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Pattern Formation of Antifreeze Glycoproteins via Solvent Evaporation Osnat Younes-Metzler,†,‡,§ Robert N. Ben,‡ and Javier B. Giorgi*,†,‡ Center for Catalysis Research and InnoVation and Department of Chemistry, UniVersity of Ottawa, 10 Marie Curie Street, Ottawa, Ontario, Canada K1N 6N5 ReceiVed May 15, 2007. In Final Form: September 19, 2007 Surface patterning of antifreeze glycoprotein fraction 8 (AFGP 8) via a solvent evaporation method is reported here. In this process, lines of AFGP 8 particles and gridlike patterns were formed as as result of the receding of the droplet contact line and the accumulation of the solute during evaporation. The solution concentration strongly affects the protein line spacing. The average height of the protein was measured to be 8.1 ( 2.5 Å, which may be attributed to the height of a single molecule.
1. Introduction Antifreeze glycoproteins (AFGPs) are unique biomolecules found in several arctic and antarctic fish. These novel compounds allow these organisms to survive cold conditions by inhibiting the growth of ice.1-4 AFGPs do not prevent the formation of ice, but instead these proteins operate by modifying the ice morphology and inhibiting the further growth of ice. Biological antifreeze proteins provide an impressive example of macromolecules with specific recognition capabilities for inorganic structures. The translation of concepts from biomineralization into strategies for the synthesis of materials has become one of the most important areas in nanobiotechnology.5,6 Generally, polypeptides capable of binding to inorganic surfaces can be used to control the assembly and formation of functional inorganic materials for nano- and nanobiotechnology applications.7-10 Gaining information on the mechanism of action of inorganic-binding proteins may lead to the development of new materials. For example, Shiba et al.6 showed that an artificial protein containing a repeated peptide sequence allows certain salts to form a variety of dendritic structures. In another example, a new polypeptide molecule was designed by Laursen et al.11 on the basis of the helix structure of the type I AFP that could bind and control the shape of the growing calcite crystal. Moreover, patterning surfaces with this type of macromolecule could serve as nucleation templates for controlling crystal growth, including the precise localization of particles, crystal sizes, and shapes.10 The self-assembly of molecules on a surface via solvent evaporation of a droplet on a solid surface can be a simple, * Corresponding author. E-mail:
[email protected]. Tel: +1-(613) 562 5800 ext. 6037. Fax: +1-(613) 562 5170. † Center for Catalysis Research and Innovation, University of Ottawa. ‡ Department of Chemistry, University of Ottawa. § Present address: Physics Department, Technical University of Munich, Munich, Germany. (1) Yeh, Y.; Feeney, R. E. Chem. ReV. 1996, 96, 601-617. (2) Davies, P. L.; Sykes, B. D. Curr. Opin. Struct. Biol. 1997, 7, 828-834. (3) Ewart, K. V.; Lin, Q.; Hew, C. L. Cell. Mol. Life Sci. 1999, 55, 271-283. (4) Harding, M. M.; Anderberg, P. I.; Haymet, A. D. J. Eur. J. Biochem. 2003, 270, 1381-1392. (5) Mann, S. Angew. Chem., Int. Ed. 2000, 39, 3393-3406. (6) Shiba, K.; Honma, T.; Minamisawa, T.; Nishiguchi, K.; Noda, T. EMBO Rep. 2003, 4, 148-153. (7) Sarikaya, M.; Tamerler, C.; Schwartz, D. T.; Baneyx, F. Annu. ReV. Mater. Res. 2004, 34, 373-408. (8) Xu, A.; Ma, Y.; Colfen, H. J. Mater. Chem. 2007, 17, 415-449. (9) Aizenberg, J. AdV. Mater. 2004, 16, 1295-1302. (10) Aizenberg, J. Bell Labs Tech. J. 2005, 10, 129-141. (11) DeOliveira, D. B.; Laursen, R. A. J. Am. Chem. Soc. 1997, 119, 1062710631.
versatile, and noninvasive approach for one-step pattern formation. This method has been used for patterning nanoparticles,12-15 polymers,16-20 dye molecules,21 and proteins22,23 on solid surfaces and is very promising for the self-assembly of 2D arrays of proteins and other macromolecules onto a suitable solid substrate. These arrays can serve as modules for the fabrication of molecular sensors and devices.24-26 Currently, surface patterning of biomolecules in the nanometer range involves techniques such as microcontact printing (µCP),27 dip-pen nanolithography (DPN),28 plasma-enhanced chemical vapor deposition (PECVD),29 critical energy electron beam lithography (CE-EBL),30 and others. However, the dewetting of thin liquid films on solid substrates may also produce patterns in the nanometer range, offers different possibilities in terms of characteristic sizes and long-range order, and presents the advantage of a one-step and higher throughput patterning technique. In this work, we applied the dewetting method for patterning antifreeze glycoprotein fraction 8 (AFGP 8) on mica surfaces. AFGPs are composed of a repeating tripeptide (L-Ala-L-AlaL-Thr)n subunit with a β-D-galactose-1,3-R-D-galactosamine (12) Adachi, E.; Dimitrov, A. S.; Nagayama, K. Langmuir 1995, 11, 10571060. (13) Shmuylovich, L.; Shen, A. Q.; Stone, H. A. Langmuir 2002, 18, 34413445. (14) Govor, L. V.; Bauer, G. H.; Reiter, G.; Shevchenko, E.; Weller, H.; Parisi, J. Langmuir 2003, 19, 9573-9576. (15) Ray, M. A.; Kim, H.; Jia, L. Langmuir 2005, 21, 4786-4789. (16) Karthaus, O.; Grasjo, L.; Maruyama, N.; Shimomura, M. Chaos 1999, 9, 308-314. (17) Kimura, M.; Misner, M. J.; Xu, T.; Kim, S. H.; Russell, T. P. Langmuir 2003, 19, 9910-9913. (18) Kim, S. H.; Misner, M. J.; Xu, T.; Kimura, M.; Russell, T. P. AdV. Mater. 2004, 16, 226-231. (19) Hong, S. W.; Xu, J.; Xia, J. F.; Lin, Z. Q.; Qiu, F.; Yang, Y. L. Chem. Mater. 2005, 17, 6223-6226. (20) Hong, S. W.; Xu, J.; Lin, Z. Nano Lett. 2006, 6, 2949-2954. (21) van Hameren, R.; Schon, P.; van Buul, A. M.; Hoogboom, J.; Lazarenko, S. V.; Gerritsen, J. W.; Engelkamp, H.; Christianen, P. C. M.; Heus, H. A.; Maan, J. C.; Rasing, T.; Speller, S.; Rowan, A. E.; Elemans, J.; Nolte, R. J. M. Science 2006, 314, 1433-1436. (22) Adachi, E.; Nagayama, K. AdV. Biophys. 1997, 34, 81-92. (23) Jacquemart, I.; Pamula, E.; De Cupere, V. M.; Rouxhet, P. G.; DupontGillain, C. C. J. Colloid Interface Sci. 2004, 278, 63-70. (24) Lowe, C. R. Curr. Opin. Struct. Biol. 2000, 10, 428-434. (25) Astier, Y.; Bayley, H.; Howorka, S. Curr. Opin. Chem. Biol. 2005, 9, 576-584. (26) Niemeyer, C. M. Angew. Chem., Int. Ed. 2001, 40, 4128-4158. (27) Feng, C. L.; Embrechts, A.; Vancso, G. J.; Schonherr, H. Eur. Polym. J. 2006, 42, 1954-1965. (28) Salazar, R. B.; Shovsky, A.; Schonherr, H.; Vancso, G. J. Small 2006, 2, 1274-1282. (29) Slocik, J. M.; Beckel, E. R.; Jiang, H.; Enlow, J. O.; Zabinski, J. S. J.; Bunning, T. J.; Naik, R. R. AdV. Mater. 2006, 18, 2095-2100. (30) Joo, J.; Chow, B. Y.; Jacobson, J. M. Nano Lett. 2006, 6, 2021-2025.
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disaccharide linked to the secondary hydroxyl group of the L-threonine residue. Eight distinct fractions of these proteins have been isolated that differ in the number of the tripeptide repeat units (n ) 4-50). AFGP 8 is the smallest isoform (n ) 4, 2.6 kDa), and AFGP 1 (n ) 50) is the largest at 34 kDa. During the past decade, a great deal of effort has been expended to more thoroughly understand the mechanism by which biological antifreeze proteins bind to ice and inhibit its growth. Although it has been proposed that the hydrophilic interactions between polar hydroxyl groups on the disaccharides and the water molecules on the ice surface are extremely important,31-37 others believe that entropic and enthalpic contributions from hydrophobic residues are crucial in the binding of AFGP to the ice surface.38-42 The solution conformation of these proteins was examined by several groups; however, the relationship between activity and conformation is still not well understood. Previous work suggested that AFGP adopts a random coil conformation in solution,43-45 but other reports support the existence of a more ordered helical structure.46-48 Furthermore, recent work suggests that AFGPs are dynamically disordered and do not have long-range order.49,50 In recent years, a few research groups have studied the interaction of antifreeze proteins with hydrophilic and hydrophobic surfaces, such as mica and graphite, with the hope of gaining insight into the nature of this adsorption process. Atomic force microscopy (AFM) has been shown to be a powerful technique for the direct characterization of the surface absorption affinity of these proteins and their molecular structure on the nanometer scale.51,52 Here we report the spontaneous pattern formation of antifreeze glycoprotein fraction 8 (AFGP 8, 2.6 kDa) deposited on mica by the solution droplet evaporation technique. Periodic lines of single proteins with different line spacing and a 2D single-protein gridlike structure were observed. The line spacing was found to be dependent on the concentration of the solution. The observed patterned surfaces may be used as templates for studying the effect of AFGP on the nucleation and growth of ice, in contrast to most previous studies done in solution. (31) Wierzbicki, A.; Taylor, M. S.; Knight, C. A.; Madura, J. D.; Harrington, J. P.; Sikes, C. S. Biophys. J. 1996, 71, 8-18. (32) Knight, C. A. Nature 2000, 406, 249-251. (33) Madura, J. D.; Baran, K.; Wierzbicki, A. J. Mol. Recognit. 2000, 13, 101-113. (34) Sicheri, F.; Yang, D. S. C. Nature 1995, 375, 427-431. (35) Hew, C. L.; Yang, D. S. C. Eur. J. Biochem. 1992, 203, 33-42. (36) Davies, P. L.; Hew, C. L. FASEB J. 1990, 4, 2460-2468. (37) Yang, D. S. C.; Sax, M.; Chakrabartty, A.; Hew, C. L. Nature 1988, 333, 232-237. (38) Chao, H.; Houston, M. E., Jr.; Hodges, R. S.; Kay, C. M.; Sykes, B. D.; Loewen, M. C.; Davies, P. L.; Sonnichsen, F. D. Biochemistry 1997, 36, 1465214660. (39) Haymet, A. D. J.; Ward, L. G.; Harding, M. M. J. Am. Chem. Soc. 1999, 121, 941-948. (40) Dalal, P.; Knickelbein, J.; Haymet, A. D. J.; So¨nnichsen, F. D.; Madura, J. D. PhysChemComm 2001, 7, 1-5. (41) Jia, Z.; Davies, P. L. Trends Biochem. Sci. 2002, 27, 101-106. (42) Jorov, A.; Zhorov, B. S.; Yang, D. S. C. Protein Sci. 2004, 13, 15241537. (43) Franks, F.; Morris, E. R. Biochim. Biophys. Acta 1978, 540, 346-356. (44) DeVries, A. L.; Komatsu, S. K.; Feeney, R. E. J. Biol. Chem. 1970, 245, 2901-2908. (45) Raymond, J. A.; Radding, W.; DeVries, A. L. Biopolymers 1977, 16, 2575-2578. (46) Rao, B. N.; Bush, C. A. Biopolymers 1987, 26, 1227-1244. (47) Bush, C. A.; Feeney, R. E.; Osuga, D. T.; Ralapati, S.; Yeh, Y. Int. J. Pept. Protein Res. 1981, 17, 125-129. (48) Bush, C. A.; Feeney, R. E. Int. J. Pept. Protein Res. 1986, 28, 386-397. (49) Lane, A. N.; Hays, L. M.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H. Protein Sci. 1998, 7, 1555-1563. (50) Lane, A. N.; Hays, L. M.; Tsvetkova, N.; Feeney, R. E.; Crowe, L. M.; Crowe, J. H. Biophys. J. 2000, 78, 3195-3207. (51) Sarno, D. M.; Murphy, A. V.; DiVirgilio, E. S.; Jones, E. W. J.; Ben, R. N. Langmuir 2003, 19, 4740-4744. (52) Lavalle, P.; DeVries, A. L.; Cheng, C. C.; Scheuring, S.; Ramsden, J. J. Langmuir 2000, 16, 5785-5789.
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Figure 1. AFM image and cross-section profile of lines of AFGP 8 deposited from aqueous solution (1.0 × 10-10 g/mL) on mica. Drop, 25 µL; scan rate, 1 line/s.
2. Experimental Section AFGP8 was generously donated by A/F Protein Inc. as a lyophilized powder after extraction and purification from the rock or Greenland cod (Gadus ogac). Muscovite mica was bought from Electron Microscopy Sciences. Prior to protein deposition, mica surfaces were cleaved using adhesive tape. All protein solutions were prepared in doubly distilled deionized water. The concentration range of the protein solutions used in the various experiments was in the range of 1.0 × 10-10-1.0 × 10-9 g/mL. For the deposition of proteins by solvent evaporation, the samples were prepared by applying a 10-25 µL drop of solution on freshly cleaved mica and drying inside a closed desicator, which was pumped down for 30 min. All experiments were performed at room temperature. Imaging of AFGP 8 on mica was performed by atomic force microscopy with a Molecular Imaging PicoPlus SPM system (Agilent, Tempe, AZ). Images were obtained in magnetic ac mode (MAC mode) using type II MAClevers. The scan rate was typically 1 line/s. Image resolution was 512 pixels per line.
3. Results and Discussion Figure 1 shows an AFM image of periodic lines of proteins formed by drying a 25 µL drop of a 1.0 × 10-10 g/mL protein solution. The dimensions of the features in this image were measured by cross-section analysis of each individual particle. A cross-sectional profile is shown in Figure 1. The average particle height was found to be 8.1 ( 2.5 Å. The features appear to have a globular shape with a large range of diameter (75-250 nm). The typical spacing between AFGP 8 in a line is 300-400 nm, whereas the spacing between the lines is about 5 µm. In previous work,51 AFGP 8 was deposited on mica and HOPG surfaces in an attempt to discern whether the driving force for adsorption onto ice was hydrophilic or hydrophobic in nature.
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Figure 2. AFM image and cross-section profile of lines of AFGP 8 deposited from aqueous solution (1.0 × 10-9 g/mL) on mica. Drop, 10 µL; scan rate, 1 line/s.
It was found that the proteins appear to bind preferentially to hydrophilic step edges of HOPG, whereas the adsorption on mica appeared to be randomly distributed. Individual globules of proteins were found to have a height of 4 to 7 nm and a width of 80 to 100 nm, which appeared to be aggregates of proteins. In the present work, the concentration of the solutions was 2 to 5 orders of magnitude lower, which allowed the observation of smaller protein features. The observed height of 8.1 ( 2.5 Å for protein features can be assigned to the height of a single protein layer because this value has been measured for several protein coverages. The measured height is also consistent with the reported diameter of the 3-fold helical rodlike structure of the AFGP.53-55 However, the observed width of a single AFGP 8 particle is wider than what is expected for a single molecule, suggesting that these particles are 2D aggregates of AFGP 8. Because the image is not tip-deconvoluted, the lateral dimensions of these aggregates cannot be reliably determined. Furthermore, variations in height are to be expected and may be due to different surface conformations of the adsorbed molecules. When the experiment was repeated at higher solution concentration, 1.0 × 10-9 g/mL, similar patterns of AFGP 8 were observed. However, the lines of protein particles were denser than in the previous case. An AFM image and a cross-section profile are shown in Figure 2. The cross-section analysis of the protein particles shows a similar height distribution as observed at the lower concentration. However, these particles appear to be much wider, 400 to 700 nm, which again shows that these (53) Knight, C. A.; Cheng, C. C.; DeVries, A. L. Biophys. J. 1991, 59, 409418. (54) Li, Q.; Luo, L. Chem. Phys. Lett. 1996, 263, 651-654. (55) Krishnan, V. V.; Fink, W. H.; Feeney, R. E.; Yeh, Y. Biophys. Chem. 2004, 110, 223-230.
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are 2D aggregates of AFGP 8 molecules. The spacing between the molecules in the line is 400-800 nm whereas the spacing between the lines is 800-1200 nm, although a few lines appear to be even closer. The formation of regular patterns or lines during the evaporation of a drop was described by Deegan56-58 as a self-pinning phenomenon. In this process, the solutes are thought to accumulate close to the contact line of the drop as a result of convection inside the drop, preventing it from retraction. As the solvent continues to evaporate, the solute deposits on the surface and leads to the self-pinning of the liquid contact line. The contact line will then recede to a depinned state. The switch between pinned and depinned states of the contact line, together with the deposition of the solute, leads to the formation of a highly ordered regular pattern,56-58 which in our case is composed of parallel lines of protein particles. We observed that increasing the concentration of AFGP 8 in the solution shrinks the line spacing on the surface. That implies that when a critical concentration of the protein at the contact line is reached, the proteins will be deposited on the surface. At higher solution concentration, the deposition of AFGP 8 as the contact line recedes will be more frequent, thereby producing denser lines. Also, the fact that the proteins were deposited parallel to the contact line indicates that the proteins have a strong affinity for the mica surface. We should note that the density of the lines in Figure 2 appears to be more than 10 times higher than in Figure 1, as expected for a 10-fold increase in protein concentration. This could be due to the difference in the local concentration of the protein when the drop is spreading on the mica surface. However, the density of lines does follow the expected concentration trend. In a similar fashion, periodic lines of positively charged latex particles were deposited on hydrophilic surfaces.15 This strong interaction is consistent with the previous HOPG-mica report showing the preference of AFGP 8 for hydrophilic sites.51 This observation is emphasized by the 2D aggregates, suggesting a dynamic process in which the proteins have enough time to find a stable configuration at the contact line prior to becoming immobilized on the surface. The fact that 2D instead of 3D islands are again observed indicates the strong binding of the protein to the mica surface. Deegan’s model does not take viscosity into account, which may become important for smaller drops. An alternative model that can be used to describe the line formation has been described by Snoeijer et al.59,60 The general result of this approach is that if the capillary number (the relative velocity of the receding line corrected for liquid viscosity and surface tension) exceeds a critical entrainment value then a thin film is left behind on the surface. The dynamics of the process are such that a ridge structure develops in the liquid and propagates away from the contact line. Because of the difference in velocity, the drying structure can be described in three parts: a ridge at the outer edge, a capillary film, and ultimately a Landau-Levich film connected to the liquid reservoir. One can then envision that it is within the ridge structure that precipitation takes place, leaving lines of deposits on the surface. The model has the advantage of explaining the 2D aggregation of AFGP 8 because the evaporation of the ridge structure would allow time for 2D aggregation. However, the (56) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Nature 1997, 389, 827-829. (57) Deegan, R. D. Phys. ReV. E 2000, 61, 475-485. (58) Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A. Phys. ReV. E 2000, 62, 756-765. (59) Snoeijer, J. H.; Delon, G.; Fermigier, M.; Andreotti, B. Phys. ReV. Lett. 2006, 96, 174504 174501-174504. (60) Snoeijer, J. H.; Andreotti, B.; Delon, G.; Fermigier, M. J. Fluid Mech. 2007, 579, 63-83.
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Figure 3. AFM images and cross-section profile of lines of AFGP 8 deposited from aqueous solution (5.0 × 10-10 g/mL) on mica. Drop, 10 µL; scan rate, 1 line/s.
model would also predict a line separation independent of protein concentration, in contrast with our observations. Although the exact mechanism of pattern formation in our experiments is unclear, it appears that the simpler Deegan model better reflects the experimental observations. The exact pattern within the original droplet area varies significantly with the exact conditions of the drying process, such as the evaporation rate and the direction of evaporation for pumped environments.56-58 In most cases, deposits along the rim of the initial shape of the droplet are expected. In our experiments, we were unable to see the curvature of the ring of the drying droplet by AFM. The large-area scans (>50 × 50 µm2) necessarily have low z resolution, and the noise level prevented the observation of the 8-Å-high AFGP 8 clusters. Occasionally, a different type of pattern morphology was observed. Figure 3 shows zones of polygonal networks of mostly single-molecule heights that were observed over an area of several micrometers. A close look at the height cross-section profile reveals that this is mostly a single AFGP 8 layer with some higher regions, possibly indicative of a second layer. The width of the features is about 200 nm, which again corresponds to a 2D protein layer deposited on the mica surface. According to Deegan,57 direct observation of the contact line under conditions where gridlike patterns are formed shows that parts of it move steadily and other parts move in a stick-slip motion. The steadily moving segments lie along the radial lines of the grid, whereas the stick-slip segments produce nothing when moving but produce a ring at rest. The combination of radial lines and rings forms the gridlike pattern. An alternative explanation is that as the drop evaporates its radius and height become smaller, and depending on the rate of evaporation, the big drop could then split into a number of smaller drops before complete evaporation occurs. This could lead to the formation of small rings attached to each other, which could also form a gridlike structure. It is interesting to compare the observed patterns of AFGP 8 on mica with other organic moieties of similar size. For example, van Harmeren et al.21 showed the spontaneous formation of periodic patterns of porphyrin trimer dye molecules via self-
assembly and dewetting. These molecules self-organize on the surface into small columnar stacks of submicrometer length. When a small droplet (3 µL) of dilute solution was evaporated on the mica surface, very large domains containing a highly ordered pattern of equidistant, nearly parallel, wirelike architectures were observed. The lines were one molecule thick (4.5 nm) with a periodicity of 0.5-1 µm. The lines were oriented parallel to the local solvent front. When a larger droplet (10 µL) of the same solution was evaporated under similar conditions, the longer evaporation time formed porphyrin lines with a larger height (55 nm) and a periodicity of 13 µm; however, the orientation of the lines was now orthogonal to the solvent front. In the case of the small droplet, the contact line was pinned several times, leaving behind thin layers of deposited molecules at this position. In the case of a large droplet, there was not enough material at the contact line to pin it completely. The partial pinning caused a flow of molecules orthogonal to the contact line, giving rise to the orthogonal direction of the patterns.21 The effect of line formation is also well known in LangmuirBlodgett film formation. Fuchs et al.61-63 have utilized this technique to transfer monolayers of L-R-dipalmitoyl-phosphatidycholine (DPPC) on mica, thereby generating a structured surface with a channel lattice that exhibits a high wettability contrast. This structure can be obtained by rapidly withdrawing a mica substrate (1000 µm/s) from the film at low monolayer surface pressure and constant temperature. Under these conditions, film adsorption becomes unstable, leading to periodic interruptions in the molecular deposition and therefore to the formation of striplike patterns. The dynamic behavior of the meniscus height at the contact line is governed by two counter-reacting processes. On one hand, the contact line between the solution and the solid surface normally exceeds the planar water surface as a result of surface tension. On the other hand, a strong interaction of the amphiphilic molecule with the surface will result in a rapid adherence of the molecules, causing a reduction in the surface (61) Lenhert, S.; Gleiche, M.; Fuchs, H.; Chi, L. ChemPhysChem 2005, 6, 2495-2498. (62) Chen, X.; Hirtz, M.; Fuchs, H.; Chi, L. AdV. Mater. 2005, 17, 28812885. (63) Gleiche, M.; Chi, L. F.; Fuchs, H. Nature 2000, 403, 173-175.
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energy and an increase in the contact angle, leading to a decrease in the meniscus height. These two processes may lead to oscillations of the meniscus height and result in regions of transfer depletion perpendicular to the dipping direction.61-63 Clearly, the pattern formation of AFGP 8 on mica follows from the dewetting and deposition at the contact line, although the exact mechanism of line formation cannot be determined at this time. In contrast to other work where lines of molecules have been observed, AFPG 8 forms regularly aligned patterns of 2D aggregates with single-molecule height.
4. Conclusions We have shown that AFGP 8 can be conveniently patterned on a mica surface by the solvent evaporation technique, requiring little surface preparation. This patterning method is simple and noninvasive, which is especially important for patterning biological materials. Periodic lines of AFGP 8 and gridlike morphology were obtained by solvent evaporation of dilute protein solutions. The line spacing was found to be dependent on the concentration of the solution. The average height of the AFGP 8 particles was found to be 8.1 ( 2.5 Å, which can be attributed to the height of single-molecule features. The adsorption of a single-molecule layer confirms that, at these very low solution concentrations, the protein exists as single molecules in solution.64 The wide lateral dimensions of the features indicate that the protein particles are 2D aggregates adsorbed onto the mica surface. (64) Bouvet, V. R.; Lorello, G. R.; Ben, R. N. Biomacromolecules 2006, 110, 223-230.
In contrast, previous work51 showed 3D aggregation at the surface but with a protein solution concentration that was 2 to 5 orders of magnitude higher than those used here. Surface patterning of biological macromolecules is of great importance in the development of new technological strategies for nanobiotechnology applications. AFGP belongs to a group of proteins involved in directing the shape of biominerals by recognizing and binding selectively to one or more faces of the growing crystal. Because of this fascinating process and its clear applications, research into the design of organic assemblies to assist the growth of inorganic crystals has increased significantly in recent years.5 The use of functionalized surfaces to control nucleation and crystal growth was demonstrated by Aizenberg,9,10 and patterning of nanocrystal calcite was achieved by control crystallization over patterned surfaces.65 Following these ideas, the crystal growth of ice over patterned surfaces with AFGP, which are known to have a strong effect on the shape and size of the ice crystal, may offer a new way to study the interactions between the antifreeze proteins and the ice surfaces. Work in this direction is currently underway. Acknowledgment. We gratefully acknowledge funding from the Natural Sciences and Engineering Research Council of Canada (NSERC), the Premier’s Research Excellence Award (PREA), and the Center for Catalysis Research and Innovation (CCRI) at the University of Ottawa. LA701408M (65) Aizenberg, J.; Black, A. J.; Whitesides, G. M. Nature 1999, 398, 495498.
