1 Proteins for Surface Structuring COPYRIGHTED MATERIAL · 1 Proteins for Surface Structuring Alexander Schulz, Stephanie Hiltl, Patrick van Rijn, and Alexander B¨oker 1.1 Introduction

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1Proteins for Surface StructuringAlexander Schulz, Stephanie Hiltl, Patrick van Rijn, and Alexander Böker

1.1Introduction

The use of proteins as an alternative for synthetic structures for the formationof new materials is a highly active topic in the research field [1, 2]. Propertiesand structures of proteins are generally well understood and this enables theiruse for other systems and in different settings/environments than the ones theyare originally designed for [3]. Proteins themselves already display interestingproperties with respect to catalytic activity, storage capabilities, and in beingavailable in a wide variety of shapes and sizes. When introduced into systemsnot comprising a natural setting for these structures, the properties can be used,for example, to influence interfacial properties, for serving as a template for thedeposition of inorganic materials, in modifications with synthetic moieties, or incombination with other biological structures.

Here we show different approaches and highlights of proteins at interfacesand the utilization in producing novel hybrid structures using their catalytic orcoordinating properties for mineralization processes at liquid–liquid as well asliquid–solid interfaces. Additionally, at liquid–solid interfaces, a more localizeddegree of organization can be achieved via various deposition processes into awide variety of patterns. The creation of patterns of biological species, includingproteins, peptide fragments, antibodies, nucleotides, and so on, on solid surfacesallows for the development of biosensors and affinity essays.

1.2Structuring and Modification of Interfaces by Self-Assembling Proteins

Nature offers a great diversity of proteins building complex superstructures thatserve as a matrix for the growth of different materials. The process of biomin-eralization differs from organism to organism. In many cases, organisms buildtheir biominerals by preorganizing a proteinous matrix that is subsequently min-eralized. The mineralization can be guided by the insoluble matrix (by binding to

Biomaterials Surface Science, First Edition.Edited by Andreas Taubert, João F. Mano, and J. Carlos Rodrı́guez-Cabello.© 2013 Wiley-VCH Verlag GmbH & Co. KGaA. Published 2013 by Wiley-VCH Verlag GmbH & Co. KGaA.

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4 1 Proteins for Surface Structuring

crystals as well as by constraining the available space) and by soluble proteins andlow-molecular-weight agents binding to the growing crystallites. We only discusssome very special systems in this chapter; a more general overview about biomin-eralization is given in the literature [4, 5]. Classical examples of proteins involvedin biomineralization are collagen on the one hand, a protein assembling into fibrilsand fibers [6, 7], and chitin on the other hand, assembling into different phasesand also forming nematic phases [8, 9]. But there are also proteins that do notself-assemble in solution but at interfaces.

Self-organized protein structures on solid and liquid interfaces can be used for thetailored production of materials. The protein can serve as a starting point for nucle-ation, but it can also be a part of the forming material, yielding a composite material.

We discuss the assembly of long-chain polyamines occurring together withsilaffins, the assembly of silicateins and hydrophobins, as well as some examplesof possible modifications of the adsorbed proteins.

1.2.1Formation and Modification of Protein Structures at Liquid Interfaces

In general, proteins are constructed from amino acids, some of them possessingapolar side chains, while others have polar or charged side chains. The best way tokeep the apolar amino acids away from the surrounding aqueous phase is in mostcase the creation of a hydrophobic core. This core enables most of the apolar sidechains to interact with each other via van der Waals forces, while the other aminoacids can interact with surrounding water molecules. This construction is stable insolution – but it is not necessarily stable when the protein approaches an interface.The apolar phase might be a much more favorable surrounding to many apolargroups compared to the hydrophobic core. The resulting surface activity is differentcompared to classical surfactants as the protein does not necessarily present apolargroups to the surrounding phases in its native state. Thus, the protein often hasto rearrange itself at the interface, so that the hydrophobic core turns inside outinto the apolar phase, with the other groups remaining in contact with the aqueousphase. This leads to an energetically favored state of the protein that also reducesthe interfacial tension. Obviously, this process often leads to dramatic changes inthe secondary structure, making the adsorption irreversible or leading at least toa high activation energy for desorption. Adsorption can be analyzed with differentmodels, which often distinguish between the diffusion to the interface and theprocess of rearrangement, sometimes including different conformations at theinterface [10, 11].

1.2.1.1 SilaffinsWe discuss silaffins as the first protein taking part in biomineralization processes.Silaffins consist of a phosphorylated backbone and polyamine side chains. Thesemolecules occur in diatoms, in which they help to build various structures of silicabeing as beautiful as highly organized. These silaffins occur together as a mixturewith other substances in nature, and the accompanying long-chain polyamines are

1.2 Structuring and Modification of Interfaces by Self-Assembling Proteins 5

especially important. These long-chain polyamines cannot be classified as proteins,but we discuss them in this chapter as they have functions similar to proteinsthat take part in the biomineralization of diatoms. While proteins such as collagenassemble into solid structures, the long-chain polyamine (most likely the crucialfactor for typical structure formation of silica in diatoms [12]) phase separatesinto small droplets in aqueous solution. Silica precipitates on the surface of thesedroplets, embedding a fraction of the polyamines. When a critical amount ofpolyamine is co-precipitated within the silica, the droplet breaks down into smallerdroplets because of the changes in phosphate concentration and pH value, andthe precipitation proceeds afterwards at the freshly built surfaces. The silaffins donot give structure to the material, but accelerate the precipitation of silica. By thisprocess, a hierarchical hexagonal material is built [12–14].

1.2.1.2 HydrophobinsHydrophobins are proteins capable of forming organized structures at interfacesvia self-assembly. Filamentous fungi excrete these small, globular proteins thatassemble into various structures. Many different hydrophobins exist, showing onlya weak similarity in sequence, but exhibiting a typical pattern of eight cysteineresidues building four disulfide bridges. These bridges also stabilize the secondarystructures as some of the cysteines lie within helices or sheets [17]. Hydrophobinsare commercially available in large amounts [18]. Despite their different sequences,fungi use different hydrophobins to lower the surface tension against water as wellas to hydrophobize their spores and fruitbodies. This enables them to grow theirfruitbodies out of the substrate into air or to infect new substrates coming from air[19, 20].

Hydrophobins are divided into two classes differing in terms of aggregatestability, as shown in Figure 1.1. Hydrophobins of class I build typical rod-shapedaggregates, termed rodlets, having a width of around 10 nm and a length of100–250 nm [21]. These rodlets assemble into films at interfaces being extremelyrobust to detergents and fluctuations in pH; solubilization of the aggregates andtheir films is only possible via treatment with trifluoroacetic acid. In contrast, classII hydrophobins do not build rodlets, and their films are less stable and can easilybe dissolved. These films also form characteristic patterns, although on a smallerlengthscale compared to the rodlets formed by class I hydrophobins [17, 20, 21].

Furthermore, all hydrophobins possess a hydrophobic patch that is importantfor their surface activity. While most proteins have a hydrophobic core and ahydrophilic surface, the hydrophobic patch of hydrophobins is located on thesurface. This leads to enhanced surface activity, as the protein is an amphiphile inits native state [17, 21]. Additionally, hydrophobins can rearrange at the interfacelike any other protein and they therefore adsorb irreversibly at the interface or needat least a much higher amount of energy to desorb in comparison to commonsurfactants [10, 11].

The quick formation of stable layers for different hydrophobins is followed bythe decrease in interfacial tension and the increase in the dilatational modulus [18,22]. The underlying processes can be understood by molecular dynamics (MD)


(a) (b)

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Figure 1.1 (a) Atomic force microscopic(AFM) image of rodlets formed by the HGFIhydrophobin from Grifola frondosa. Rodletformation is characteristic of class I hy-drophobins. The rodlets were formed at theair–water interface in a Langmuir trough bymultiple compression and lifted on a solidsupport for imaging, as described in [15].

(b) A surface membrane of HFBI imaged byAFM showing an organized structure. Thefilm was formed at the air–water interfaceand lifted onto a mica support, as describedin [16]. Source: Figure and description aretaken from [17], reprinted with permission ofElsevier, Copyright 2009.

simulations [23], and also by visualizing the structure via AFM and SEM, as canbe seen in Figure 1.1 [17, 19]. The structures of HFBI show regular and nearlyhexagonal features at liquid interfaces. The lattice parameters can be varied by thepreparation technique of the interface or by protein engineering [24].

Films of the artificial hydrophobin H*Protein B on silica can serve as a templatefor the growth of layers of TiO2, consisting of polycrystalline anatase. The proteinfilms are prepared by immersing a piranha-cleaned silicon wafer into a bufferedhydrophobin solution at different temperatures for various periods. Afterwards,the coated wafer is transferred to an aqueous solution of titania at a controlledtemperature to grow the titanium layer. The protein film does not only serve as anucleation point, but as IR spectra show, it also gets incorporated into the layer oftitanium dioxide. The roughness of the film can be controlled by the depositiontime, and the mechanical strength in terms of hardness and Young’s moduluswas found to be much greater compared to layers prepared by chemical bathdeposition [25]. This shows clearly that the use of proteins is not just anotherroute to prepare materials, but rather a route to produce composite materials withsuperior properties.

Films of hydrophobin on liquid interfaces can serve as a matrix for subsequentmineralization (an example structure is shown in Figure 1.2). For example, anoil-in-water emulsion stabilized with the artificial hydrophobin H*Protein B canserve as a template for the creation of mineral microcapsules. In the first step,the protein adsorbs to the oil–water interface. Several oils are applicable for thisprocess, and many of them work in the subsequent mineralization. The interfacial


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Figure 1.2 Capsules synthesized from ahydrophobin-stabilized emulsion: (a) in-tact mineral capsule after 68 days of min-eralization from a perfluorooctane/wateremulsion; (b) capsules prepared from aperfluorooctane/water emulsion, sinteredfor 1 h at 900 ◦C. Structure of the mineralshell of capsules in dependence of oil: (c)

perfluorooctane/water (50 days of mineral-ization); (d) silicone oil/water after partialwashing with heptane, remains of siliconeoil cover the surface at the right-hand sideof the image (17 days of mineralization).Source: Figures and descriptions are takenfrom [22], reprinted with permission of TheRoyal Society of Chemistry, Copyright 2011.

tension between oil and water is the important parameter that determines whethermineralization will take place or not. This knowledge enables to choose an oil thatwill match the desired properties of each process without having to test differentoils in a screening. The protein is mineralized by a saturated solution of calciumphosphate with a suitable pH of 7.4 for the precipitation of hydroxyapatite, yieldingoil-filled mineral capsules with a shell of hydroxyapatite. This process has severaladvantages. In most cases, the oil can be removed easily after the synthesis of thecapsules, but it can also be used to solubilize compounds and keep them insidethe capsules. Moreover, the process works under mild reaction conditions, and theresulting mineral phase is the same as in bones (nanocrystalline hydroxyapatite);consequently, the probability of getting a biocompatible material is high. Thecapsules can also withstand high temperatures up to 900 ◦C, and in addition to


that, their morphology is tunable by thermal treatment. The morphology changesin two ways. First, the small crystallites begin to sinter together – this affects themechanical properties as well as the porosity. Second, the mineral phase seemsto change at high temperatures, accompanied by a drastic change in morphology(shown in Figure 1.2b). This enhances the scope of these capsules, as they could alsobe used as microreactors in processes that take place at elevated temperatures [22].

The biomimetic character of this approach of synthesis is not just the useof the protein as a simple matrix to start the mineralization. Proteins refold atinterfaces to optimize the contact of hydrophobic groups with the apolar phaseand the contact of the hydrophilic groups with the polar phase. It is feasible thatthe reorganization depends on the character of the apolar phase. The experimentsshow that the morphology of the mineral changes for different oils. This indicatesthat the protein does not just start the mineralization by heterogeneous nucleation,but also influences the mineral growth – a concept that is frequently used by matrixproteins in nature [5, 22].

1.2.2Formation and Modification of Protein Structures at Solid Interfaces

Hydrophobins form stable films on hydrophobic solid as well as on liquid surfaces.The adsorption onto solid surfaces is often characterized by contact angle measure-ments. The contact angle of the hydrophobin EAS dissolved in water on Teflon(pure system: 108 ± 2◦) changes to 48 ± 10◦ by adsorption of hydrophobin. Thebinding to the surface is strong, as the contact angle is still 62 ± 8◦ after washingwith a hot solution of sodium dodecyl sulfate [19]. A commercially available classI hydrophobin can build remarkable stable layers on oxidized silica, changing thecontact angle of water from 0◦ to 67◦. This behavior emphasizes the amphiphilicproperties of the molecule, as it is able to turn an apolar surface into a much morepolar one and vice versa. The film withstands temperatures up to 90 ◦C withoutdissolving and shows a regular structure [25]. Films made of hydrophobin are alsofeasible to protect silicon against etching by alkaline solutions; hydrophobins cantherefore serve as an alternative to classical lithography masks [26]. The microscopicstructure of hydrophobin films was already explored by MD simulations. Thesesimulations identified the important parts for the binding to hydrophobic surfacesfor SC3 [23] and HFBII (a snapshot of the stable conformation at a silicone surfaceis shown in Figure 1.3) [27]. This knowledge enables molecular engineering totailor the adsorption properties of these proteins to specific requirements.

1.2.2.1 SilicateinsSilicateins are enzymes extracted from marine sponges, in which they hydrolyzedifferent silica precursors under ambient conditions and physiological pH, withoutbeing very substrate-specific [28]. Special care has to be taken when immobilizingsilicateins, as they become inactive if their secondary structure changes because ofadsorption or constrictions in mobility of the active center. These constraints can befulfilled using a spacing layer between matrix and silicatein layer. A quite general


(a) (b)

Figure 1.3 (a,b) Representative tightlybound HFBII/Si(1 1 1) interface. The full hy-drophobic patch is colored blue with themost adhesive residues colored red. Figure(b) gives a zoomed-in perspective view with

the near-silicon methyl carbons shown astransparent van der Waals’s spheres. Source:Figure and description are taken with modifi-cations from [27], reprinted with permissionof Springer, Copyright 2011.

approach is to use a polymer layer together with a spacer. In the original system,nitrilotriacetic acid (NTA) binds to a gold surface via its thiol groups. The acidicgroups, localized at the other end of the molecule, complex a nickel ion, whichcan be subsequently complexed by a His-tag attached to a silicatein (see Figure 1.4for the adapted modification of WS2 rods) [29]. The silicatein keeps its catalyticactivity, which is discussed in detail later. This system has also been adapted to usepolymer layers [30], Fe2O3 [31], WS2 [32], or TiO2 [33] as a matrix. The variety ofmatrix materials shows that this system is well studied for many cases. There arealso more straightforward ways that also preserve the catalytic activity of silicatein:gold surfaces can be modified with cystamine or cysteamine via their thiol groups.Afterwards, glutardialdehyde is added to link the silicatein covalently to the aminelayer. Subsequently, the surface is mineralized by the addition of silica precursors[34]. All these processes share the need for a spacer in contrast to the systems atliquid/liquid interfaces described previously.

We described several ways to bind silicatein to various surfaces without dimin-ishing its activity. This remaining catalytic activity can be used to precipitate variousmaterials from precursors, giving rise to several hybrid materials under mild reac-tion conditions: WS2 nanotubes coated with a layer of titania [32], gold nanocrystalsgrown on TiO2 nanorods (see also Figure 1.5) [33], magnetite particles covered witha layer of silica [31], and zirconium or titanium deposited onto a polymer substrate[30]. Furthermore, some of the materials are deposited in rather unusual shapes –such as triangular gold crystals on TiO2 nanorods (shown in Figure 1.5). Thereason for this uncommon shape supposedly lies in the chiral surrounding of thereaction center of silicatein [33]. Furthermore, the layer thickness and roughnessare determined by the specific reaction conditions. When these parameters arecontrolled carefully, the layers are smooth and can be adapted to the desired values,even when a simple system is used for the immobilization of the protein [34].


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1.3 Structuring and Modification of Solid Surfaces via Printing of Biomolecules 11

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Figure 1.5 HRSEM image demonstrating the hierarchical structure of the TiO2nanowire/Au nanoparticle composite. Overview images of the TiO2/Au nanocomposites(top left) and a magnified view (right bottom) are given. Source: Figure and description aretaken from [33], reprinted with permission of WILEY-VCH, Copyright 2006.

In addition to the induction of mineralization processes by surface-boundproteins, the control of the exact protein position might be of interest. For thispurpose, printing of proteins is a suitable technique that is discussed in thefollowing section.

1.3Structuring and Modification of Solid Surfaces via Printing of Biomolecules

The focus of this section is on different approaches used for structuring and mod-ifying solid surfaces [35, 36] with biomolecules [37]. We address intaglio printing(IntP) [38] by means of wrinkling as a lithography-free method for the preparation ofnanostructured substrates and its application in the assembly of bionanoparticles.Besides particle assembly on nanostructured corrugated substrates, the elastomerictemplates serve as stamps in microcontact printing (μCP) and affinity contact print-ing (αCP) processes [39, 40] and also for fabrication of microfluidic devices [41, 42].In general, IntP differs from μCP concerning the area of the stamp where from theink is transferred. For IntP, particles are printed from wrinkle grooves, while forμCP, the ink is located at stamp protrusions. In contrast, in αCP, a target protein insolution complexes with a probe protein adsorbed on the stamp forming a proteincomplex, subsequently being transferred to a substrate.

1.3.1Intaglio Printing Using Nanostructured Wrinkle Substrates

1.3.1.1 Wrinkling: Nanostructured TemplatesSurface wrinkles form by release of strain in a bilayer system composed of a stiffthin layer resting on top of an elastic substrate. The long axis of the wrinkles


develops perpendicular to the direction of the strain. Depending on the elasticproperties of the system, wrinkle dimensions – wavelength and amplitude – rangefrom nanometer to kilometer scale [43]. A large variety of materials is used to buildup those bilayer systems with controlled solvent diffusion [44], deposition of metalfilms [45], and plasma oxidation of soft materials [46, 47] as prominent examples.

Here we address lithography-free preparation of wrinkles by plasma oxidationof polydimethylsiloxane (PDMS). The sample is uniaxially stretched in a custom-made apparatus according to Genzer et al. [48]. Subsequent plasma treatmentconverts the top layer of the elastomer into glasslike SiOx with variable thickness hldepending on the plasma treatment time. Strain release leads to surface wrinklingbecause of different Young’s moduli of the components. Figure 1.6a shows anAFM height image of wrinkles and a cross-section revealing the sinusoidal shapeof the structure, which is supported by the TEM cross-section (Figure 1.6b).

In the case of PDMS, the thickness hl of the top layer is tunable by duration ofthe plasma treatment and directly influences the characteristic wrinkle dimensions(wavelength λ and amplitude A). λ is proportional to hl and the Young’s modulusof the top layer as well as indirectly proportional to the Young’s modulus of thesubstrate. The amplitude A is proportional to hl and the compressive strain [49].Besides systems composed of a variety of materials, a large number of wrinklegeometries is available, including linear, radial [50], and random [51] surfacewrinkles as well as herringbone patterns [52] and wrinkle gradients [53, 54].

1.3.1.2 Assembly of Bionanoparticles on WrinklesIn addition to the concept of wrinkling, we focus on the assembly of bionanopar-ticles, for example, the tobacco mosaic virus (TMV), on nanostructured wrinkledsubstrates as performed by Horn et al. [55]. TMV is a rod-like virus of 300 nm

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A = 59 ± 8 nm). (c) AFM height image(�z = 50 nm) with corresponding heightprofile showing adsorption of virus (TMV,tobacco mosaic virus) in the wrinkle grooves(λ = 299 ± 18 nm and A = 27 ± 1 nm) [55].Source: Reprinted with permission of TheRoyal Society of Chemistry, Copyright 2009.


length and 18 nm diameter. The coat protein self-assembles into a helical structurewith an inner channel of 4 nm. This work aims at optimized assembly conditionsof TMV in wrinkle grooves and to avoid typical liquid crystal-like clustering of TMVduring self-assembly to form uniform lines. Therefore, a set of parameters, namely,virus concentration and spin speed, were scanned to obtain statistic data on the as-sembly quality. As shown in Figure 1.6c, TMV aligns selectively in wrinkle grooves,presumably because of discontinuous dewetting during the spin coating processused for assembly. The easily tunable wavelength of the wrinkles predeterminesthe distance of the virus strings after the assembly.

Figure 1.7 combines SEM images from TMV assembly at different concentrations(a–c) and the virus occupancy � and deviator � parameters for the assembly at

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eyes). Light and dark blue symbols belongto concentration axis, while orange and redsymbols belong to the spin speed axis, asthe black arrows also indicate [55]. Source:Reprinted with permission of The Royal Soci-ety of Chemistry, Copyright 2009.


various concentrations and spin speeds (d). � defines the number of particlesadsorbed in the grooves, with 1 representing 100% occupation. The number ofviruses adsorbed outside the grooves compared to the total virus number provides�. With increasing concentration, the virus occupancy � increases. In relation tothe total number of viruses, the number of particles outside the grooves decreases,leading to a decrease of �. With 90% of wrinkles filled with TMV and a minimumof viruses adsorbed outside the grooves, Horn et al. determined an optimalconcentration of 0.9 mg ml−1 for the TMV assembly. A screening of different spinspeeds revealed maximum occupation (�) of wrinkles with TMV and a minimumof � at 3000 rpm.

Dewetting of a continuous water film present on the substrate surface is supposedto be crucial for proper virus assembly. The thickness of the film influenced bythe spin speed regulates the assembly quality. A thin water film starts dewettingon top of the wrinkles and locally raises the virus concentration in the wrinklegrooves and directs the virus arrangement. For a film thickness smaller than thewrinkle amplitude, a controlled dewetting is inhibited by hole formation in thefilm. Consequently, TMV sticks to the surface without preferential alignment.

Owing to swelling of PDMS in contact with organic solvents, the TMV linesneed to be transferred to suitable plane substrates for further modification. Thedevelopment of an appropriate printing process is summarized in the followingsection.

1.3.1.3 Intaglio Printing of Tobacco Mosaic VirusAs mentioned previously, the incision of an image into a surface-holding inkdefines IntP. Horn et al. [38] transferred this definition to their system of TMValigned in wrinkle grooves, with TMV acting as ink. For the printing process, aninked stamp (alignment of virus in wrinkle grooves by spin coating [55]) is pressedon a silicon wafer for 30 s. IntP results in regular virus stripes with line spacingsfrom ≈300 nm to ≈1 μm characterized by SEM and AFM (Figure 1.8).

Factors influencing pattern quality are prealignment of the virus (ink) on thestamp, hydrophilicity of the stamp and smoothness of the substrate to ensurequantitative particle transfer, and the stamp amplitude. With a stamp amplituderanging from 20 to 65 nm, IntP is successful. Higher amplitudes result in ahigher defect density of the pattern, or no pattern forms at all. During printing,a water film present after the spin coating presumably acts as transfer mediumfor the viruses. Water wets the channels build up of the Si wafer at the bottomand wrinkles on top inducing sufficient mobility of the viruses to move to the Sisurface. Beyond an amplitude limit of 65 nm, incomplete wetting of the channelsleads to incomplete or no particle transfer. Besides the amplitude limit, one hasto hydrophilize the Si surface to obtain good wetting properties and successfulprinting. Treatment of the sample with oxygen plasma is a fast and convenientmethod for the hydrophilization.

Owing to addressable reactive groups on the virus surface, the TMV patterns canserve as templates for further mineralization and metallization reactions for theproduction of nanowires.


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[38]

.


1.3.2Microcontact Printing for Bioinspired Surface Modification

Besides IntP, microcontact printing [35, 39] (μCP) is a general approach to patternself-assembled monolayers (SAMs) of functional molecules. During conformalcontact between a topographically structured elastomeric stamp and a flat substrate,ink transfers to the flat substrate. Reactive inks bind covalently to the surface, whilesurface-active inks can also attach noncovalently. The topography of the stampdefines the resulting molecule pattern. Besides patterning of SAMs, μCP includespatterning of biomolecules, polymers, and colloids. Herein we focus on structuringof biomolecules on solid supports. The resolution of μCP is in the submicrometerrange. Additionally, the process is carried out under ambient conditions and withonly mild chemical treatments.

1.3.2.1 Microcontact Printing onto Self-Assembled MonolayersInitially, μCP was shown for proteins on empirically chosen solid supports. Chenet al. [56] examined the influence of surface wettability of support and stampon the quality of the resulting pattern. They printed proteins on two-componentSAMs composed of polar and aliphatic alkanethiols. The fraction of polar groupsincorporated into the SAM controls the wettability.

They found a minimum wettability necessary for successful μCP of protein ontothe substrate (Figure 1.9a). Below a certain threshold, incomplete or no proteintransfer occurs, while for ≥65% COOH SAM, the pattern transfers completely.The interaction between protein and substrate increases with increasing densityof polar groups on the surface. To prove a relationship among wettability ofthe SAM, transferred protein amount, and chemical nature of functionality, theysubstituted polar COOH– groups with OH– (Figure 1.9b) and EG6OH groups(Figure 1.9c) (EG6OH: hexa(ethylene glycol)-terminated alkanethiol). The resultingthreshold value is slightly increased (70%) for OH SAMS, as the functional groupprovides only one site for polar interactions compared to COOH. EG6OH withsix interaction sites reduces the value to 4%. The threshold wettability, allowingcomplete protein transfer and performance of μCP under ambient conditionswith proteins resistant to adsorption in aqueous conditions, suggest differentmechanisms for adsorption and μCP. Additionally, the wettability of the stampinfluences μCP results. Minimum SAM wettability for protein patterning increasesfor increasing stamp wettability. A model of competing attractive forces betweenstamp and substrate describes the results [56]. Polar groups on one of the surfacesincrease the attractive forces, and increasing the hydrophobicity of the stamp allowspatterning of proteins resistant to μCP from untreated stamps.

1.3.2.2 Microcontact Printing with Wrinkle StampsIn the above-mentioned approach, PDMS stamps are replicas of a silicon masterproduced by photolithography with features on the microscale. Fery et al. [57] usedPDMS wrinkles as stamps for μCP of fluorescently labeled macromolecules and


(a)

(b)

(c)

50% COOH 55% COOH 60% COOH 65% COOH

55% OH

2% EG 3% EG 4% EG 5% EG

60% OH 65% OH 70% OH

Figure 1.9 Printing of proteins on three dif-ferent types of mixed SAMs. Micrographsof fluorescently labeled protein printedonto mixed SAMs of alkanethiol present-ing –CH3 and (a) –COOH, (b) –OH, and(c) –EG6OH functionalities. Each image is

a representative image from experimentsrepeated a minimum of three times withsimilar results. Scale bar: 100 μm. Source:Reprinted with permission of AmericanChemical Society, Copyright 2002 [56].

bovine serum albumin (BSA) onto substrates coated with polyelectrolyte multilay-ers. Figure 1.10 shows an AFM image of printed BSA stripes with correspondingcross-section.

By using PDMS wrinkles as stamps, the spacing between the protein linesdecreases to nanometer scale. Additionally, the spacing is easily tunable by thewavelength of the wrinkled stamp, and expensive lithographic master productionbefore the stamp casting is redundant. Fery et al. found limits below whichstamping failed. The critical stamp dimensions are 40 nm in amplitude and 335 nmin wavelength, that is, below an aspect ratio A/λ of 0.11, μCP is not successful.Analysis of cross-sections from AFM images shows a mean height of 6–7 nm ofthe printed structure for stamp amplitudes exceeding 80 nm (λ ≥ 435 nm). Smallerstamp dimensions reduce the pattern height to zero, leading to disappearance of thefeatures. With reduced stamp dimensions, the protrusions of the stamp approacheach other accompanied by a loss in height/amplitude. Consequently, duringprinting, more protrusions contact the substrate and ink from the protrusions, andthe wrinkle grooves are transferred.


0 1 2 3 4 5

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ghit

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ile o

f prin

t (nm

) (b)

1μm

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Figure 1.10 (a) AFM height image of printed BSA on a poly(ethylene imine) (PEI) acti-vated glass surface. (b) Height profile of the printed BSA structures averaged perpendicularto the obtained pattern over all cross-sections is found to be 8 nm. Source: Reprinted withpermission of American Chemical Society, Copyright 2008 [57].

As the hydrophobic PDMS surface is hydrophilized during plasma treatment,aqueous solutions are printable and the process is applicable to proteins or biologicalmaterials in general.

1.3.2.3 Microcontact Printing with Porous StampsBesides stamps produced by casting photolithographic masters and wrinkledstamps, porous stamps gained increasing attention for structuring dendrimers,nanoparticles [58], and proteins [58, 59]. Huskens et al. [58] produced porousstamps by one-step phase separation micromolding. Phase separation micromold-ing is a microfabrication technique to structure block copolymers, conductive, andbiodegradable polymers under ambient conditions. The method takes advantageof the phase separation of polymer solutions occurring in contact with a struc-tured mold. The microstructured polymer replica exhibits porosity. Figure 1.11shows SEM images of porous stamps consisting of poly(etherimide) (PEI),poly(vinylpyrrolidone) (PVP), and poly(ethersulfone) (PES). N-methylpyrrolidone(NMP) serves as solvent for the polymers during stamp fabrication. Water-solublePVP renders the stamp hydrophilic and provides a connected pore network withoutfurther modifications. Polymer composition and polymer concentration influencethe degree of porosity and pore morphology.

The pores showing a maximum size of 2 μm act as ink reservoirs. Owing totheir dimensions, proteins adhere only to the outer surface of common PDMSstamps. During printing, the larger part of the adsorbed protein remains on thesubstrate making reinking necessary after every printing step. A porous stampovercomes this drawback. Huskens et al. [58] performed multiple printing stepswith porous PES/PVP stamps and fluorescently labeled human immunoglobulinas ink. Proteins are trapped inside the pores of the stamp and subsequentlytransferred to the substrate upon conformal contact. Three consecutive printingcycles provide protein patterns with good quality.


(a) (b)

2 μm

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Figure 1.11 (a, b) Scanning electron micro-graphs of porous stamps replicated from asilicon mold with 20 μm wide lines: Surfaceand cross-section images of PEI/PVP (in so-lution PEI:PVP:NMP = 18%:12%:70% w/w).(c–e) Fluorescence microscopy images (scalebars indicate 40 μm) and intensity profiles(insets) of HIgG–Fe (10–5 M, phosphate

buffer saline (PBS) buffer) printed on N-[3-(trimethoxysilyl)propyl]ethylenediamine(TPEDA) functionalized glass slides in firstto third prints using an oxidized PDMSstamp. Printing time was 5 min, and noreinking was used [58]. Source: Reprintedwith permission of American Chemical So-ciety, Copyright 2009.

Besides fabrication of porous polymeric stamps, modification of PDMS stampswith porous polyelectrolyte multilayers employing layer-by-layer (LbL) depositionwas performed [59]. Post-treatment with base and cross-linking leads to porousstamps with pores acting as ink reservoirs. The polyelectrolyte multilayers consistof alternating layers of poly(4-vinylpyridine) (P4VP) and poly(acrylic acid) (PAA).The group studied multiple printing and the stability of the multilayer architectureduring printing. The pore diameter ranges from several tens of nanometers to200 nm. Contact angle measurements proved the stamp to be hydrophilic andtherefore suitable for multiple printing steps of aqueous biological samples such asfluorescently labeled immunoglobulin. Cross-linking of the porous stamp structure,obtained by dissolution of PAA with base, is crucial as otherwise the film is partiallyor fully transferred onto the substrate upon μCP. BrC3H6Br acted as cross-linkerin the gas phase and bound the multilayer covalently to the PDMS.


1.3.2.4 Enhanced Microcontact PrintingIn this section, we focus on modified μCP processes utilizing porous surfaces,reactive μCP, and affinity microcontact printing (αCP). Additionally, we showexamples for bioparticle assembly on printed surface patterns.

Zhang et al. [60] developed nanoporous silica surfaces for enhanced μCP ofproteins. They prepared silica thin films on solid substrates via spin coating.Triblock copolymers act as directing agents and create porosity in the films. Theporous layer is biocompatible resulting in minimal protein damage during printing.Conventional chemical surface modifications interact electrostatically/covalentlywith protein surface groups enhancing adsorption. Porous silica surfaces leadto more complete protein transfer compared to chemically modified surfaces.Especially, silica with a pore size similar or smaller than the dimensions of theprotein yielded effective protein transfer concerning pattern completeness, proteinlayer thickness, and roughness. Protein layers on porous silica are thicker andmore uniform compared to modified and untreated substrates. As protein functionis retained after immobilization, porous silica serves as a basis for patternedimmunoassays. Incubation with a secondary protein leads to deposition of thisprotein exclusively in areas of the primary protein [60].

With reactive μCP, chemical reactions are induced when ink is printed on asubstrate, even when the reaction partners are unreactive under standard conditions[61]. On one hand, a feature of μCP is the short contact time necessary to forma dense monolayer of ink on the substrate, while on the other hand, it takeshours to prepare SAMs by adsorption from solutions. So the question was raisedas to whether μCP is useful for acceleration of surface reactions. Generally, μCPreactions follow the rules of ‘‘click chemistry’’ showing high yields, mild conditions,and short reaction times. The scope of reactions ranges from condensations tocycloadditions, nucleophilic substitutions, and deprotections. The reactions benefitfrom several effects. Nanoscale confinement results in concentrated reagents in thecontact area of stamp and substrate (concentration effect). The preorganization effectof one reacting group constrained and aligned on a surface accelerates the reaction.Reactions benefit from the pressure effect during conformal contact between stampand substrate. The reactions are influenced by the micropolarity of the contact area(medium effect) [61]. An example for reactive μCP is the immobilization of peptidenucleic acids (PNAs) on aldehyde-functionalized surfaces [62]. Aldehyde groupson the substrate react with amino derivatives of the PNAs under slight pressureon the stamp. Reduction of the imine bond between PNA and substrate enablestesting of the hybridization properties of the patterns with oligonucleotides. PNAsrecognizing mutations in DNA are applicable to further biosensor developments.

While μCP transfers particles from stamp to substrate in dry state, αCP [40]transfers proteins from solution to solid surfaces. Probe proteins are immobilizedon a stamp to capture target proteins from protein solutions. If a probe proteinrecognizes a target protein, a complex forms on the stamp. Subsequently, thestamp is brought into contact with a substrate, resulting in three scenarios afterstamp removal (Figure 1.12):


Remove stamp

αCP

(a) (b) (c)

Figure 1.12 Possible scenarios of αCPwhen the stamp is peeled off the solid sur-face: (a) breaking the interaction betweenprobe and target protein, only the target pro-tein is transferred onto the solid surface.(b) Breaking the interaction between thestamp and the protein complex, the entire

protein complex is transferred onto the solidsupport. (c) Breaking the interaction be-tween protein complex and the support,the target protein remains on the stampsurface. Source: Reprinted with permissionof American Chemical Society, Copyright2011 [40].

Depending on the interaction strength among protein complex and support,protein complex and stamp, as well as target protein and probe protein, either thetarget protein (Figure 1.12a) or the protein complex (Figure 1.12b) is transferred orno transfer takes place (Figure 1.12c). Therefore, exact control of protein surfaceinteraction is indispensable and easily obtained by changing the surface propertiesof stamp and substrate (e.g., by chemical modifications).

Besides direct patterning of protein via μCP, it is important for the prepara-tion of biochemical assays to preserve protein function and steric accessibility.The main drawback of μCP lies in protein denaturation upon adsorption on hy-drophobic PDMS stamps, drying, and stamping [63, 64]. Martinez et al. [65] exploitSNAP-tag protein functionalities to overcome the problems. They developed aSNAP–FLAG–HIS 10 (SFH) cassette with three tags (SNAP, FLAG, and HIS-tags)useful for fluorescence labeling or surface immobilization. SNAP-tag, a mutant ofa DNA repair protein, preserves functionalities of fused proteins. Patterned SFHacts as immobilization vehicle for patterning of any protein expressed in the SFHcassette. Successful immunostaining demonstrates the versatility of SFH for gentlehigh-resolution patterning.

Hlady et al. [66] patterned fibrinogen for controlled platelet adhesion and activa-tion by μCP. Platelets, cellular fragments circulating in blood, form clots at vascularinjuries. When platelet activation occurs on devices, clotting causes complicationsor failure. If a device contacts blood, plasma proteins adsorb on the surface deter-mining the platelet response. This motivated Hlady et al. to immobilize randomfibrinogen patterns on reactive substrates by μCP. Varying size and extension ofthe fibrinogen islands control the overall adhesion and activation of the platelets.Also platelet morphology is influenced by the underlying fibrinogen pattern. Theapproach is applicable to other physiologically relevant protein models to investi-gate the mechanisms of platelet adhesion and activation on synthetic materials andimprove medical devices.


1.4Conclusion and Outlook

More sophisticated interface-based systems are being prepared with proteins asthe active species inducing and introducing new surface confined properties andphenomena. The ability of proteins to stabilize, recognize, and alter interfaces whileretaining their native properties is crucial for the success of the development ofnew systems. While the structuring of the protein layer at liquid–liquid interfacesis determined by the assembly properties of the protein itself, at solid–liquidinterfaces, this can be better controlled via soft printing techniques. Althoughthe deposition on solid interfaces can be controlled and directed using specificinteractions between a protein and the surface, structures at liquid interfaces canpossibly be directed as well in combination with genetic engineering. As wasmentioned, different classes of hydrophobins assemble differently at interfaces,which can potentially be redirected by blocking certain sites to prevent rodletformation or add additional structures to enhance film stability. Some examples ofthis have already been shown with other protein structures in solution [1].

Improved methods for protein deposition, the better controllable structuralfeatures of stamps used in different soft printing techniques, protein engineering,and use of native catalytic or chemical properties will provide new and interestingmaterials and devices; especially when the different disciplines associated with thevarious aspects of these biologically inspired hybrid materials (chemistry, biology,materials science, and physics) cooperate and communicate efficiently across theborders of their single disciplinary competences.

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1 Proteins for Surface Structuring COPYRIGHTED MATERIAL · 1 Proteins for Surface Structuring Alexander Schulz, Stephanie Hiltl, Patrick van Rijn, and Alexander B¨oker 1.1 Introduction