sign in sign up
<<
Home , Biomimetics

ARTICLE IN PRESS

Phil. Trans. R. Soc. A 1 doi:10.1098/rsta.2009.0018 2 3 4 5 R EVIEW 6 7 8 Molecular biomimetics: and 9 bionanotechnology using genetically 10 11 engineered peptides 12 1,3,* 1,2 13 BY CANDAN TAMERLER AND MEHMET SARIKAYA 14 1Genetically Engineered Materials Science and Engineering Center, and 15 2Department of Materials Science and Engineering, University of Washington, 16 Seattle, WA 98195, USA 17 3MOBGAM and Molecular -Genetics, Istanbul Technical University, 18 34469 Istanbul, Turkey 19 20 Nature provides inspiration for designing materials and systems, which derive their 21 functions from highly organized structures. Biological hard tissues are hybrid materials 22 having both inorganics within a complex organic matrix, the molecular scaffold 23 controlling inorganic structures. Biocomposites incorporate both biomacromolecules 24 such as , lipids and polysaccharides, and inorganic materials, such as 25 hydroxyapatite, silica, magnetite and calcite. The ordered organization of hierarchical 26 structures in organisms begins via the molecular recognition of inorganics by proteins 27 that control interactions and followed by the highly efficient self-assembly across scales. 28 Following the molecular biological principle, proteins could also be used in controlling 29 materials formation in practical engineering via self-assembled, hybrid, functional 30 materials structures. In molecular biomimetics, material-specific peptides could be the 31 key in the molecular engineering of biology-inspired materials. With the recent 32 developments of nanoscale engineering in physical sciences and the advances in 33 , we now combine genetic tools with synthetic nanoscale constructs 34 to create a novel methodology. We first genetically select and/or design peptides with 35 specific binding to functional solids, tailor their binding and assembly characteristics, 36 develop bifunctional peptide/ genetic constructs with both material binding and 37 biological activity, and use these as molecular synthesizers, erectors and assemblers. 38 Here, we give an overview of solid-binding peptides as novel molecular agents coupling 39 bio- and nanotechnology. 40 Keywords: bioinspiration; material-specific peptides; molecular recognition; 41 biological materials ; binding and assembly; bionanotechnology 42 43 44 45 46 * Author and address for correspondence: Department of Materials Science and Engineering, 47 University of Washington, Roberts Hall, Box 352120, Seattle, WA 98195, USA ([email protected]). 48 49 One contribution of 9 to a Theme Issue ‘Biomimetics II: fabrication and applications’.

1 This journal is q 2009 The Royal Society

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 2 C. Tamerler and M. Sarikaya

50 1. Introduction 51 52 (a ) Inspiration and lessons from biology 53 Nature provides inspiration for engineering structural and processing design 54 criteria for the fabrication of practical materials to perform ’s functions 55 Q8 56 (Sarikaya et al. 1990; Sarikaya 1994; Mann & Calvert 1998). During the last two Q9 57 decades, the realization that nanoscale inorganic materials have interesting 58 physical characteristics based on their nanometre-scale size (1–100 nm)-driven 59 promises and expectations from nanotechnology with potential applications in 60 both engineering and medical systems. Although there have been significant 61 advances in the applications of nanotechnology, there have also been serious 62 limitations mostly based on the problems associated with the assembly of 63 nanoscale objects. These stem from the limitations in nanotechnological systems 64 in controlling surface forces, inability to synthesize homologous sizes or shapes, 65 and limitations in their higher scale, controlled organizations. In biological 66 systems, on the other hand, inorganic materials are always in the form of 67 nanometre-scale objects, which are self-assembled into ordered structures for full 68 benefits of their function, that derive from their controlled size, morphology and 69 organization into two- and three-dimensional constructions. Recently, this 70 realization, therefore, brought biomimetics back into the forefront for renewed 71 inspiration for solving nanotechnological problems (Sarikaya 1999; Ball 2001; 72 Q10 Seeman & Belcher 2002). Biological materials are highly organized from the 73 molecular to the nano-, micro- and the macroscales, often in a hierarchical 74 manner with intricate nanoarchitectures that ultimately make up a myriad of 75 different functional elements, soft and hard tissues (Alberts et al. 2008). Hard 76 tissues such as , dental tissues, spicules, shells, bacterial nanoparticles are 77 examples that all have one or more protein-based organic components that 78 control structural formation as well as become an integral part of the biological 79 Q11 composites (Lowenstam & Weiner 1989; Sarikaya & Aksay 1995). These include 80 slaffins and silicateins in silica-based structures, amelogenin in enamel and 81 morphogenesis proteins or collagen in mammalian bone-, calcite- or aragonite- 82 forming proteins in mollusc shells and magnetite-forming proteins in magneto- 83 Q12 tactic bacteria (Berman et al. 1988; Cariolou & Morse 1988; Schultze et al. 1992; 84 Paine & Snead 1996). The inorganic component could be of various types of 85 materials (traditionally called ‘minerals’) with highly regular morphologies and 86 three-dimensional organizations. These include piezoelectric aragonite platelets 87 in (figure 1a), precipitation-hardened single-crystal calcite with a complex 88 architecture in sea urchin spines (figure 1b), optically transparent silica layers in 89 sponge spicules (figure 1c) and superparamagnetic nanoparticles in magnetotac- 90 tic bacteria (figure 1d ). 91 The types of inorganics chosen by the organism have precursors or raw 92 ingredients that are either in the soil, water or air that can relatively easily be 93 accessed to (Lowenstam & Weiner 1989; Sarikaya & Aksay 1995; Mann 1996). 94 In addition to the intrinsic physical properties, the overall function and 95 of the biological material, therefore, is derived by the high degree 96 of control that the organisms have over the formation of the structure of the 97 material produced. The traditionally used term, ‘’, therefore, is 98 misnomer, as the inorganics produced are not minerals but are materials with

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 3

99 (a) organic (c) 100 matrix CaCO 101 3 SEM 102 103 SEM 104 TEM 105 200 µm 106 10 nm 250 nm 107 5–10 µm 108 109

110 organic arganite platelets 111 thin film matrix (d) TEM 112 (b) SEM 113 114 115 116 a 200 µm 117 118 TEM HREM 119 120 121 µ 8 nm 50 nm 122 b 50 m 123 Q1 Q2 Figure 1. Examples of biologically fabricated, hierarchically structured (proteinCinorganic solid) 124 Q3 hybrid, functional nanomaterials. (a) Layered nanocomposite: growth edge of nacre (mother-of-pearl) 125 of abalone (Haliotis rufescens). Nacre is made of aragonite platelets separated by a thin film of 126 organic matrix. (b) Sea urchin spine is a single-crystal calcite with complex architecture containing 127 internal nanometre-scale MgCO3 precipitates. (c)Spongespicule(Rosella) is an optical fibre made of 128 layered amorphous silica with the central proteinaceous core. The apex of the spicule is a star-shaped 129 lens, a light collector. (d ) Magnetotactic bacteria (Aquaspirillum magnetotacticum) contain 130 superparamagnetic magnetite (Fe3O4) particles aligned to form a nano-compass that senses the 131 Earth’s magnetic field. 132 133 134 ‘unique’ architectures with detailed micro- and nanostructures, including the 135 defect structures such as dislocations and mechanical or crystallographic twins, 136 all specific to the organism that is producing them (Sarikaya 1994). For example, 137 even in the case of mother-of-pearl, each of the organisms, e.g. pinctada, nautilus 138 or abalone, producing it has different single-crystal aragonite platelets that are 139 different from each other and each different from that of geological aragonite 140 single crystal, both in term of the crystal itself, morphology and, more 141 significantly, intrinsic physical property, such as elastic modulus. From this 142 point of view, these materials fabrication processes could be called biomater- 143 ialization to give the true meaning to the biological processes. The biological 144 processing or fabrication (different from bioprocessing or biomimetic processing) 145 is accomplished at ambient conditions of (near) room temperature, pH 146 approximately 7.0 and in aqueous environments (Lowenstam & Weiner 1989; 147 Coelfen & Antonietti 2008).

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 4 C. Tamerler and M. Sarikaya

148 As we see in figure 1a, nacre has a brick-and-mortar architecture that is a 149 layered segmented aragonitic (orthorhombic CaCO3) tiles separated by an organic 150 matrix. The organics is in the form of a 10 nm or thinner film that contains both 151 proteins and polysaccharides, such as . Either within the layer or on the 152 surface of the organic film or within the particles themselves, the proteins possibly 153 nucleate the inorganic, aragonite, establish its crystallography and control the 154 growth. The resultant architecture, mother-of-pearl, is one the most durable hybrid 155 composites with excellent specific toughness/strength combinations (Mayer & 156 Q13 Sarikaya 2002). In figure 1b, sea urchin spines are single crystals of calcites 157 (rhombohedral CaCO3) with complex architectures. The spicule has high 158 toughness and elastic modulus, unusual for a mineral calcite. Despite its single 159 crystallinity, excellent mechanical property combinations in the spicule is probably 160 due to the presence of nanoscale MgCO3 precipitates, of which each associated with 161 a strain field, toughening the, otherwise, brittle calcite matrix through microcrack 162 closure (H. Fong & M. Sarikaya 2008, unpublished data). Both the formation of 163 the complex architecture of the calcite and the presence of precipitates must, again, 164 be due to the control that proteins have over these essential structural formations. 165 Another example (figure 1c), the spicules of the sponge species, Rosella are known 166 to have excellent light collection (via the lens-shaped tip) and transmission (via the 167 stem) properties with interesting layered structure made up of non-crystalline silica 168 (Sarikaya et al.2001), all controlled by the silica-binding proteins known as 169 silicatein (Morse 1999; Muller 2001). Finally, in magnetotactic bacteria (figure 1d), 170 superparamagnetic single particles of magnetite (Fe3O4) form a string of particles 171 aligned to sense the Earth’s magnetic field, aligning the bacteria and directing its 172 motion via magnetotaxis (Frankel & Blakemore 1991). Each of the magnetite 173 particles forms within a proteinacous magnetosome membrane, a component of 174 which directs the magnetite formation (Sakaguchi et al.1993). 175 In each of the examples above, through materialization, the resultant hybrid 176 composite structures, incorporating inorganic and proteinaceous components, are 177 organized at the nanometre and higher dimensions, resulting in viable 178 mechanical, magnetic and optical devices and each offer unique design, not yet 179 seen in man-made engineered systems. These functional biological systems are 180 simultaneously self-organized, dynamic, complex, self-healing and multifunc- 181 tional, and have characteristics difficult to achieve in purely synthetic systems 182 even with the recently developed bottom-up processes that use molecules and 183 nanocomponents. Under genetic control, biological tissues are synthesized in 184 aqueous environments in mild physiological conditions using biomacromolecules, 185 primarily proteins but also carbohydrates and lipids. Proteins both collect and 186 transport raw materials, and consistently and uniformly self- and co-assemble 187 subunits into short- and long-range ordered nuclei and substrates (Tamerler & 188 Sarikaya 2007). Whether in controlling formation or being an integral part 189 of the tissue in its biological functions and physical performance, proteins are an 190 indispensable part of the biological structures and systems. A simple conclusion 191 is that any future biomimetic system, whether for or nanotech- 192 nology, should include protein(s) in its assembly and, perhaps, in its final hybrid 193 structure (Sarikaya et al. 2003). 194 In traditional materials systems, the final product is a result of a balance of 195 interactions, dictated by the kinetics and thermodynamics of the system, that are 196 often achieved through ‘heat-and-beat’ approaches of the traditional materials

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 5

197 science and engineering, which provide the energy for structural formations 198 (Kingery 1976; Reed-Hill 1991). In biological systems, on the other hand, the 199 same balance, and the energy, is achieved through evolutionary selection 200 processes that result in the emergence of a specific molecular recognition using 201 peptides and proteins (Pauling 1946). As we discussed below, and throughout the 202 paper with examples, our approach is to engineer peptides with materials 203 selectivity and use these as molecular building blocks in organizing functional 204 materials systems in practical proof-of-principle demonstrations. Availability of 205 new platforms will bring to the forefront new materials functionalities provided 206 by the solid-binding peptides that will extend current technology via coupling 207 nanoentities using the principles of biosorption beyond those provided by the 208 traditional chemisorption or physisorption. 209 210 (b ) Molecular biomimetics pathways to nano- and bionanotechnology 211 212 Molecular biomimetics is using biology’s molecular ways in genetic selection or 213 design of proteins and peptides that can control the synthesis of nanoscale 214 objects and self-assembly of higher ordered multifunctional materials systems 215 (Sarikaya et al. 2003). In the development of the molecular biomimetics protocols 216 in nanotechnology, therefore, one uses solid-binding peptides and control the 217 formation, assembly and organization of functional nanoentities towards building 218 useful technologies. To accomplish the overarching task, we integrate recent 219 developments in molecular- and nanoscale engineering in physical sciences 220 (nanoparticle formation, nano- and micropatterning such as dip-pen nanolitho- 221 graphy and microcontact printing, and self and directed assemblies), and the 222 advances in molecular biology, genetics and towards materials 223 fabrication all at the molecular and nanometre scales (Sarikaya 1999; Sarikaya 224 et al. 2003). Using closely controlled molecular, nano- and microstructures 225 through molecular recognition, templating and self-assembly properties in 226 biology, this field is evolving from the true marriage of physical and biological 227 sciences towards providing practical application platforms (Niemeyer 2001; 228 Sarikaya et al. 2004). The advantage of the new approach for nanotechnology is 229 that inorganic surface-specific proteins could be used as couplers, growth 230 initiators and modifiers, bracers and molecular erector sets, i.e. simply as 231 building blocks for the self-assembly of materials with controlled organization 232 and desired functions from the bottom-up. 233 The realization of heterofunctional nanostructure materials and systems could 234 be at three levels (Sarikaya et al. 2004), all occurring simultaneously with a 235 closely knit feedback similar to the biological materials formation mechanisms 236 (Alberts et al. 2008). The first is that the inorganic-specific peptides are identified 237 and peptide/protein templates are designed at the molecular level through 238 directed evolution using the tools of molecular biology. This ensures the 239 molecular scale and up processing for nanostructural control at the lowest 240 practical dimensional scale possible. The second is that these peptide building 241 blocks can be further engineered to tailor their recognition and assembly 242 properties similar to the biology’s way of successive cycles of mutation and 243 generation can lead to progeny with improved features eventually for their usage 244 as couplers or molecular erector sets to join synthetic entities, including 245 nanoparticles, functional or other nanoentities on to molecular

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 6 C. Tamerler and M. Sarikaya

246 templates (molecular and nanoscale recognition). Finally, the third is that the 247 biological molecules self- and co-assemble into ordered nanostructures. This 248 ensures an energy-efficient robust assembly process for achieving complex 249 nano, and possibly hierarchical structures, similar to those found in biology 250 (self-assembly; Sarikaya et al. 2004). 251 Q14 In the following sections, we provide an overview of molecular biomimetics 252 approaches to achieve the premises of bionanotechnology with specific 253 applications, mostly, in medicine, and summarize their potentials and 254 limitations. Here, we first summarize the protocols, adapted from molecular 255 biology to materials science and engineering, for selecting polypeptides that 256 recognize and bind to solids, and describe the protocols of combinatorial biology 257 for identifying, characterizing and genetically engineering peptides for practical 258 use. We emphasize surface and phage display approaches that are well 259 adapted for the identification of solid material-specific peptides and to explain 260 ways to further tailor peptides using post-selection engineering and bioinfor- 261 matics pathways. The protocols, established over years in this group, are 262 presented in the quantitative binding characterization of the peptides using 263 various spectroscopic techniques. We also briefly discuss possible mechanisms 264 through which a given peptide might selectively bind to a material. Finally, we 265 present extensive practical examples of current achievements in the usage of the 266 solid-binding polypeptides as building blocks to demonstrate their wide range of 267 applications and, finally, discuss future prospects. 268 269 2. Genetic selection and directed evolution of solid-binding peptides 270 271 (a ) Biocombinatorial selection of peptides 272 273 Genetically engineered peptide for inorganics (GEPI) is selected through affinity- 274 based biopanning protocol (Sarikaya et al. 2003). Biopanning steps consist of 275 contacting the library with the material of interest, then washing out weak or 276 non-binders and repeating the process to enrich for tight binders to select a 277 subset of the original library exhibiting the ability to tightly interact with the 278 desired surface. During the biopanning step, a minimum of three to five cycles of 279 enrichment is usually performed. Generally in early rounds, low-affinity binders 280 can be accessed if the selection is performed under mild conditions. In later 281 rounds, as the conditions get harsher, tight binders are also recovered. Because 282 the chimera is encoded within the phage genome or on a carried by the 283 cell, the identity of the selected sequences (e.g. their compositions) 284 can be deduced by DNA sequencing (figure 2). 285 We selected peptides for a variety of materials including noble metals (such as 286 Au, Pt and Pd), metals (Ag and Ti), oxide and nitride 287 (e.g. Cu2O, ITO, GaN, ZnO), minerals (such as mica, hydroxyapatite, calcite, 288 aragonite, sapphire and graphite) or biocompatible substrates (such as silica, 289 titania and alumina) that were selected by using either phage display 290 (specifically filamentous phage strain M13) or cell surface display (specifically 291 flagellar display) (Sarikaya et al. 2004). There are also a number sequences 292 selected for various materials by other groups. The ones selected via cell surface 293 display includes (Brown 1997) and zinc oxide (Kjærgaard et al. 2000), 294 whereas phage display selected ones are for their affinity towards gallium

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 7

295 296 297 selection 298 engineering 299 300 301 302 first generation peptides 303 computational 304 biomimetics 305 306 polypeptide binding 307 characteristics 70 6.0 OCM SPR 308 60 5.0 50 3.9 µg/ml 4.0 2.2 µg/ml genetic design 40 309 3.0 30 and tailoring 20 2.0

310 signal (nm) 10 1.0 311 0 20 40 60 80 0 500 1500 2500 frequency difference (Hz) difference frequency time (min) time (min) 312 313 molecular second generation 314 constructs polypeptides 315 316 317 318 engineered 319 peptide constructs Huntington antibody 320 321 bioscaffold fibrillation 322 engineering AuBP 323 designer proteins 324 gold 325 protein-based nano- and 326 nanobio-technology 327 328 Figure 2. Standardized steps in the selection, binding characterization, designing/tailoring of solid- 329 Q15 binding peptides and their usefulness as bifunctional molecular constructs. 330 331 332 arsenide (Whaley et al. 2000), silica (Naik et al. 2002a), silver (Naik et al. 333 Q16 2002b), zinc sulphide (Lee et al. 2002a,b), calcite (Li et al. 2002), cadmium 334 sulphide (Mao et al. 2003) and titanium oxide (Sano et al. 2005). Some of 335 biocombinatorially selected peptides have been used to assemble inorganic 336 particles (Whaley et al. 2000; Lee et al. 2002a,b; Mao et al. 2003) or to control 337 nucleation of the compounds that they were selected for (Li et al. 2002; Naik 338 et al. 2002a,b). 339 When one is focusing on the material-specific peptide interactions, finding a 340 consensus sequence might lead to a misleading result. This could be due to the 341 high potential that a genetic bias in the selection by the organism may produce 342 the same sequence without the diversity. As it is well known, the health of 343 genetic diversity leads to an assortment of sequences, which presumably reflects

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 8 C. Tamerler and M. Sarikaya

344 the heterogeneity of the inorganic substrates at the atomic, topographic, 345 chemical and crystallographic levels. Chemical diversity of the surfaces alone 346 could produce a variety of sequences due to the different binding strategies that 347 the peptide library could entail that are derived from the shape and lattice 348 complementarities, electrostatic interactions, van der Waal’s interactions or 349 various combinations of these mechanisms (Kulp et al. 2004; Evans et al. 2008; 350 Seker et al. in print). The ultimate robust usage of the inorganic-binding 351 peptides for the fabrication and assembly of hybrid materials and systems 352 requires fundamental studies towards better insights into peptide–solid 353 molecular interactions and their incorporation into the design of desired 354 material-specific peptides. 355 356 (b ) Structural design concepts: mutation, multimerization, conformational 357 constraints 358 359 Both the amino acid content (chemistry) as well as the sequence of the amino 360 acids (molecular conformation) in a given selected set of peptides could affect their 361 binding characteristics. We have recently demonstrated that the molecular 362 constraints can be used to tune the architectural features and, consequently, the 363 binding properties of the first generation of selected peptides. Specifically, we used 364 a high-affinity 7-amino acid Pt-binding sequence, PTSTGQA, to build two 365 different constructs: one is a Cys–Cys constrained ‘loop’ sequence (CPTSTGQAC) 366 that mimics the domain used in the pIII tail sequence of the phage library 367 construction, and the second is the linear form, a septapeptide, without the loop 368 (Seker et al.2007). By incorporating surface plasmon resonance (SPR, measuring 369 binding) and circular dichroism (CD, determining molecular architecture), one is 370 able to analyse the consequence of the loop constraint on peptide adsorption and 371 kinetics and the conformation of peptides. These studies are related to each other 372 with a comparative approach (as determined in figure 2). 373 One may also modify the binding activity of a given selected peptide by simply 374 increasing the number of repeats of the original sequence. This multimerization 375 could be accomplished using the simple tandem repeat, i.e. sequential 376 attachment of the original sequence. We applied multiple-repeat-based strategy 377 on both phage display selected and quartz binder (7 and 12 amino acid 378 sequences each, respectively) and cell surface selected gold binders (14 amino 379 acids each). One would expect that, as the number of repeats increased, there 380 would be an increase in the binding activity of a given peptide. Surprisingly, 381 however, not in all cases, the increase in the number of repeating peptide was 382 reflected in the enhancement of binding activity. In addition, material selectivity 383 behaviour of each of the single peptides also changed when they were used in 384 multiple-repeat forms. These results indicate that, rather than the amino acid 385 content in a given material-binding sequence, it is the molecular conformation 386 (secondary structure) that is more relevant, which dictates the solid-binding 387 function. These preliminary results, therefore, show that there is a correlation 388 between conformational instability (or adaptability) and binding ability (Seker 389 et al. in print). It is imperative that, in the next stage of multimerization studies, 390 one could incorporate designed linkers between successive sequences to 391 intentionally conform the overall multiple-repeat second-generation peptides 392 for desired binding and other biological functions.

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 9

393 (c ) Binding and assembly of peptides on solids 394 In the design and assembly of functional inorganic solids, it is essential to 395 understand the nature of polypeptide recognition and binding on to solid materials. 396 Although considerable research has been directed in the literature towards 397 understanding peptide binding to solids, it is not yet clear how proteins recognize 398 an inorganic surface and how it could be manipulated to enhance or reduce this 399 binding activity. This problem is similar to protein–protein recognition in biology 400 (Pauling 1946); in the current hybrid systems, the problem reduces to one of 401 peptide–solid interface. Here, the peptide is relatively small, perhaps approximately 402 10 amino acids long (1 kD), and the inorganic solid is relatively flat but with 403 atomic and molecular features with mostly crystallographic lattice organization. 404 The specificity of a protein for a surface may originate from both chemical 405 (e.g. H-bonding, polarity and charge effects) and physical (conformation, size and 406 Q17 morphology) recognition mechanisms (Izrailev et al.1997;Daiet al. 2000; Evans 407 2003; Evans et al. 2008). Recent studies have also demonstrated that the peptide 408 overall molecular architecture (i.e. constraint versus linear) plays a key role in the 409 solid recognition (Hnilova et al.2008). For a given system, these mechanisms may 410 be all significant, but with varying degrees depending on the peptide sequence, 411 chemistry and topology of the solid surface, and the conditions of the solvent 412 (water). Therefore, each, with a certain degree, would contribute towards a 413 collective behaviour. Similar to the molecular recognition in biomacromolecular 414 systems, the major contribution, however, comes from amino acid sequences that 415 lead to a specific molecular conformation on the surface of the solid, and to a lesser 416 extent on composition and overall amino acid content of the peptide, as 417 demonstrated in the example below (see §2e). 418 419 (d ) Peptide binding to solids and kinetics 420 421 Among the experimental approaches to rapidly monitor the protein adsorption 422 and binding on inorganics is fluorescence microscopy (FM), which now become a 423 routine tool as a first step in the qualitative evaluation of these sequences with 424 respect to their affinity and selectivity (figure 3). The FM imaging is an essential 425 part of the screening protocol in our laboratory. However, this type of 426 characterization does not provide quantitative information of polypeptide 427 adsorption or detailed binding kinetics or mechanism(s). Another frequently 428 used technique in molecular biology binding assays is ELISA, an immuno- 429 fluorescence labelling detection using monoclonal antibody conjugated with 430 Q18 secondary antibody fragments (Brown 1992; Whaley et al. 2000; Naik et al. 2002; 431 Dai et al. 2004; Sarikaya et al. 2004). Although time consuming and statistically 432 less significant, scanning probe microscopy (SPM) protocols could also be used, 433 which require the integration of sample preparation, self-assembly, tip design, 434 observation conditions, data analysis and interpretations of specific polypeptides 435 binding on to inorganic surfaces (Whitesides et al. 1991). Both atomic force 436 microscopy (AFM) and scanning tunnel microscopy (STM) techniques have been 437 used to acquire static information of peptide binding to solids. The quantitative 438 data towards determining kinetic parameters of binding could, however, be 439 obtained using more established techniques such as quartz crystal microbalance 440 (QCM; Murray & Deshaires 2000; Bailey et al. 2002) and SPR 441 Q19 (Czenderna & Lu 1984; Homola et al. 1999).

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 10 C. Tamerler and M. Sarikaya

442 (a) Au (b) 443 Si/SiO2 444 Pt 445 100 µm 446 447 448 Au Pt 449 100 µm 450 IgG 451 (c) specific DNA antibody Au segment (d) Au 452 DNA Si 453 sequence zenon label reagent 454 displayed Pt binding Pt Pt 455 polypeptides Ti/Pt 456 SiO2 Si substrate 457 458 Q4 Figure 3. Material selectivity of inorganic-binding peptides (a) peptide alone, the case of quartz- 459 binding peptide conjugated with a fluorescein molecule and (b) peptide displayed on the host organism, i.e. Pt-mutant phage, PtBP1, fluorescently labelled. The contrast reversal, as visualized 460 using a fluorescence microscope in both cases, indicates the material specificity of QBP for silica 461 against Au or Pt, and PtBP for Pt against Si and Au, respectively. 462 463 Both QCM and SPR (figure 2) have been successively used to quantitatively 464 analyse peptide adsorption kinetics under various protein concentrations, solution 465 466 properties, such as pH and salinity, and solid surface conditions (Sarikaya et al. 2004; 467 Sano et al.2005; Seker et al. 2007; Hnilova et al. 2008). Recently, conventional 468 spectroscopy techniques, such as X-ray photoelectron spectroscopy and time- 469 of-flight-secondary ion mass spectroscopy techniques, have also proven to provide 470 the fingerprint of peptide adsorption on to the surfaces (Coen et al.2001; Suzuki et al. 471 2007). Although difficult to carry out, the application of solid and liquid state NMR 472 could provide quantitative information of molecular conformations of peptides, 473 essential information towards the understanding of the mechanism of polypeptide 474 binding on to solids (Evans 2003). Finally, molecular modelling that studies interface 475 interactions between a peptide and a solid will lead to rapid evaluations of various 476 types of hybrid interfaces. These studies, e.g. molecular dynamics, that make use of 477 computational chemistry, biology and physics, are still in their infancy, but are 478 expected to provide protocols in the near future through the implementation of model 479 experimental systems coupled with theoretical approaches (Evans et al.2008). 480 A detailed understanding of the peptide recognition and assembly processes 481 will inevitably lead to better insights into the design of peptides for tailored 482 binding. A better knowledge of the mechanisms of the quantitative adsorption 483 may become possible through high-resolution surface microscopy (e.g. AFM and 484 STM), molecular spectroscopy and surface diffraction studies as well (such as 485 small angle X-ray diffraction). Many of these techniques, with their advantages 486 and pitfalls, have been discussed extensively in the literature; in this review, we 487 will discuss one technique, SPR, which provides the most practical information 488 on binding kinetics and materials selectivity of peptides for solid and, therefore, 489 frequently used in our research in the identification of the most promising 490 peptides that are in frequent use today for practical implementations (§3).

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 11

491 (c) linear constrained 492 (a) 4 (i) 5 (ii) 2.00 µM 3 µ 4 493 2.00 M 0.92 µM 0.92 µM 2 3 494 0.46 µM 2 µ 1 0.46 M 495 0.23 µM 1 0.23 µM 496 0 123450 12345 4 (iii) 5 (iv) 497 2.00 µM 2.00 µM RC+PPII RC 3 4 µ I-AuBP1 c-AuBP1 498 µ 0.92 M K =K 2 0.46 µM 0.92 M 3 eq eq 2 499 dip position shift (nm) 1 µ µ 1 0.23 µM 0.46 M 500 0.23 M (d) linear constrained 0 123450 12345 501 time (min) time (min) 502 (b) 0.05 (i)0.05 (ii) 0.04 ) 0.04

503 –1 0.03 0.03

504 (S 0.02 0.02 obs

K 0.01 0.01 505 RC+PPII RC 506 0 0.5 1.01.5 2.0 2.5 0 0.51.0 1.5 2.0 2.5 I-AuBP2 c-AuBP2 concentration (µM) concentration (µM) Keq < Keq 507 508 Q5 Figure 4. Effect of GEPI conformation on binding, the case study with gold-binding peptides. 509 (a,b) SPR studies of the binding of AuBP1 and AuBP2, constraint and linear, respectively, are 510 given ((a) (i) l-AuBP1, (ii) l-AuBP2, (iii) c-AuBP1, (iv) c-AuBP2; (b) (i) diamonds, l-AuBP1; 511 squares, c-AuBP1; (ii) diamonds, l-AuBP2; squares, c-AuBP2). (c,d ) Molecular architectures of 512 the linear and constraint forms, respectively, are given ((c) AuBP1, (d ) AuBP2). Note that the 513 linear and constraint forms of AuBP2 have the same molecular conformation and, therefore, the same binding property while AuBP1 has two different conformations in two architectures and, 514 therefore, the binding strengths are different. 515 516 (e ) Peptide adsorption via molecular architectural control 517 518 Most studies on the adsorption behaviour of combinatorially selected 519 inorganic-binding peptides on to solids have focused mainly on their amino 520 acid compositions (Naik et al. 2002; Mao et al. 2003). Only recently some studies 521 have addressed the peptide structural constraints on the adsorption behaviour 522 Q20 and affinity to solids (Tamerler et al. 2006a,b; Makrodimitris et al. 2007; Seker 523 et al. 2007; Gungormus et al. 2008; M. Gungormus, D. Khatayevich, C. So, 524 C. Tamerler & M. Sarikaya 2008, unpublished data). It is well known in protein 525 engineering that the protein molecular architecture affects its function (Alberts 526 et al. 2008). In this example, we hypothesized that the structure–function 527 relationship also persists in peptide binding to inorganic materials (figure 4). 528 To assess the hypothesis, we used two gold-binding peptides that were originally 529 selected in a cyclic form, i.e. constraint architecture, and compared their 530 adsorption and conformational behaviours to those of their linear, free, forms 531 using, respectively, SPR and CD spectroscopy and computational modelling. We 532 used two gold-binding sequences that were originally selected using the FliTrx 533 cell surface approach (Hnilova et al. 2008). These two peptides, AuBP1 534 (WAGAKRLVLRRE) and AuBP2 (WALRRSIRRQSY), were synthesized 535 using solid-state technique in an open dodecapeptide version, called linear 536 (l ) as well as in constraint form, i.e. through an 18-aa Cys–Cys constrained loops, 537 called cyclic (c), to mimic the original FliTrx displayed peptide conformations. 538 We first carried out the CD spectroscopy to assess the molecular conformations 539 and found that the cyclic versions of AuBPs have mainly random coil structures;

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 12 C. Tamerler and M. Sarikaya

540 however, the linear versions of AuBPs also have some degree of polyproline type 541 II (PPII) rigid structures in addition to the random coil structures (Hnilova 542 et al. 2008). The percentage of PPII structure in l-AuBP2 is greater than that in 543 l-AuBP1, and, thus, the structural differences between the l- and c-versions of 544 AuBP2 are much bigger than the structural differences between the l- and 545 c-versions of AuBP1. 546 The SPR analysis showed that both the linear and cyclic forms of AuBPs have K1 547 Q21 high affinities to gold (e.g. DGadsZK8.7 kcal mol ). We also found that both 548 the linear and cyclic forms of AuBPs have random coil and PPII structures, 549 which cooperatively promote unfolded, conformationally labile peptides that 550 may enhance their adaptability to interfacial features that exist on gold surfaces. 551 One would expect differences in the binding characteristics between the cyclic 552 and linear forms as the structure may change. In fact, we found that AuBP2 has 553 an order of magnitude higher affinity in the cyclic version than the linear one 554 (figure 4). This difference is consistent with the observation of significant 555 structural change in the molecular conformations of the cyclic and linear versions 556 of AuBP2 in solution. On the other hand, the binding affinities of AuBP1 in the 557 cyclic and linear forms are quite similar. In this case, the molecular structures of 558 this peptide in the two architectures are similar, as we show both experimentally 559 (CD) and via modelling. On the basis of all the evidence, we show that the 560 sequence of the amino acids in a given peptide and its molecular conformation 561 may be the key determinants that facilitate peptide-selective binding on solid 562 Q22 materials (Hinlova et al. 2008). 563 564 3. Implementations of solid-binding peptides in bionanotechnology 565 566 Once a bank of fully characterized solid-binding peptides becomes available, then 567 it could be used as a ‘molecular toolbox’ for a wide range of applications from 568 solid synthesis to molecular and nanoscale assemblies. Here, the peptide is not 569 only be useful in linking one nanomaterial to another, but a GEPI could also be 570 used for genetically fusing it on to another functional protein and use the system 571 as bifunctional molecular construct, where peptide would be the ligand. 572 Alternatively, a GEPI could be fused, chemically, on to a synthetic , 573 to create multifunctional hybrid polymeric structures. Below, we will 574 demonstrate a few uses of various GEPIs in generating new functional materials 575 systems to understand their potential usage as molecular building blocks. 576 577 (a ) GEPI-assisted synthesis of nanoinorganics 578 579 Given that these genetically engineered peptides recognize and bind to 580 minerals, there may also be an inherent capability within the sequences to 581 influence the morphology of these minerals as well, a prospect that has not yet 582 been fully explored in great detail so far. Once this is achieved, peptide-based 583 molecular scaffolds developed may have great potential for applications in tissue 584 regeneration. An example from our recent work on biomineralization using 585 hydroxyapatite (HA)-binding peptides (Gungormus et al. 2008; M. Gungormus, 586 D. Khatayevich, C. So, C. Tamerler & M. Sarikaya 2008, unpublished data) is 587 shown in figure 5a,b. We demonstrated that the biocombinatorially selected 588 HA-binding peptides could offer a route for regulating calcium phosphate-based

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 13

589 (a) (b) 590 591 592 593 594 595 596 597 598 599 400 nm 600 601 (c) (d) 602 603 604 605 606 607 608 609 610 100 nm 611 612 Q6 Figure 5. Peptide-assisted biomaterialization using GEPIs. (a,b) Hydroxyapatite synthesis in the 613 presence of phage display selected HABP1 with respect to a control containing no peptide. 614 (c,d ) Au nanoparticle synthesis in the presence of AuBP with respect to a control prepared by a 615 non-specific peptide (a) control, (b) w/HABP1, (c) control, (d ) w/AuBP1. 616 617 nanocrystal formation within a biomedical context. Specifically, a successful 618 generation of cysteine-constrained M13 bacteriophage heptapeptide library were 619 screened against HA powder. Using the library, we selected 49 sequences and two 620 were identified for further investigation. One of these peptides exhibited the highest 621 binding affinity (HABP1), and the other, a much lower binding affinity (HABP2) to 622 HA, for subsequent calcium phosphate formation and biophysical characterization 623 studies. Here, we were interested in learning whether HA-binding polypeptide 624 sequences could also regulate calcium phosphate formation in vitro, and likewise, 625 determine the contributions of primary sequence and secondary structural 626 properties that are associated with HA affinity as well as calcium phosphate 627 formation capability. We found that both peptides affected calcium 628 phosphate formation, with the former exhibiting a higher inhibitory activity over 629 the latter, inducing a desired morphology on the formed Ca-phosphate mineral 630 (figure 5a). The resulting nanoparticles are plate shaped, several 10s of nanometres 631 in length and only a few nanometres in thickness. These particles resemble 632 hydroxyapatite particles in dentine in tooth (Fong et al. 2000). These results 633 reveal a possibility of peptides in controlling particle morphology that is the major 634 difference in differentiating the dental hard tissues (dentine, cementum and enamel) 635 as well as the bone architectures. Peptide-controlled morphogenesis of Hap 636 nanoparticles could be used in regulating materialization in hard-tissue regeneration 637 or filler design for tissue restoration.

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 14 C. Tamerler and M. Sarikaya

638 (a) (b) (d) (c) 639 (i) 640 (i) 641 QBP1-bio 642 50 µm 643 (ii) 644 (e) (ii) 645 646 (iii) 647 648 649 (iii) 650 651 652 653 (iv) 50 µm 654 655 Q7 Figure 6. (a–e) Targeted co-assembly of molecular functional entity (fluorescein attached to QBP1- 656 bio, silica-binding peptide) and nanoparticle (a QD) functionalized with streptavidin targeting 657 biotinylated QBP on a microcontact-printed micropatterned Si substrate (containing native silicon 658 oxide) (a)(i) incubation, (ii) washing and drying, (iii) stamping, (iv) incubation; (c) (i) SA-QD 659 micropattern, (ii) self-assembly of QBP1-F, (iii) fluorescein and QD micropatterns on quartz. 660 661 Another example is in the morphology control of gold particles using gold- 662 binding peptides (figure 5c,d ). Gold nanoparticles with 12 nm diameter monosize 663 can be formed at ambient conditions using the well-known Faraday’s technique 664 Q23 by reducing AuCl3 by Na-citrate (or other reducing agents; Turkevich et al. 665 1951). In the presence of peptide, reducing the gold concentration and lowering 666 temperature allow particle formation at a slower rate, giving the protein time to 667 interact with surfaces during the growth and provides conditions to examine the 668 effect of gold binding during colloidal gold formation. We conducted a search for 669 mutants that modulated the architecture, i.e. particle versus thin film, of gold 670 crystallites (Hnilova et al. 2008). The selection of mutants was based on the 671 change of colour of the gold colloid (from pale yellow to a red colloid), which was 672 related to altered rate of . Forty gold mutants were tested this 673 way, and the sequence analysis showed that two separate mutants that 674 accelerated the crystal growth also changed the particle shape from cubo- 675 octahedral (the usual shape of the gold particles under equilibrium growth 676 conditions) to flat, thin films (figure 6c,d ). This new observation is interesting 677 from the point of enzymatic effect of protein in crystal growth rather than 678 traditionally assumed templating effect. The polypeptides, in spite of being 679 slightly basic, may have caused the formation of gold crystals similar to those 680 formed in acidic conditions. This suggests that the role of the polypeptides in 681 gold crystallization is to act as an acid, a common mechanism in enzyme 682 function, and the protocol could be used to regulate the shape of metal 683 nanoparticles for photonic and electronic applications. 684 As demonstrated with the examples above, biocombinatorially selected 685 peptides can have enzymatic effects in the synthesis, morphogenesis and 686 fabrication of inorganic nanomaterials. Similar to biological systems, it may be

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 15

687 expected that the solid-binding peptides may have further potential for size, 688 crystallography and mineral selectivity, with potential usage in a variety of 689 practical applications, from filler material in papers to paints, as well as 690 specialized coatings (Sarikaya et al. 2004). 691 692 (b ) Directed and mediated assembly of functional nanoentities 693 694 Protein microarray technologies, used in proteomics and clinical assays, 695 require efficient patterning of biomolecules on selected substrates (Gristina 1987; 696 Blawas & Reichert 1998; Chicurel & Dalma-Weiszhausz 2002; Cutler 2003; 697 Min & Mrksich 2004; Cretich et al. 2006), which is possible provided that the 698 proteins are spatially immobilized on solid substrate via various lithography 699 techniques, e.g. soft lithography (Xia & Whitesides 1998), dip-pen lithography 700 (Lee et al. 2002a,b) and photolithography (Revzin et al. 2001). Recently, protein 701 immobilization has become a key issue in bionanotechnology since immobili- 702 zation provides physical support to the molecule, resulting in improved stability 703 and activity and, furthermore, helps to separate proteins from solution, 704 rendering them reusable (Castner & Ratner 2002; Bornscheuer 2003) The 705 approaches for biomolecule immobilization on glass or metal (e.g. gold) 706 substrates generally require surface functionalization by self-assembled mono- 707 layers (SAMs) of bifunctional molecules, such as amino-terminated aminoalkyl- 708 alkoxysilanes for silica and carboxyl-terminated alkanethiols for gold 709 substrates (Mrksich & Whitesides 1996; Ostuni et al. 1999). Despite their 710 widespread usage, these traditionally available linkers have certain limitations, 711 such as causing random orientation of the protein on solid surface and requiring 712 multistep chemical reactions and, furthermore, the assembled monolayers can be 713 unstable during immobilization (Fujiwara et al. 2006; Park et al. 2006). 714 To overcome these limitations, it is preferable to have molecules as direct 715 linkers to the solid substrate of interest, which not only have all the desired 716 features of the conventional chemically prepared SAMs but also have specificity 717 to a given solid substrate and assemble on to it efficiently. In addition, the 718 molecule used as the linker could be amenable to genetic manipulation for 719 selecting the best linker site to the displayed protein or nanoentity without 720 causing any effect in reducing the binding activity. Solid-binding peptides can 721 provide the multifunctionality as a preferred linker with high structural stability 722 incorporating a target molecule aligned consistently to carry out a desired 723 function (Sarikaya et al. 2003). 724 Here we demonstrate the solid-binding peptide as a molecular assembler for 725 two different nanoentities, quantum dots (QDs) and fluorescent molecules, and 726 sequentially assemble them on a micropatterned surface using the material 727 specificity of the GEPI (Kacar et al.inpress). In this case, directed 728 immobilization of the QDs is followed by the GEPI-mediated assembly of the 729 fluorescent molecule using the microcontact printing and self-assembly 730 procedures schematically illustrated in figure 6a. The directed immobilization 731 of SA-QD on a QBP1-biopatterned surface is shown in figure 6b as red stripes, 732 imaged with a fluorescent microscope using a QD605 filter, revealing red 733 fluorescent contrast. Here, the dark stripes represent the regions originally 734 unoccupied, exposing the bare quartz surface (figure 6a(i)). Next, following the 735 procedure in figure 6a, the assembly of the fluorescent molecule, i.e. fluorescein,

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 16 C. Tamerler and M. Sarikaya

736 is mediated using the QBP1-F molecular conjugate. The assembled conjugate 737 molecules are imaged in green, as shown in figure 6c, using a FITC filter. At this 738 step, the QBP1-F molecular conjugate diffuses towards the regions of the 739 substrate previously unoccupied, after the initial directed immobilization of QDs 740 (figure 6d ). Both images in figure 6e,c were recorded from the same area of the 741 sample, showing regular alternating lines of red and green stripes, corresponding 742 to the directed-assembled QDs and mediated-assembled fluorescein molecules, 743 respectively. This result demonstrates that the QBP1 is active as an efficient 744 molecular linker as well as a versatile PDMS ink. Furthermore, we demonstrate 745 here the co-assembly of two diverse nanoentities without the involvement of 746 complex surface modification, often involved in the silane-based traditional 747 procedures (Fujiwara et al. 2006). The patterning protocol developed here would 748 be useful as microscale platforms for wide range of applications from generating 749 photonic lattices to co-assembling multi-enzyme or multi-protein assays. 750 751 752 4. Future prospects of solid-binding peptides as molecular building 753 blocks in bionanotechnology 754 755 The joining of biology with materials requires an ability to design, engineer and 756 control interfaces at the materials/bio intersections as these sites are significant 757 in the implementation of nanotechnology, developments of new materials and 758 protocols in molecular engineering, and realization of bionanotechnology 759 (figure 7). Biology controls all interfaces between molecular materials, tissues 760 and organs using peptides and proteins which are also the agents of molecular 761 communication. In a sense, proteins are the workhorses in biology carrying out 762 the chemical, physical and biological functions of the organisms. Similar to 763 biology, in engineering and technological systems, we can genetically select 764 peptides with an ability to bind to inorganic materials to create a new 765 fundamental building block to couple bio and synthetic entities. As we describe 766 here, genetically engineered polypeptides for inorganics (GEPI) have short 767 amino acid sequences with material selective binding and self-assembling 768 properties. Once selected using combinatorial mutagenesis, GEPIs can be 769 further tailored to enhance/modify their binding ability and multifunctionality. 770 The multifunctionality could be introduced either using two or more material- 771 binding peptides to create novel ways of making dissimilar materials 772 thermodynamically compatible, or by genetically fusing a functional protein, 773 e.g. enzyme or an antibody, to develop heterofunctional molecular constructs. 774 Solid-binding peptides coupled with solid substrates form a new generation of 775 novel hybrid materials systems (Sarikaya et al. 2003). Genetic control of the 776 coupling and the resulting function of the hybrid material are new approaches 777 with potential to overcome limitations encountered in the progress of wide range 778 of applications in which traditionally synthetic linkers, such as either thiol or 779 silane, have been used. The attachment of biomolecules, in particular proteins, 780 on to solid supports is fundamental in the development of advanced , 781 , affinity chromatographic separation materials and many diagnostics 782 such as those used in cancer therapeutics (Blawas & Reichert 1998; He et al. 783 2006; Behrens & Behrens 2008). Protein adsorption and macromolecular 784 interactions at solid surfaces play key roles in the performance of implants and

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 17

785 786 implant nano- 787 surface magnetics 788 bone engineering 789 and tooth nano- 790 regeneration 791 792 nano- bionano 793 technology technology 794 nano- 795 electronics 796 molecular 797 molecular biomimetics 798 matrices 799 NEMS/ nano- 800 MEMS medicine 801 lab 802 -on-a-chip probing and multitargeted 803 drug assays 804 phage and delivery cancer cell sorting 805 probing 806 807 808 Figure 7. Potential application areas of GEPI in molecular biomimetics field, which include 809 molecular probing, separation, nanotechnology and nanomedicine, with potential of growing in to 810 new areas (dotted hexagons). 811 812 hard-tissue regeneration (Gottlieb et al. 2008; Ma 2008). Proteins adsorbed 813 specifically on to probe substrates are used to build protein microarrays suitable 814 Q24 for modern proteomics (Cuttler 2003; Cretich et al. 2006). Enzyme immobili- 815 zation on substrates (e.g. nanoparticles in a colloid) will greatly enhance the 816 817 usage of industrial enzymes (Kasemo 2002). Designing bifunctional peptides 818 (e.g. attached to a probe) coupled to nanoparticles, e.g. QDs or fluorescent 819 molecules will provide new avenues for multicomponent design 820 Q25 (Li et al. 2007). The same (nanoparticle/GEPI-probe) platform, where the 821 probe is an antibody and the nanoparticle is a therapeutic or imaging entity, will 822 provide new molecular platform for cancer probing (Weissleder 2006; Tamerler 823 & Sarikaya 2007). The examples given above illustrate only a part of achievable 824 goals by these new classes of functional molecular linkers. All these and a wide 825 variety of other applications form the core of biological materials science and 826 engineering (Sarikaya et al. 2003) which can be designed and genetically 827 engineered (figure 7). Based on its recognition and self-assembly characteristics, 828 the role of GEPI in these hybrid structures would be to provide the essential 829 molecular linkage between the inorganic components, and, at the same time, be 830 an integral component of the overall structure providing to it the functional 831 (e.g. mechanical) durability. Owing to the intrinsic properties mimicked after 832 natural proteins, in the coming years and decades, we are likely to see engineered 833 inorganic-binding polypeptides to be used more and in wide range of applications

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 18 C. Tamerler and M. Sarikaya

834 from particles synthesis and assembly with genetically controlled physical and 835 chemical characteristics in materials science to probing for biological targets Q26 836 Q27 in biology and medicine (Eisledder 2006; Sengupta & Sasisekharan 2007; 837 Tamerler & Sarikaya 2008). 838 839 840 5. Uncited references 841 Q28 842 Gaskin et al. (2000), Eteshola et al. (2005), Sanchez et al. (2005), Feldheim & 843 Eaton (2007), Guo et al. (2007), Rusmini et al. (2007), Matthes et al. (2008), Shu 844 et al. (2008), Tomzcak et al. (2008) and Tullman et al. (2008). 845 We thank our collaborators for their invaluable contribution through their ideas, discussions and 846 results. Among them are Prof. R. Samudrala, M. Somerman, F. Baneyx and J. E Evans; Dr E. E. 847 Oren, Dr H. Fong and Dr M. Hnilova; and graduate students T. Kacar, M. Gungormus, B. Wilson, 848 Q29 D. Khateyevich and U. O. S. Seker. The research is supported, mainly, by NSF/MRSEC, NSF- 849 BioMat and NSF-IRES, and also by TR-SPO, EU-FW6 and TUBITAK-NSF joint programs. 850 851 References 852 853 Alberts, B., Johnson, A., Lewis, J., Raff, M., Roberts, K. & Walter, P. (eds.) 2008 Molecular 854 biology of the cell. New York, NY: Garland Science. Bailey, L. E., Kambhampati, D., Kanazawa, K. K., Knoll, W. & Frank, C. W. 2002 Using surface 855 plasmon resonance and the quartz crystal microbalance to monitor in situ the interfacial 856 behaviour of thin organic films. Langmuir 18, 479–489. (doi:10.1021/la0112716) 857 Ball, P. 2001 Life’s lessons in design. Nature 409, 413–416. (doi:10.1038/35053198) 858 Behrens, S. S. & Silke, S. 2008 Synthesis of inorganic nanomaterials mediated by protein 859 assemblies. J. Mater. Chem. 18, 3788–3798. (doi:10.1039/b806551a) 860 Berman, A., Addadi, L. & Weiner, S. 1988 Interactions of sea-urchin skeleton macromolecules with 861 growing calcite crystals: a study of intracrystalline proteins. Nature 331, 546–548. (doi:10.1038/ 862 331546a0) 863 Q30 Blawas, A. S. & Reichert, W. M. 1998 Protein patterning. Biomaterials 19, 595–609. (doi:10.1016/ 864 S0142-9612(97)00218-4) 865 Bornscheuer, U. T. 2003 Immobilizing enzymes: how to create more suitable biocatalysts. Angew. Chem. Int. Ed. 42, 3336–3337. (doi:10.1002/anie.200301664) 866 Brown, S. 1997 Metal recognition by repeating polypeptides. Nat. Biotechnol. 15, 269–272. (doi:10. 867 1038/nbt0397-269) 868 Cariolou, M. A. & Morse, D. E. 1988 Purification and characterization of calcium-binding 869 conchiolin shell peptides from the mollusk, Haliotis rufescens, as a function of development. 870 J. Comp. Physiol. B 157, 717–729. (doi:10.1007/BF00691002) 871 Castner, D. G. & Ratner, B. D. 2002 Biomedical surface science: foundations to Frontiers. Surf. 872 Sci. 500, 28–35. (doi:10.1016/S0039-6028(01)01587-4) 873 Chicurel, M. E. & Dalma-Weiszhausz, D. D. 2002 Microarrays in pharmagenomics—advances and 874 future promise. 3, 589–601. (doi:10.1517/14622416.3.5.589) 875 Coelfen, H. & Antonietti, M. 2008 Mesocrystal: new self assembled structures. New York, NY: 876 Wiley. Coen, M. C., Lehman, R., Groning, P., Bielmann, M., Galli, C. & Schlapbach, L. 2001 Adsorption 877 and bioactivity of protein A on silicon surfaces studied by AFM and XPS. J. Colloid Interface 878 Sci. 233, 180–189. (doi:10.1006/jcis.2000.7240) 879 Cretich, M., Damin, F., Pirri, G. & Chiari, G. 2006 Protein and peptide arrays: recent trends and 880 new directions. Biomol. Eng. 23, 77–88. (doi:10.1016/j.bioeng.2006.02.001) 881 Cutler, P. 2003 Protein arrays: the current state-of-the-art. Proteomics 3, 3–18. (doi:10.1002/pmic. 882 200390007)

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 19

883 Czenderna, A. W. & Lu, C. 1984 Applications of piezoelectric quartz crystal microbalances, 884 methods and phenomena. New York, NY: Elsevier. 885 Dai, H. X., Thai, C. K., Sarikaya, M., Baneyx, F. & Schwartz, D. T. 2004 Through-mask anodic 886 patterning of copper surfaces and film stability in biological media. Langmuir 20, 3483–3486. 887 (doi:10.1021/la0350711) 888 Eteshola, E., Brillson, L. J. & Lee, S. C. 2005 Selection and characteristics of peptides that bind thermally grown silicon dioxide films. Biomol. Eng. 22, 201–204. 889 Evans, J. S. 2003 ‘Apples’ and ‘oranges’: comparing the structural aspects of biomineral- and 890 ice-interaction proteins. Curr. Opin. Colloid Interface Sci. 8, 48–54. (doi:10.1016/S1359- 891 0294(03)00009-8) 892 Evans, J. S., Samudrala, R., Walsh, T., Oren, E. E. & Tamerler, C. 2008 Molecular design of 893 inorganic binding polypeptides. MRS Bull. 33, 514–518. 894 Feldheim, D. L. & Eaton, B. E. 2007 Selection of biomolecules capable of mediating the formation 895 of nanocrystals. ACS Nano 1, 154–159. (doi:10.1021/nn7002019) 896 Fong, H., Sarikaya, M., , S. & Snead, M. L. 2000 Nanomechanical properties profiles across 897 dentin–enamel junction of human incisor teeth. Mater. Sci. Eng. C 7, 119–128. (doi:10.1016/ 898 S0928-4931(99)00133-2) 899 Frankel, R. B. & Blakemore, R. P. 1991 Iron biominerals. New York, NY: Plenum. 900 Fujiwara, K., Watarai, H., Itoh, H., Nakahama, E. & Ogawa, N. 2006 Measurement of antibody 901 binding to protein immobilized nanoparticles by localized surface plasmon spectroscopy. Anal. 902 Bioanal. Chem. 386, 639–644. (doi:10.1007/s00216-006-0559-2) 903 Gaskin, D. J. H., Strack, K. & Vulfson, E. N. 2000 Identification of inorganic crystal-specific 904 sequences using phage display combinatorial library of short peptides: a feasibility study. 905 Biotechnol. Lett. 22, 1211–1216. (doi:10.1023/A:1005603117023) Gottlieb, D., Morin, S. A., Jin, S. & Raines, R. T. 2008 Self-assembled collagen-like peptide 906 fibers as templates for metallic nanowires. J. Mater. Chem. 18, 3865–3870. (doi:10.1039/ 907 b807150k) 908 Gristina, A. G. 1987 Biomaterials centered infaction—microbial adhesion versus tissue integration. 909 Science 237, 1588–1595. (doi:10.1126/science.3629258) 910 Gungormus, M., Fong, H., Kim, I. W., Evans, J. S. E., Tamerler, C. & Sarikaya, M. 2008 911 Regulation of in vitro calcium phosphate mineralization by combinatorially selected 912 hydroxyapatite-binding peptides. Biomacromolecules 9, 966–973. (doi:10.1021/bm701037x) 913 Guo, Y., Zhuang, J. Q. & Yang, W. S. 2007 Application of surface display peptides in syntheses 914 and assembly of inorganic nanomaterials. Prog. Chem. 19, 51–58. 915 He, L. Z., Dexter, A. F. & Middelberg, A. P. 2006 at interfaces. Chem. 916 Eng. Sci. 61, 989–1003. (doi:10.1016/j.ces.2005.05.064) 917 Hnilova, M., Oren, E. E., Seker, U. O. S., Wilson, B. R., Collino, S., Evans, J. S., Tamerler, C. & 918 Sarikaya, M. 2008 Effect of molecular conformations on the adsorption behavior of gold-binding 919 peptides. Langmuir 24, 12 440–12 445. (doi:10.1021/la801468c) 920 Homola, J., Sinclair, S. Y. & Gauglitz, G. 1999 Surface plasmon resonance : a review. Sens. 921 Actuators B 54, 3–15. (doi:10.1016/S0925-4005(98)00321-9) Izrailev, S., Stepaniants, S., Balsera, M., Oono, Y. & Schulten, K. 1997 Molecular dynamics study 922 of unbinding of avidin–biotin complex. Biophys. J. 72, 1568–1581. (doi:10.1016/S0006- 923 3495(97)78804-0) 924 Q31 Kacar, T., Ray, J., Gungormus, M., Oren, E. E., Tamerler, C. & Sarikaya, M. In press. Quartz 925 binding peptides as molecular linkers towards fabricating multifunctional micropatterned 926 substrates. Adv. Mater. 927 Kasemo, B. 2002 Biological surface science. Surf. Sci. 500, 656–677. (doi:10.1016/S0039- 928 6028(01)01809-X) 929 Kingery, W. D. 1976 Introduction to ceramics. New York, NY: Wiley. 930 Kjærgaard, K., Sorensen, J. K., Schembri, M. A. & Klemm, P. 2000 Sequestration of zinc oxide by 931 fimbrial designer chelators. Appl. Environ. Microbiol. 66, 10–14.

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 20 C. Tamerler and M. Sarikaya

932 Kulp, J. L., Sarikaya, M. & Evans, J. S. 2004 Molecular characterization of a prokaryotic 933 polypeptide sequence that catalyzes Au crystal formation. J. Mater. Chem. 14, 2325–2332. 934 (doi:10.1039/b401260g) 935 Lee, K. B., Park, S. J., Mirkin, C. A., Smith, J. C. & Mrksich, M. 2002a Protein nanoarrays 936 generated by dip-pen nanolithography. Science 295, 1702–1705. (doi:10.1126/science.1067172) 937 Lee, W. S., Mao, C., Flynn, C. E. & Belcher, A. M. 2002b Ordering quantum dots using genetically engineered . Science 296, 892–895. (doi:10.1126/science.1068054) 938 Li, C. M., Botsaris, G. D. & Kaplan, D. L. 2002 Selective in vitro effects of peptides on calcium 939 carbonate crystallization. Cryst. Growth Des. 2, 387–393. (doi:10.1021/cg0255467) 940 Li, Y., Paavola, C. D., Kagawa, H., Chan, S. L. & Trent, J. D. 2007 Mutant chaperonin proteins: 941 new tools for nanotechnology. Nanotechnology 18, 455 101–455 104. (doi:10.1088/0957-4484/ 942 18/45/455101) 943 Lowenstam, H. A. & Weiner, S. 1989 On biomineralization. Oxford, UK: Oxford University Press. 944 Ma, P. 2008 Biomimetic materials for . Adv. Drug Deliv. Rev. 60, 184–198. 945 (doi:10.1016/j.addr.2007.08.041) 946 Makrodimitris, K., Masica, D. L., Kim, E. T. & Gray, J. J. 2007 Structure prediction of protein– 947 solid surface interactions reveals a molecular recognition motif of statherin for hydroxyapatite. 948 J. Am. Chem. Soc. 129, 13 713–13 722. (doi:10.1021/ja074602v) 949 Mann, S. (ed.) 1996 Biomimetic materials chemistry. New York, NY: VCH Publishers, Inc. 950 Mann, S. & Calvert, P. 1998 Synthesis and biological composites formed by in-situ precipitation. 951 J. Mater. Sci. 23, 3801–3815. 952 Mao, C., Flynn, C. E., Hayhurst, A., Sweeney, R., Qi, J., Georgiou, G., Iverson, B. & Belcher, 953 A. M. 2003 Viral assembly of oriented quantum dot nanowires. Proc. Natl Acad. Sci. USA 100, 954 6946–6951. (doi:10.1073/pnas.0832310100) Matthes, J. M., Loughlin, F. E. & Mackay, J. P. 2008 Designed metal-binding sites in biomolecular 955 and bioinorganic interactions. Curr. Opin. Struct. Biol. 18, 484–490. (doi:10.1016/j.sbi.2008.04. 956 009) 957 Mayer, G. & Sarikaya, M. 2002 Rigid biological composite materials: structural examples for 958 biomimetic design. Exp. Mech. 42, 395–403. (doi:10.1007/BF02412144) 959 Min, D. H. & Mrksich, M. 2004 Peptide arrays towards routine implementation. Curr. Opin. 960 Chem. Biol. 8, 554–558. (doi:10.1016/j.cbpa.2004.08.007) 961 Morse, D. E. 1999 Silicon biotechnology; harnessing biological silica production to construct new 962 materials. Trends Biotechnol. 17, 230–232. (doi:10.1016/S0167-7799(99)01309-8) 963 Mrksich, M. & Whitesides, G. M. 1996 Using self-assembled monolayers to understand the 964 interactions of man-made surfaces with proteins and cells. Annu. Rev. Biophys. Biomol. Struct. 965 25, 55–78. (doi:10.1146/annurev.bb.25.060196.000415) 966 Muller, B. 2001 Natural formation of nanostructures: from fundamentals in metal heteroepitaxy to 967 applications in and biomaterials science. Surf. Rev. Lett. 8, 169–228. (doi:10.1016/S0218- 968 625X(01)00085-9) 969 Murray, B. S. & Deshaires, C. 2000 Monitoring protein fouling of metal surfaces via a quartz 970 crystal microbalance. J. Colloid Interface Sci. 227, 32–41. (doi:10.1006/jcis.2000.6882) Naik, R. R., Brott, L., Carlson, S. J. & Stone, M. O. 2002a Silica precipitating peptides isolated 971 from a combinatorial phage display libraries. J. Nanosci. Nanotechnol. 2, 1–6. (doi:10.1166/jnn. 972 2002.074) 973 Naik, R. R., Stringer, S. J., Agarwal, G., Jones, S. E. & Stone, M. O. 2002b Biomimetic synthesis 974 and patterning of silver nanoparticles. Nat. Mater. 1, 169–172. (doi:10.1038/nmat758) 975 Niemeyer, C. M. 2001 Nanoparticles, proteins, and nucleic acids: biotechnology meets materials 976 science. Angew. Chem. Int. Ed. 40, 4128–4158. (doi:10.1002/1521-3773(20011119)40:22! 977 4128::AID-ANIE4128O3.0.CO;2-S) 978 Ostuni, E., Yan, L. & Whitesides, G. M. 1999 The interactions of proteins and cells with self- 979 assembled monolayers of alkanethiolates on gold and silver. Colloids Surf. B 15, 3–30. (doi:10. 980 1016/S0927-7765(99)00004-1)

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS Review. Peptide-based nanotechnology 21

981 Paine, M. L. & Snead, M. L. 1996 Protein interactions during assembly of the enamel organic 982 extracellular matrix. J. Bone Miner. Res. 12, 221–226. (doi:10.1359/jbmr.1997.12.2.221) 983 Park, T. J. et al. 2006 Protein nanopatterns and sensors using gold binding polypeptide as a fusion 984 partner. Anal. Chem. 78, 7197–7205. (doi:10.1021/ac060976f) 985 Pauling, L. 1946 Molecular architecture and biological reactions. Chem. Eng. News 24, 1375–1377. Reed-Hill, R. E. 1991 Physical metallurgy principles. Boston, MA: CANGAGE-Engineering Press. 986 Revzin, A., Russell, R. J., Yadavalli, V. K., Koh, W. G., Deister, C., Hile, D. D., Mellott, M. B. & 987 Pishko, M. V. 2001 Fabrication of poly (ethylene glycol) hydrogel microstructures using 988 photolithography. Langmuir 17, 5440–5447. (doi:10.1021/la010075w) 989 Rusmini, F., Zhong, Z. Y. & Feijen, J. 2007 Protein immobilization strategies for protein biochips. 990 Biomacromolecules 8, 1775–1789. (doi:10.1021/bm061197b) 991 Sakaguchi, T., Burgess, J. G. & Matsunaga, T. 1993 Magnetite formation by a sulfate reducing 992 bacterium. Nature 365, 47–49. (doi:10.1038/365047a0) 993 Sanchez, C., Arribart, H. & Madeleine, G. G. 2005 Biomimetism and bioinspiration as tools for the 994 design of innovative materials and systems. Nat. Mater. 4, 277–288. (doi:10.1038/nmat1339) 995 Sano, K., Sasaki, H. & Shiba, K. 2005 Specificity and biomineralization activities of Ti-binding 996 peptide- (TBP-1). Langmuir 21, 3090–3095. (doi:10.1021/la047428m) 997 Sarikaya, M. 1994 An introduction to biomimetics: a structural viewpoint. Microsc. Res. Tech. 27, 360–375. (doi:10.1002/jemt.1070270503) 998 Sarikaya, M. 1999 Biomimetics: materials fabrication through biology. Proc. Natl Acad. Sci. USA 999 96, 14 183–14 185. (doi:10.1073/pnas.96.25.14183) 1000 Sarikaya, M. & Aksay, I. A. (eds.) 1995. Biomimetics: design & processing of materials. New York, 1001 NY: American Institute of Physics. 1002 Sarikaya, M., Gunnison, K. E., Yasrebi, M. & Aksay, I. A. 1990 Mechanical property– 1003 microstructural relationships in abalone shell. In Proc. Materials Research Society Symposium, 1004 vol. 174, pp. 109–116. Pittsburgh, PA: MRS. 1005 Sarikaya, M., Fong, H., Sunderlan, N., Flinn, B. D., Mayer, G., Mescher, A. & Gaiona, E. 2001 1006 Biomimetic model of a sponge-spicular optical-fiber–mechanical properties and structure. 1007 J. Mater. Res. 16, 1420–1428. (doi:10.1557/JMR.2001.0198) 1008 Sarikaya, M., Tamerler, C., Jen, A. K. Y., Schulten, K. & Baneyx, F. 2003 Molecular 1009 biomimetics—nanotechnology through biology. Nat. Mater. 2, 577–585. (doi:10.1038/nmat964) Sarikaya, M., Tamerler, C., Schwartz, D. T. & Baneyx, F. 2004 Materials assembly and formation 1010 using engineered polypeptides. Annu. Rev. Mater. Res. 34, 373–408. (doi:10.1146/annurev. 1011 matsci.34.040203.121025) 1012 Schultze, S., Harauz, G. & Beveridge, T. I. 1992 Participation of a cyanobacterial S layer in fine- 1013 grain mineral formation. J. Bacteriol. 174, 7971–7981. 1014 Seeman, N. C. & Belcher, A. M. 2002 Emulating biology: building nanostructures from the bottom 1015 up. Proc. Natl Acad. Sci. USA 99, 6452–6455. (doi:10.1073/pnas.221458298) 1016 Seker, U. O. S., Wilson, B., Dincer, S., Kim, I. W., Oren, E. E., Evans, J. S., Tamerler, C. & 1017 Sarikaya, M. 2007 Adsorption behavior of linear and cyclic genetically engineered platinum 1018 binding peptides. Langmuir, 237 895–237 900. 1019 Q32 Seker, U. O. S., Wilson, B., Sahin, D., Tamerler, C. & Sarikaya, M. In print. Quantitative affinity 1020 of genetically engineered repeating polypeptides to inorganic surfaces. Biomacromolecules. 1021 Sengupta, S. & Sasisekharan, R. 2007 Exploiting nanotechnology to target cancer. Br. J. Cancer 1022 96, 1315–1319. Shu, J. Y., Tan, C., DeGrado, W. F. & Xu, T. 2008 New design of helix bundle peptide–polymer 1023 conjugates. Biomacromolecules 9, 2111–2117. (doi:10.1021/bm800113g) 1024 Suzuki, N., Gamble, L., Tamerler, C., Sarikaya, M., Castner, D. G. & Ohuchi, F. S. 2007 1025 Adsorption of genetically engineered proteins studied by time-of-flight secondary ion mass 1026 spectrometry (TOF-SIMS). Part A: data acquisition and principal component analysis (PCA). 1027 Surf. Interface Anal. 39, 419–426. (doi:10.1002/sia.2536) 1028 Tamerler, C. & Sarikaya, M. 2007 Molecular biomimetics: utilizing nature’s molecular ways in 1029 practical engineering. Acta Biomater. 3, 289–299. (doi:10.1016/j.actbio.2006.10.009)

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24 ARTICLE IN PRESS 22 C. Tamerler and M. Sarikaya

1030 Tamerler, C. & Sarikaya, M. 2008 Molecular biomimetics: genetic synthesis, assembly, and 1031 formation of materials using peptides. MRS Bull. 33, 504–511. 1032 Tamerler, C., Duman, M., Oren, E. E., Gungormus, M., Xiong, X. R., Kacar, T., Parviz, B. A. & 1033 Sarikaya, M. 2006a Materials specificity and directed assembly of a gold-binding peptide. Small 2, 1372–1378. (doi:10.1002/smll.200600070) 1034 Tamerler, C., Oren, E. E., Duman, M., Venkatasubramanian, E. & Sarikaya, M. 2006b Adsorption 1035 kinetics of an engineered gold binding peptide by surface plasmon resonance spectroscopy and a 1036 quartz crystal microbalance. Langmuir, 27 712–27 718. 1037 Tomzcak, M. M., Slocik, J. M., Stone, M. O. & Naik, R. R. 2008 Biofunctionlizaed nanoparticles 1038 and their uses. MRS Bull. 33, 519–523. 1039 Tullman, J. A., Finney, W. F., Lin, Y. J. & Bishnoi, S. W. 2008 Tunable assembly of peptide- 1040 coated gold nanoparticles. Plasmonics 2, 119–127. (doi:10.1007/s11468-007-9033-z) 1041 Turkevich, J., Stevenson, P. C. & Hillier, J. 1951 A study of the nucleation and growth of processes 1042 in the synthesis of colloidal gold. Trans. Faraday Soc. Discuss. 11, 55–75. (doi:10.1039/ 1043 df9511100055) 1044 Weissleder, R. 2006 Molecular imaging in cancer. Science 312, 1168–1171. (doi:10.1126/science. 1125949) 1045 Whaley, S. R., English, D. S., Hu, E. L., Barbara, P. F. & Belcher, M. A. 2000 Selection of peptides 1046 with semiconducting binding specificity for directed nanocrystal assembly. Nature 405, 665–668. 1047 (doi:10.1038/35015043) 1048 Whitesides, G. M., Mathias, J. P. & Seto, C. T. 1991 Molecular self-assembly and nanochemistry— 1049 a chemical strategy for the synthesis of nanostructures. Science 254, 1312–1319. (doi:10.1126/ 1050 science.1962191) 1051 Xia, Y. N. & Whitesides, G. M. 1998 Soft lithography. Annu. Rev. Mater. Sci. 28, 153–184. (doi:10. 1052 1146/annurev.matsci.28.1.153) 1053 1054 1055 1056 1057 1058 1059 1060 1061 1062 1063 1064 1065 1066 1067 1068 1069 1070 1071 1072 1073 1074 1075 1076 1077 1078

Phil. Trans. R. Soc. A

RSTA 20090018—31/1/2009—19:31—PARANDAMAN—324119—XML RSA – pp. 1–24