Pattern Formation of Antifreeze Glycoproteins via SolventEvaporation

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 dropletcontact line and the accumulation of the solute during evaporation. The solution concentration strongly affects theprotein line spacing. The average height of the protein was measured to be 8.1( 2.5 Å, which may be attributed tothe height of a single molecule.

1. Introduction

Antifreeze glycoproteins (AFGPs) are unique biomoleculesfound in several arctic and antarctic fish. These novel compoundsallow these organisms to survive cold conditions by inhibitingthe growth of ice.1-4 AFGPs do not prevent the formation of ice,but instead these proteins operate by modifying the ice morphol-ogy and inhibiting the further growth of ice.

Biological antifreeze proteins provide an impressive exampleof macromolecules with specific recognition capabilities forinorganic structures. The translation of concepts from bio-mineralization into strategies for the synthesis of materials hasbecome one of the most important areas in nanobiotechnology.5,6

Generally, polypeptides capable of binding to inorganic surfacescan be used to control the assembly and formation of functionalinorganic materials for nano- and nanobiotechnology applica-tions.7-10 Gaining information on the mechanism of action ofinorganic-binding proteins may lead to the development of newmaterials. For example, Shiba et al.6 showed that an artificialprotein containing a repeated peptide sequence allows certainsalts to form a variety of dendritic structures. In another example,a new polypeptide molecule was designed by Laursen et al.11 onthe basis of the helix structure of the type I AFP that could bindand control the shape of the growing calcite crystal. Moreover,patterning surfaces with this type of macromolecule could serveas nucleation templates for controlling crystal growth, includingthe precise localization of particles, crystal sizes, and shapes.10

The self-assembly of molecules on a surface via solventevaporation of a droplet on a solid surface can be a simple,

versatile, and noninvasive approach for one-step pattern forma-tion. This method has been used for patterning nanoparticles,12-15

polymers,16-20dye molecules,21and proteins22,23on solid surfacesand is very promising for the self-assembly of 2D arrays ofproteins and other macromolecules onto a suitable solid substrate.These arrays can serve as modules for the fabrication of molecularsensors and devices.24-26 Currently, surface patterning ofbiomolecules in the nanometer range involves techniques suchas microcontact printing (µCP),27 dip-pen nanolithography(DPN),28 plasma-enhanced chemical vapor deposition (PECVD),29

critical energy electron beam lithography (CE-EBL),30and others.However, the dewetting of thin liquid films on solid substratesmay also produce patterns in the nanometer range, offers differentpossibilities in terms of characteristic sizes and long-range order,and presents the advantage of a one-step and higher throughputpatterning technique. In this work, we applied the dewettingmethod for patterning antifreeze glycoprotein fraction 8 (AFGP8) on mica surfaces.

AFGPs are composed of a repeating tripeptide (L-Ala-L-Ala-L-Thr)n subunit with a â-D-galactose-1,3-R-D-galactosamine

* Corresponding author. E-mail: jgiorgi@uottawa.ca. Tel:+1-(613) 5625800 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,

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10.1021/la701408m CCC: $37.00 © 2007 American Chemical SocietyPublished on Web 10/10/2007

disaccharide linked to the secondary hydroxyl group of theL-threonine residue. Eight distinct fractions of these proteinshave been isolated that differ in the number of the tripeptiderepeat 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. Duringthe past decade, a great deal of effort has been expended to morethoroughly understand the mechanism by which biologicalantifreeze proteins bind to ice and inhibit its growth. Althoughit has been proposed that the hydrophilic interactions betweenpolar hydroxyl groups on the disaccharides and the watermolecules on the ice surface are extremely important,31-37othersbelieve thatentropicandenthalpiccontributions fromhydrophobicresidues are crucial in the binding of AFGP to the ice surface.38-42

The solution conformation of these proteins was examined byseveral groups; however, the relationship between activity andconformation is still not well understood. Previous work suggestedthat AFGP adopts a random coil conformation in solution,43-45

but other reports support the existence of a more ordered helicalstructure.46-48 Furthermore, recent work suggests that AFGPsare dynamically disordered and do not have long-range order.49,50

In recent years, a few research groups have studied theinteraction of antifreeze proteins with hydrophilic and hydro-phobic surfaces, such as mica and graphite, with the hope ofgaining insight into the nature of this adsorption process. Atomicforce microscopy (AFM) has been shown to be a powerfultechnique for the direct characterization of the surface absorptionaffinity of these proteins and their molecular structure on thenanometer scale.51,52

Here we report the spontaneous pattern formation of antifreezeglycoprotein fraction 8 (AFGP 8, 2.6 kDa) deposited on micaby the solution droplet evaporation technique. Periodic lines ofsingle proteins with different line spacing and a 2D single-proteingridlike structure were observed. The line spacing was found tobe dependent on the concentration of the solution. The observedpatterned surfaces may be used as templates for studying theeffect of AFGP on the nucleation and growth of ice, in contrastto most previous studies done in solution.

2. Experimental Section

AFGP8 was generously donated by A/F Protein Inc. as alyophilized powder after extraction and purification from the rockor Greenland cod (Gadus ogac). Muscovite mica was bought fromElectron Microscopy Sciences. Prior to protein deposition, micasurfaces were cleaved using adhesive tape.

All protein solutions were prepared in doubly distilled deionizedwater. The concentration range of the protein solutions used in thevarious experiments was in the range of 1.0× 10-10-1.0 × 10-9

g/mL. For the deposition of proteins by solvent evaporation, thesamples were prepared by applying a 10-25 µL drop of solutionon freshly cleaved mica and drying inside a closed desicator, whichwas pumped down for 30 min. All experiments were performed atroom temperature.

Imaging of AFGP 8 on mica was performed by atomic forcemicroscopy with a Molecular Imaging PicoPlus SPM system (Agilent,Tempe, AZ). Images were obtained in magnetic ac mode (MACmode) 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 proteinsformed by drying a 25µL drop of a 1.0× 10-10 g/mL proteinsolution. The dimensions of the features in this image weremeasured by cross-section analysis of each individual particle.A cross-sectional profile is shown in Figure 1. The average particleheight was found to be 8.1( 2.5 Å. The features appear to havea 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 5µm.

In previous work,51AFGP 8 was deposited on mica and HOPGsurfaces in an attempt to discern whether the driving force foradsorption onto ice was hydrophilic or hydrophobic in nature.

(31) Wierzbicki, A.; Taylor, M. S.; Knight, C. A.; Madura, J. D.; Harrington,J. P.; Sikes, C. S.Biophys. J.1996, 71, 8-18.

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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.Biochemistry1997, 36, 14652-14660.

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(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, 1524-

1537.(43) Franks, F.; Morris, E. R.Biochim. Biophys. Acta1978, 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.Biopolymers1977, 16,

2575-2578.(46) Rao, B. N.; Bush, C. A.Biopolymers1987, 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.

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Figure 1. AFM image and cross-section profile of lines of AFGP8 deposited from aqueous solution (1.0× 10-10 g/mL) on mica.Drop, 25µL; scan rate, 1 line/s.

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It was found that the proteins appear to bind preferentially tohydrophilic step edges of HOPG, whereas the adsorption onmica appeared to be randomly distributed. Individual globulesof proteins were found to have a height of 4 to 7 nm and a widthof 80 to 100 nm, which appeared to be aggregates of proteins.In the present work, the concentration of the solutions was 2 to5 orders of magnitude lower, which allowed the observation ofsmaller protein features. The observed height of 8.1( 2.5 Å forprotein features can be assigned to the height of a single proteinlayer because this value has been measured for several proteincoverages. The measured height is also consistent with thereported diameter of the 3-fold helical rodlike structure of theAFGP.53-55 However, the observed width of a single AFGP 8particle 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 dimensionsof these aggregates cannot be reliably determined. Furthermore,variations in height are to be expected and may be due to differentsurface conformations of the adsorbed molecules.

When the experiment was repeated at higher solutionconcentration, 1.0× 10-9 g/mL, similar patterns of AFGP 8were observed. However, the lines of protein particles were denserthan in the previous case. An AFM image and a cross-sectionprofile are shown in Figure 2. The cross-section analysis of theprotein particles shows a similar height distribution as observedat the lower concentration. However, these particles appear tobe much wider, 400 to 700 nm, which again shows that these

are 2D aggregates of AFGP 8 molecules. The spacing betweenthe molecules in the line is 400-800 nm whereas the spacingbetween the lines is 800-1200 nm, although a few lines appearto be even closer.

The formation of regular patterns or lines during the evaporationof a drop was described by Deegan56-58 as a self-pinningphenomenon. In this process, the solutes are thought to accumulateclose to the contact line of the drop as a result of convectioninside the drop, preventing it from retraction. As the solventcontinues to evaporate, the solute deposits on the surface andleads to the self-pinning of the liquid contact line. The contactline will then recede to a depinned state. The switch betweenpinned and depinned states of the contact line, together with thedeposition of the solute, leads to the formation of a highly orderedregular pattern,56-58 which in our case is composed of parallellines of protein particles. We observed that increasing theconcentration of AFGP 8 in the solution shrinks the line spacingon the surface. That implies that when a critical concentrationof the protein at the contact line is reached, the proteins will bedeposited on the surface. At higher solution concentration, thedeposition of AFGP 8 as the contact line recedes will be morefrequent, thereby producing denser lines. Also, the fact that theproteins were deposited parallel to the contact line indicates thatthe proteins have a strong affinity for the mica surface. We shouldnote that the density of the lines in Figure 2 appears to be morethan 10 times higher than in Figure 1, as expected for a 10-foldincrease in protein concentration. This could be due to thedifference in the local concentration of the protein when the dropis spreading on the mica surface. However, the density of linesdoes follow the expected concentration trend. In a similar fashion,periodic lines of positively charged latex particles were depositedon hydrophilic surfaces.15 This strong interaction is consistentwith the previous HOPG-mica report showing the preferenceof AFGP 8 for hydrophilic sites.51This observation is emphasizedby the 2D aggregates, suggesting a dynamic process in whichthe proteins have enough time to find a stable configuration atthe contact line prior to becoming immobilized on the surface.The fact that 2D instead of 3D islands are again observed indicatesthe strong binding of the protein to the mica surface.

Deegan’s model does not take viscosity into account, whichmay become important for smaller drops. An alternative modelthat can be used to describe the line formation has been describedby Snoeijer et al.59,60The general result of this approach is thatif the capillary number (the relative velocity of the receding linecorrected for liquid viscosity and surface tension) exceeds a criticalentrainment value then a thin film is left behind on the surface.The dynamics of the process are such that a ridge structuredevelops in the liquid and propagates away from the contact line.Because of the difference in velocity, the drying structure canbe described in three parts: a ridge at the outer edge, a capillaryfilm, and ultimately a Landau-Levich film connected to theliquid reservoir. One can then envision that it is within the ridgestructure that precipitation takes place, leaving lines of depositson the surface. The model has the advantage of explaining the2D aggregation of AFGP 8 because the evaporation of the ridgestructure would allow time for 2D aggregation. However, the

(53) Knight, C. A.; Cheng, C. C.; DeVries, A. L.Biophys. J.1991, 59, 409-418.

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2004, 110, 223-230.

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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.

Figure 2. AFM image and cross-section profile of lines of AFGP8 deposited from aqueous solution (1.0× 10-9 g/mL) on mica.Drop, 10µL; scan rate, 1 line/s.

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model would also predict a line separation independent of proteinconcentration, in contrast with our observations.

Although the exact mechanism of pattern formation in ourexperiments is unclear, it appears that the simpler Deegan modelbetter reflects the experimental observations. The exact patternwithin the original droplet area varies significantly with the exactconditions of the drying process, such as the evaporation rateand the direction of evaporation for pumped environments.56-58

In most cases, deposits along the rim of the initial shape of thedroplet are expected. In our experiments, we were unable to seethe curvature of the ring of the drying droplet by AFM. Thelarge-area scans (>50 × 50 µm2) necessarily have lowzresolution, and the noise level prevented the observation of the8-Å-high AFGP 8 clusters.

Occasionally, a different type of pattern morphology wasobserved. Figure 3 shows zones of polygonal networks of mostlysingle-molecule heights that were observed over an area of severalmicrometers. A close look at the height cross-section profilereveals that this is mostly a single AFGP 8 layer with somehigher regions, possibly indicative of a second layer. The widthof the features is about 200 nm, which again corresponds to a2D protein layer deposited on the mica surface.

According to Deegan,57 direct observation of the contact lineunder conditions where gridlike patterns are formed shows thatparts of it move steadily and other parts move in a stick-slipmotion. The steadily moving segments lie along the radial linesof the grid, whereas the stick-slip segments produce nothingwhen moving but produce a ring at rest. The combination ofradial lines and rings forms the gridlike pattern. An alternativeexplanation is that as the drop evaporates its radius and heightbecome smaller, and depending on the rate of evaporation, thebig drop could then split into a number of smaller drops beforecomplete evaporation occurs. This could lead to the formationof small rings attached to each other, which could also form agridlike structure.

It is interesting to compare the observed patterns of AFGP 8on mica with other organic moieties of similar size. For example,van Harmeren et al.21 showed the spontaneous formation ofperiodic patterns of porphyrin trimer dye molecules via self-

assembly and dewetting. These molecules self-organize on thesurface into small columnar stacks of submicrometer length.When a small droplet (3µL) of dilute solution was evaporatedon the mica surface, very large domains containing a highlyordered pattern of equidistant, nearly parallel, wirelike archi-tectures were observed. The lines were one molecule thick (4.5nm) with a periodicity of 0.5-1 µm. The lines were orientedparallel to the local solvent front. When a larger droplet (10µL)of the same solution was evaporated under similar conditions,the longer evaporation time formed porphyrin lines with a largerheight (55 nm) and a periodicity of 13µm; however, the orientationof the lines was now orthogonal to the solvent front. In the caseof 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 atthe contact line to pin it completely. The partial pinning causeda flow of molecules orthogonal to the contact line, giving riseto the orthogonal direction of the patterns.21

The effect of line formation is also well known in Langmuir-Blodgett film formation. Fuchs et al.61-63 have utilized thistechnique to transfer monolayers ofL-R-dipalmitoyl-phospha-tidycholine (DPPC) on mica, thereby generating a structuredsurface with a channel lattice that exhibits a high wettabilitycontrast. This structure can be obtained by rapidly withdrawinga mica substrate (1000µm/s) from the film at low monolayersurface pressure and constant temperature. Under these conditions,filmadsorptionbecomesunstable, leading toperiodic interruptionsin the molecular deposition and therefore to the formation ofstriplike patterns. The dynamic behavior of the meniscus heightat the contact line is governed by two counter-reacting processes.On one hand, the contact line between the solution and the solidsurface normally exceeds the planar water surface as a result ofsurface tension. On the other hand, a strong interaction of theamphiphilic molecule with the surface will result in a rapidadherence of the molecules, causing a reduction in the surface

(61) Lenhert, S.; Gleiche, M.; Fuchs, H.; Chi, L.ChemPhysChem2005, 6,2495-2498.

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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.

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energy and an increase in the contact angle, leading to a decreasein the meniscus height. These two processes may lead tooscillations of the meniscus height and result in regions of transferdepletion perpendicular to the dipping direction.61-63

Clearly, the pattern formation of AFGP 8 on mica followsfrom the dewetting and deposition at the contact line, althoughthe exact mechanism of line formation cannot be determined atthis time. In contrast to other work where lines of moleculeshave been observed, AFPG 8 forms regularly aligned patternsof 2D aggregates with single-molecule height.

4. Conclusions

We have shown that AFGP 8 can be conveniently patternedon a mica surface by the solvent evaporation technique, requiringlittle surface preparation. This patterning method is simple andnoninvasive, which is especially important for patterningbiological materials. Periodic lines of AFGP 8 and gridlikemorphology were obtained by solvent evaporation of dilute proteinsolutions. The line spacing was found to be dependent on theconcentration of the solution. The average height of the AFGP8 particles was found to be 8.1( 2.5 Å, which can be attributedto the height of single-molecule features. The adsorption of asingle-molecule layer confirms that, at these very low solutionconcentrations, the protein exists as single molecules in solution.64

The wide lateral dimensions of the features indicate that theprotein particles are 2D aggregates adsorbed onto the mica surface.

In contrast, previous work51showed 3D aggregation at the surfacebut with a protein solution concentration that was 2 to 5 ordersof magnitude higher than those used here.

Surface patterning of biological macromolecules is of greatimportance in the development of new technological strategiesfor nanobiotechnology applications. AFGP belongs to a groupof proteins involved in directing the shape of biominerals byrecognizing and binding selectively to one or more faces of thegrowing crystal. Because of this fascinating process and its clearapplications, research into the design of organic assemblies toassist the growth of inorganic crystals has increased significantlyin recent years.5 The use of functionalized surfaces to controlnucleation and crystal growth was demonstrated by Aizenberg,9,10

and patterning of nanocrystal calcite was achieved by controlcrystallization 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 sizeof the ice crystal, may offer a new way to study the interactionsbetween the antifreeze proteins and the ice surfaces. Work in thisdirection is currently underway.

Acknowledgment. We gratefully acknowledge funding fromthe 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.

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(64) Bouvet, V. R.; Lorello, G. R.; Ben, R. N.Biomacromolecules2006, 110,223-230.

(65) Aizenberg, J.; Black, A. J.; Whitesides, G. M.Nature1999, 398, 495-498.

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