pubs.acs.org/Langmuir © 2010 American Chemical Society
Evaporative Deposition Patterns of Bacteria from a Sessile Drop: Effect of Changes in Surface Wettability Due to Exposure to a Laboratory Atmosphere Kyle F. Baughman,† Raina M. Maier,‡ Theresa A. Norris,‡ Brooke M. Beam,§ Anoma Mudalige,§ Jeanne E. Pemberton,§ and Joan E. Curry*,‡ †
Department of Materials Science and Engineering, The University of Arizona, Tucson, Arizona 85721, Department of Soil, Water and Environmental Science and BIO5 Institute, The University of Arizona, Tucson, Arizona 85721, and §Department of Chemistry and Biochemistry, The University of Arizona, Tucson, Arizona 85721
‡
Received July 16, 2009. Revised Manuscript Received April 7, 2010 Evaporative deposition from a sessile drop is a simple and appealing way to deposit materials on a surface. In this work, we deposit living, motile colloidal particles (bacteria) on mica from drops of aqueous solution. We show for the first time that it is possible to produce a continuous variation in the deposition pattern from ring deposits to cellular pattern deposits by incremental changes in surface wettability which we achieve by timed exposure of the mica surface to the atmosphere. We show that it is possible to change the contact angle of the drop from less than 5° to near 20° by choice of atmospheric exposure time. This controls the extent of drop spreading, which in turn determines the architecture of the deposition pattern.
Introduction Spontaneous ordering of materials is of importance for numerous technological applications and has captured the interest of scientists for decades. Patterns in nature have inspired the development of new materials and have helped advance understanding of the fundamental physics and chemistry of materials. Selfassembled patterns of molecules and particles on surfaces offer ways to create new surfaces and templates for future materials.1-19 A well-known technique to deposit materials on surfaces is through evaporative deposition from a drying drop.1,6,9,11,12,14,17-19 While this deposition technique is simple, it has been shown that by varying system parameters, such as particle and surfactant concentration, it is possible to produce a variety of particulate residue architectures.1,11 Evaporative deposition is rich in possibilities *To whom correspondence should be addressed. E-mail:
[email protected]. (1) Deegan, R. D. Phys. Rev. E 2000, 61, 475–485. (2) Moriarty, P.; Taylor, M. D. R.; Brust, M. Phys. Rev. Lett. 2002, 89, 248303/ 1–248303/4. (3) Ge, G.; Brus, L. J. Phys. Chem. B 2000, 104, 9573–9575. (4) Nagayama, K. Colloids Surf., A 1996, 109, 363–374. (5) Nguyen, V. X.; Stebe, K. J. Phys. Rev. Lett. 2002, 88, 164501. (6) Truskett, V. N.; Stebe, K. J. Langmuir 2003, 19, 8271–8279. (7) Fan, F.; Stebe, K. J. Langmuir 2005, 21, 1149–1152. (8) Aizenberg, J.; Braun, P. V.; Wiltzius, P. Phys. Rev. Lett. 2000, 84, 2997–3000. (9) Park, J.; Moon, J. Langmuir 2006, 22, 3506–3513. (10) Jarai-Szabo, F.; Astilean, S.; Neda, Z. Chem. Phys. Lett. 2005, 408, 241– 246. (11) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. Rev. E 2000, 62, 756–765. (12) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827–829. (13) Weibel, D. B.; Lee, A.; Mayer, M.; Brady, S. F.; Bruzewicz, D.; Yang, J.; DiLuzio, W. R.; Clardy, J.; Whitesides, G. M. Langmuir 2005, 21, 6436–6442. (14) Nellimoottil, T. T.; Rao, P. N.; Ghosh, S. S.; Chattopadhyay, A. Langmuir 2007, 23, 8655–8658. (15) Sommer, A. P.; Zhu, D. Langmuir 2007, 23, 11941. (16) Denkov, N.; Velev, O.; Kralchevski, P.; Ivanov, I.; Yoshimura, H.; Nagayama, K. Langmuir 1992, 8, 3183–3190. (17) Maheshwari, S.; Zhang, L.; Zhu, Y.; Chang, H. C. Phys. Rev. Lett. 2008, 100, 044503. (18) Shmuylovich, L.; Shen, A. Q.; Stone, H. A. Langmuir 2002, 18, 3441–3445. (19) Kuncicky, D. M.; Velev, O. D. Langmuir 2008, 24, 1371–1380.
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for controlled production of two-dimensional particulate films on surfaces. Deegan and co-workers12 evaporated a drop of microspheres suspended in an aqueous solution on mica and showed how contact line pinning leads to formation of ring or “coffee stain” deposits on drying. Multiring deposits form if the contact line repeatedly depins and repins during drying.17,18 The surface area covered by the drop can be manipulated by controlling evaporation rate and interior deposition is influenced by particle, salt, and surfactant concentrations.1,11 Rings, arches, radial lines, and gridlike patterns1 that resemble honeycomb or cellular patterns20-22 are possible. Careful control of system parameters makes it possible to manipulate the deposition patterns. Stebe and co-workers5-7 deposited drops of microspheres with varying amounts of surfactant at the drop air-water interface. In the absence of surfactant, ring deposits were produced on solvent evaporation. Honeycomb or cellular patterns were produced when the surfactant was in the liquid expanded-liquid condensed phase state. Kuncicky and Velev19 used surfaces of different wettability to systematically vary microsphere patch deposition from ring deposits on hydrophilic surfaces (θ=4°) to spherical cap formations on more hydrophobic surfaces (θ = 101°). There is interest in controlled deposition of living materials (bacteria, viruses, cells) on surfaces.14,23,24 In a report of evaporative deposition of bacteria on glass,14 it was determined that motile bacteria create uniform deposits and that nonmotile bacteria produce ring stains. In this work we use evaporative deposition to (20) Weaire, D.; Rivier, N. Contemp. Phys. 1984, 25, 59–99. (21) Balint, Z.; Nagy, K.; Laczko, I.; Bottka, S.; Vegh, G. A.; Szegletes, Z.; Varo, G. J. Phys. Chem. C 2007, 111, 17032–17037. (22) Curry, J. E.; Heo, C. H.; Maier, R. M. In Microbial Surfaces; Structure, Interactions and Reactivity; Camesano, T. A., Mello, C. M.; Eds.; Oxford University Press: New York, 2007; pp 217-229. (23) Chiu, D. T.; Jeon, N. L.; Huang, S.; Kane, R. S.; Wargo, C. J.; Choi, I. S.; Ingber, D. E.; Whitesides, G. M. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2408–2413. (24) Klem, M. T.; Willits, D.; Young, M.; Douglas, T. J. Am. Chem. Soc. 2003, 125, 10806–10807.
Published on Web 04/20/2010
DOI: 10.1021/la100932k
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deposit motile bacteria from drops of aqueous salt solutions and show that the range of deposition patterns is much more complex. We produce a continuous variation of patterns from multirings to cellular patterns depending on the extent of drop spreading which we show can be controlled through timed exposure of the mica surface to the atmosphere. We employ the well-known phenomenon that the contact angle of water on many surfaces such as oxides, mica, and glass increases with time if the surface is exposed to a laboratory atmosphere.25,26 As previous research has shown, a number of adsorptive atmospheric components such as water, carbon dioxide, and molecular and particulate organic materials are likely involved.27-34 This has implications for deposition of living materials but also for colloidal materials in general. While most work in this area has focused on using this technique to controllably arrange particles on surfaces for engineered applications there is also potential to use this technique to probe the underlying particle-particle and particle-surface interactions. This could open a new avenue for characterizing the colloidal interactions of environmentally important materials such as bacteria. Direct bacteria-surface interactions play a key role in biofilm formation.35 Methods to probe these interactions without potentially perturbing the bacteria are limited. Evaporative deposition can be performed directly from aqueous solutions with little to no perturbation of the bacterial environment. Potentially, analysis of patterns produced through evaporative deposition may be useful for understanding the basic bacteria-bacteria and bacteria-surface interactions governing the deposition process important in initiation of biofilm formation.
Figure 1. Contact angle (0, ) and surface area ([) covered by a drop of ultrapure water on mica as a function of mica exposure time. Each square represents an individual mica sheet, and the error bars mark the standard deviation of at least three measurements. The symbol represents single measurements on samples on which the drops spread to such an extent that the contact angle was not measurable.
Materials and Methods A standard bacterial suspension was prepared as follows. Pseudomonas aeruginosa PAO1 was precultured in 30 mL of R2B growth media (composition below) under aerobic conditions at 23 °C for 24 h with gyratory shaking at 200 rpm. R2B consists of 0.5 g of dextrose, 0.5 g of protease peptone #3, 0.5 g of yeast extract, 0.5 g of casamino acid extract, 0.5 g of soluble starch, 0.1 g of MgSO4 3 7H2O, 0.3 g of HK2PO4, and 0.3 g of sodium pyruvate in 1 L of 18 MΩ water. The preculture (100 μL) was used to inoculate a 30 mL culture prepared using the same growth medium and conditions as described above, which was grown for a further 24 h. The bacteria were harvested between late exponential and early stationary phase. The cells were centrifuged for 10 min at 1940g (Beckman, model J2-21 centrifuge) and then washed in 30 mL of a minimal salts medium (MSM: 0.15 g of NH4H2PO4; 0.1 g of K2HPO4; 0.000 41 g of FeSO4 3 7H2O; 0.05 g of MgSO4 3 7H2O in 100 mL of water) to remove extracellular material and organic medium components. The MSM did not contain a carbon source, and the ionic strength and pH were 0.061 M and 6.5, respectively. The washed cells were centrifuged, resuspended in 30 mL of fresh MSM, dispensed into 1.8 mL microcentrifuge tubes, and stored at room (25) Birch, W.; Carre, A.; Mittal, K. L. In Developments in Surface Contamination and Cleaning; Kohli, R., Mittal, K. L., Eds.; William Andrew: Norwich, NY, 2008; pp 693-723. (26) White, M. L. In Clean Surfaces: Their Preparation and Characterization for Interfacial Studies; Goldfinger, G., Ed.; Marcel Dekker: New York, 1970; pp 361-373. (27) Israelachvili, J. N.; Alcantar, N. A.; Maeda, N.; Mates, T. E.; Ruths, M. Langmuir 2004, 20, 3616–3622. (28) Christenson, H. K. J. Phys. Chem. 1993, 97, 12034–12041. (29) Yaminsky, V. V.; Stewart, A. M. Langmuir 2003, 19, 4037–4039. (30) Beaglehole, D.; Radlinska, E. Z.; Ninham, B. W.; Christenson, H. K. Phys. Rev. Lett. 1991, 66, 2084–2087. (31) Balmer, T. E.; Christenson, H. K.; Spencer, N. D.; Heuberger, M. Langmuir 2008, 24, 1566–1569. (32) Christenson, H. K.; Israelachvili, J. N. J. Colloid Interface Sci. 1987, 117, 576–577. (33) Ohnishi, S.; Hato, M.; Tamada, K.; Christenson, H. K. Langmuir 1999, 15, 3312–3316. (34) Ostendorf, F.; Schmitz, C.; Hirth, S.; Kuehnle, A.; Kolodziej, J. J.; Reichling, M. Nanotechnology 2008, 19, 305705/1–305705/6. (35) Dunne, W. M., Jr. Clin. Microbiol. Rev. 2002, 15, 155–166.
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Figure 2. Residue area as a function of mica exposure time. Open symbols are for drop residues made from bacterial suspensions. Different open symbols are used to differentiate between sets of residues formed from separately prepared bacterial suspensions. The letters A through E correspond to the Figure 3 residues. Filled circles are for drops deposited from MSM without bacteria. temperature for drop deposition experiments. This protocol resulted in a reproducible suspension of cells with a concentration between 5 108 and 1 109 CFU/mL as determined by dilution plating. P. aeruginosa PAO1 is a motile strain, and under these conditions the bacteria are negatively charged. Ruby mica (S & J Trading Inc.) was selected as the substrate for these experiments because mica is commonly used in surface studies and is easily prepared into molecularly smooth surfaces that are clean when initially cleaved. Thick pieces of mica were initially cut into samples typically 2.5-3.0 cm wide and about 3-7 cm long. The mica was cleaved, and the samples were then placed on the working surface of the laminar flow hood until drop deposition experiments were performed. The laminar flow hood (air flow speed 0.44 m/s) was used to minimize dust contamination. The time the mica was exposed to the atmosphere (in the laminar flow hood) prior to drop deposition ranged from 0.25 min to 148 days. The drops were deposited under ambient conditions during the months of February through May and October through December, during which the laboratory humidity typically varies between 10 and 30%. In order to characterize changes in surface wettability with laboratory air exposure, the contact angle of ultrapure water was measured with a Kruss model DSA10 sessile drop contact angle system. Drops of bacterial suspension (4 μL) were deposited on the mica samples using a P100 Gilson pipetter fitted with a polypropylene tip. The number of drops per piece of mica varied Langmuir 2010, 26(10), 7293–7298
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Figure 3. Residues formed through evaporative deposition of a bacterial suspension on mica surfaces exposed to the laboratory atmosphere. Dark regions are bacteria and salt, and light regions are where residual material is minimal. Air flow (in the laminar flow hood) is from left to right. The whole residue is shown in the left column and higher magnification images of boxed regions are shown in the middle (left box) and right (right box) columns, respectively. The scale bar is 2 mm for all images in the left column and is 500 μm for all images in the middle and right columns. Mica exposure times are 7 min (A), 31 min (B), 95 min (C), 24 h (D), and 92 days (E). The arrow in panel C1 indicates a particle which disrupted the regularity of the cellular pattern during pattern formation. The arrow in the lower right panel (E2) indicates the location where the final water evaporated for this residue as discussed in the text. with length of the sample. In order to keep the bacterial suspension well-mixed, the suspension was vortexed before each drop or set of drops were deposited. The suspension age ranged from 6 to 430 min and was determined from the time of the final suspension resuspension. The dried residues were imaged using an Olympus IMT-2 inverted optical microscope with a Hamamatsu ORCA100 CCD camera. The area covered by the residue and the fraction of the interior covered with a film of bacteria and salts was determined directly from these images. Residue size and fraction of interior deposition data were analyzed as a function of cell suspension age (6-430 min), and it was found that there was no dependence on suspension age at these early times (data not shown). AFM images of dried residues were collected in tapping mode on a Langmuir 2010, 26(10), 7293–7298
Veeco Dimension 3100 Nanoscope IV scanning probe microscope (Santa Barbara, CA) with NSC15 silicon nitride cantilevers (Mikromasch). Images were plane fit to the mica surface for height measurements.
Results and Discussion Effect of Laboratory Air Exposure on Mica Surface Wettability. Under our laboratory conditions (10-30% RH) a drop of water placed on a mica surface spreads to cover a finite area. The contact line retracts smoothly without pinning as the drop evaporates. We find that the maximum area covered by the drop on initial spreading decreases if the mica surface is first exposed to DOI: 10.1021/la100932k
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the laboratory atmosphere prior to drop deposition. We have quantified this (Figure 1) by directly measuring both the maximum area covered by a 4 μL drop of ultrapure water and the contact angle of ultrapure water on mica as a function of the time the mica surface is exposed to laminar flow filtered laboratory air, hereafter referred to as “mica exposure time”. The area covered clearly decreases, and the contact angle increases to near 20° with prolonged exposure. This decrease in mica surface wettability due to exposure to laboratory air is well-known. Our results are consistent with White,26 who measured the contact angle of water on mica and showed that it increased from
