilic), or 1-undecanethiol (UDT, Sigma-Aldrich 510467) (hydrophobic) (for background and characterization of SAMS on gold surfaces, refer to Bain et al.35). Sensors were left to incubate for 60 min; thereafter, they were rinsed three times in absolute ethanol and once in deionized water. The sensor was then gently dried with nitrogen gas. The adsorption of these two thiols to the gold electrode surface of the QCM sensors provided two surfaces of greatly differing wettability. The adsorption of UDT produced a surface with a typical water contact angle of 104.9° ( 2.5°, while MUA provided a water contact angle
