ng C 27 (2007) 558–564www.elsevier.com/locate/msec

Materials Science and Engineeri

Genetically engineered polypeptides for inorganics: A utilityin biological materials science and engineering

Candan Tamerler a,c, Turgay Kacar a,c, Deniz Sahin a,c, Hanson Fong a, Mehmet Sarikaya a,b,⁎

a Materials Science & Engineering, University of Washington, Seattle, WA 98195, USAb Chemical Engineering, University of Washington, Seattle, WA 98195, USA

c Molecular Biology & Genetics, Istanbul Technical University, Maslak 80626, Istanbul, Turkey

Available online 11 July 2006

Abstract

Adapting molecular biology to materials science we developed peptide-based protocols for the assembly and formation of hybrid materials andsystems. In this approach of generating molecular scale biomimetic materials, peptides are designed, synthesized, genetically tailored and, finally,utilized as potential molecular linkers in self-assembly, ordered organization, and fabrication of inorganics for specific technological applications. Thepotential areas range from molecular and nanoscale functional materials to medical fields, e.g., from diagnosis to biosensors. Here, we describe aselection of inorganic binding polypeptides via directed evolution, post-selection modifications through genetic engineering, and utility in practicalapplications. The selection of the inorganic binders is accomplished through combinatorial biology based peptide libraries. The diversity of applicationsis highlighted in three case studies. First, the molecular and nanoscale recognition of the polypeptide is presented via nanosize gold particleimmobilization onto a molecular template by gold binding polypeptide. Second, we present that the alkaline phosphatase fused with multiple repeats ofgold binding polypeptide can still be enzymatically activewhen it is immobilized onto a solid substrate. Finally, we present silica biosynthesis in aqueousenvironment using engineered quartz-binding peptides.© 2006 Published by Elsevier B.V.

Keywords: Molecular biomimetics; Inorganic binding polypeptides; Display technologies; Immobilization; Heterofunctionality; Biofabrication

1. Introduction

In developing biomimetic materials, Mother Nature providesenormous inspiration based on her highly organized biologicalstructures controlled from the nano to macro scales [1–3].Biological hard tissues are but one example of hybrid compositematerials incorporating both inorganic and organic phases withexcellent physical functionality that include piezoelectric (bone),magnetic (bacterial particles) and optical properties (sponge spi-cules) [4,5]. Biocomposites incorporate both structural macro-molecules such as proteins, lipids and polysaccharides, andminerals such as hydroxyapatite, magnetite, and silica [4–6].Among these biomacromolecules, proteins are the most affectivebecause of their recognition, binding and self-assembly char-acteristics [7]. Consequently, future bio-inspired materials could

⁎ Corresponding author. Materials Science and Engineering, Roberts Hall,Box: 352120, University of Washington, Seattle, WA 98195, USA. Tel.: +1 206543 0724; fax: +1 206 543 3100.

E-mail address: sarikaya@u.washington.edu (M. Sarikaya).

0928-4931/$ - see front matter © 2006 Published by Elsevier B.V.doi:10.1016/j.msec.2006.05.046

be based on proteins that specifically bind to inorganics, and beused as molecular agents with which to control their synthesis,formation, and assembly as well as incorporating them as integralparts of the final hybrid composite system [1–3,6,7]. We call thisnew approach to materials formationmolecular biomimetics, i.e.,mimicking the processes of biology at the molecular scale inmaterials formation for addressable structures and geneticallytailorable properties [7].

There are several ways to obtain inorganic-specific proteins.These include extraction from hard tissue (e.g., silaffins andsilicateins [8–10]), designing them via theoretical approaches(extremely difficult, time consuming and impractical) or byrecombinant approaches (e.g., amelogenins [11,12]). Each ofthese approaches has its major limitations and may not be prac-tical for engineering applications. With the recent developmentsin recombinant DNA technology, the preferred approach to obtaininorganic surface specific proteins is to use combinatorial biologybased molecular libraries [13–15]. Here, a large number of ran-dom peptides are screened for an inorganic binding property[7,16–19]. Once selected, these inorganic surface specific

559C. Tamerler et al. / Materials Science and Engineering C 27 (2007) 558–564

proteins can also be further engineered, modified or redesignedand synthesized as molecular constructs.

In this article, first we briefly summarize the combinatorialbiology based methods on how to obtain the inorganicpolypeptides and present some of the binding sequences thatwe have identified for materials with selective functionality.Then we present three case studies to represent a variety ofpotential applications. Organization and immobilization ofinorganic nanoparticles in two- or three-dimensional geometriesare fundamental in the utilization of nanoscale effects [1,18].First example, therefore, demonstrates the assembly ofnanogold particles via the gold binding polypeptide coated onflat mica surfaces. To demonstrate the versatility of geneticmanipulation, the second example illustrates the construction ofheterofunctional molecular entity by genetic fusion of aninorganic binding polypeptide with a functional protein.Specifically, using gold binding protein as the molecular erectorwe immobilize alkaline phosphatase onto gold substrate andshow that the enzyme retains its biological activity. In the finalexample, we discuss the synthesis of silica in the presence of aphage-display selected quartz-specific polypeptide. This lastexample demonstrates the potential use of inorganic bindingpolypeptides for the biofabrication of a wide variety ofbiomimetically engineered functional inorganic materials.

2. Selection of inorganic binding polypeptides

The combinatorial biology based approaches have beenwidely used for selecting peptide sequences with specific affi-nity to biomacromolecules [13–15]. Here, the principle is to usereceptor–ligand interactions to discover ligands (binding pep-tides) for specific substrates (receptors), such as drugs, that bind

Fig. 1. A schematic illustration of cell surface (flagellar) and phage display protocsequences with binding affinity to inorganic substrates.

to surface proteins, such as on tumors and viruses). We, andothers, have adapted these approaches to selecting peptidesequences (ligands) that bind to inorganics (instead ofmacromolecules) [7,16–18,20,22]. Here, combinatorial peptidelibraries are generated by inserting randomized oligonucleotidesinto phage genomes or into plasmids in such a way that theforeign gene products (differently-sized peptide units) aredisplayed on the surface of the phage or bacterium, respectively(Fig. 1). The randomized phage genomes or plasmids are thenpacked into phage or transformed into cells, respectively todisplay a different but random peptide unit. The next step,biopanning, involves exposing the library with the substratematerial followed by several washing steps that eliminatepeptides that nonspecifically bind to the substrate. The strongbinders to the substrate are then (usually chemically) eluted andpooled together for amino acid characterization of theindividual clones by DNA sequencing. Binding characteristicsof the selected peptide sequences (usually 20 or more inorganicbinders) are then tested microscopically (e.g., immunofluores-cence microscopy) or spectroscopically (surface plasmonresonance spectroscopy and quartz crystal microbalance) [23].

The combinatorial display techniques are developed origi-nally for peptide selection for specific protein interactions [13–15]. The adaptation to the selection of peptides specific toinorganic substrates requires special care. In particular, duringbiopanning experiments, the inorganic substrates should betreated in such ways that no surface modification or dete-rioration could take place. The nature of the inorganic substratemay also disqualify a particular display technology. For ex-ample, while phage display can be robust to harvest complexesbetween binding phages and particles [23], in some casescentrifugal forces would damage the flagella from the cell

ols adapted from molecular biology in this research for selecting polypeptide

Fig. 2. Potential application areas of GEPI in the field of molecular biomimetics.In the inset, I1: inorganic-1, I2: inorganic-2, P1 and P2: GEPIs; S: substrate, LP:linker protein; FP: fusion protein; NSL: Non-specific linker.

560 C. Tamerler et al. / Materials Science and Engineering C 27 (2007) 558–564

flagellar-display [22]. On the other hand, the later approachallows the display of longer amino acids under controllableconditions.

Both in our laboratory as well as in others', cell surface andphage display approaches have been used to identify peptidesrecognizing various metals, ceramics, minerals, and semiconduc-tors [23]. In phage display approach, we used both constrainedand linear forms of hepta- or dodecapeptide libraries exposed onthe surface of filamentous phage M13 through its fusion to theminor coat protein pIII (g3p). The constrained form was cons-tructed by flanking the random sequence by two cysteine residuesthat form a disulfide bond under oxidizing conditions and lead tothe display of the heptapeptide as a loop. The peptides have beenused for synthesizing and nucleating inorganics [21], controllinggrowth morphology [20], and assembling and contracting bio-mimetic materials [23]. When working with inorganic surfaces assubstrates, the surface could be well defined, such as a singlecrystal or a nanostructure or it might be rough, or totally non-descriptive such as a powder where the sequence space is thelargest. In our studies, we also worked with powders with varioussizes (ranging from a few nms to sub-μm) and morphologies(sharp corners, rods, spherical particles, etc.) as well as the crystalface specific substrates to understand the recognition mechanismof the polypeptides. We carried out the selection always by usingmaterials that can be synthesized and stable in aqueous environ-ment under physiological conditions and have fairly inert andsmooth surfaces. We generated over 1000 sequences by using awide variety of materials including noble metals (Au, Pd, and Pt),oxides (Al2O3, SiO2, ZnO, Cu2O, TiO2), minerals (mica, calcite,graphite, sapphire, hydroxyapatite), and semiconductors (GaN,ZnS, CdS). [23,24] To demonstrate the diversity of utilizations,below we discuss three specific applications of the engineeredpeptides.

3. Potential Applications

We call combinatorially selected and post-selection tailoredpeptides as genetically engineered polypeptides for inorganics(GEPI). Here the functional (linking) properties of GEPI could beused either alone or as part of another macromolecule (such as anenzyme or DNA) via genetic fusion. Alternatively, a GEPIchemically fused to a synthetic polymer could lead to a multi-functional hybrid polymeric structure. These different types ofutilizations of GEPI form the core of applications in biologicalmaterials science and engineering by providing the required andaddressable molecular binder, linker, bracer, or self-assembler ofinorganics (Fig. 2 — inset). Our molecular toolbox contains allthe inorganic binding engineered peptides, which can be of utilityin a wide range of applications (Fig. 2). Once inorganic bindingdomains are identified, they then can be genetically fused to createheterofunctional units incorporating multiple GEPI constructslink two (or more) different materials to assemble complex nan-ocomposites and hybrid materials. Based on its recognition andself-assembly characteristics, the role of the engineered protein inthese hybrid structures would be to provide the essential mole-cular linkage among the inorganic components, and at the sametime, be an integral component of the overall structure providing

to it functional (e.g., mechanical) durability. The structural andfunctional characteristics of the peptide/nanoparticle architecturesmight be tailored to develop a new generation of molecularlyaddressable functional units.

In molecular bimimetics, inorganic binding proteins are themajor molecular building blocks based on their recognition,controlled binding, and assembly (Fig. 2). Protein adsorption andmacromolecular interactions at solid surfaces play key roles in theperformance of implants and hard-tissue engineering [25]. Theattachment of biomolecules, in particular proteins, onto solidsupports is fundamental in the development of advanced biosen-sors, bioreactors, affinity chromatographic separation materials,and many diagnostics and prognostics such as those used incancer therapeutics [26]. Proteins adsorbed specifically ontoprobe substrates are used to build protein microarrays suitable formodern proteomics [27]. Engineered polypeptides hybridizedwith functional synthetic molecules could be used as heterofunc-tional building blocks in molecular electronics and photonics[7,23,28]. Furthermore, engineered inorganic binding peptide-based systems could be new platforms for environmental treat-ment. Using inorganic binding polypeptides, one can createmolecular erector sets for potential nano- and bionanotechnolo-gical applications (Fig. 2). The examples given below demon-strate some of the typical applications.

4. Immobilization of gold nanoparticles using gold bindingpolypeptide

To achieve nanoscale effects (such as quantum conduction,tunneling, photonic signal enhancement, or spintronics) [29],inorganic nanoparticles need to be immobilized in controlled two-

561C. Tamerler et al. / Materials Science and Engineering C 27 (2007) 558–564

or three-dimensional geometries and at specific positions onselected substrates. One of the traditional approaches to producequantum dots for harvesting quantum electronic properties hasbeen to use molecular beam epitaxy (MBE). Here quantum dots(QDs) are formed at thin film strain centers on semiconductorsubstrates. An example of directed assembled GaAlAs quantumdot organization on GaAs substrate is shown in Fig. 3(a) (T.Pearsall, unpublished data). These traditional approaches arecarried out under highly stringent conditions of high temperature,ultra high vacuum and toxic environment.

The proof-of-principle utility of inorganic binding polypeptideas a linker is demonstrated in the assembly of nano-inorganicparticle. In this example, we used cell surface display selectedgold binding polypeptides, which have been one of the firstexamples of engineered binders. [16,20] The polypeptidesequences were displayed on the outer surface of E. coli as aninsertion in the gene coding for the λ-receptor, lamB. They wereisolated as extracellular loops of maltoporin which were subse-quently fused to the amino terminus of the alkaline phosphotase(AP) with retention of gold binding activity. The geneticallyengineered AP with the insert show specific binding for goldcompared to native AP. More than 50 sequences were identifiedand one of them studied in detail, GBP-1:MHGKTQATSGTIQS.Via post-selection engineering, we have produced differentrepeating units of GBP-1 and achieved an efficient gold nano-particle (Au-NP) immobilization using the seven-repeat binder(7R-GBP-1).

In Fig. 3, we display two AFM images showing 15 nmdiameter GaInAs QDs that are directed assembled on the GaAssubstrate (Fig. 3(a)) and compare it with the high density of 12 nmdiameter Au-NPs directed assembled and immobilized using the7R-GBP-1 as linker on polystyrene-coated flat mica substrate(Fig. 3(b)). Preferably, unlike the conditions of the MBE, in thiscase, the assembly was accomplished in water under a pH of 7.0.Therefore, the homogenous decoration of the surface with Au-NPs suggests that peptides could be useful in the production of

Fig. 3. The AFM images show (a) quantum dots (GaInAs) formed and directed assepitaxy) (courtesy of T. Pearsall, University of Washington), and (b) gold nanoparticovered mica substrate. (c) Schematic illustration of (b), where the mica surface is co

tailored nanostructures, which could be extended in the assemblyof semiconductor QDs. The recognition activity of the proteinwould provide the ability to control the particle distribution whilesolution preparation conditions would allow particle size control.Specific binding functionality of the inorganic binding polypep-tides offers new opportunities that would eventually help toovercome the difficulties so far encountered in nanoparticle andQD organization by working under mild conditions, includingaqueous solutions at room temperature and biofriendlyenvironment.

5. Genetically engineered bifunctional alkaline phosphataseconstructs with inorganic binding and enzymatic activities

In this section, we discuss the development of a molecularconstruct using inorganic binding polypeptide and demonstrate itsbifunctional utility. Specificallywe genetically fused gold bindingpolypeptide (GBP-1) with alkaline phosphatase (AP) and showthat the enzyme retains its biomineralization activity. The AP is amember of a group of hydrolytic enzymes catalyzing hydrolysisof phosphomonoesters as well as the phosphates termini of DNA[30,31]. In our study, using AP as the enzyme and through ahydrolysis reaction we form p-nitrophenol and inorganic phos-phate from p-nitrophenyl phosphate [32]. The activity of AP wasdetermined spectrophotometrically by measuring the rate ofreaction, which can easily be monitored, by the color change ofthe solution.

We incorporated genetic engineering strategies for increasedbinding affinity to gold surface via insertion of multiple repeats ofGBP-1 into AP, such as 5 (5R-GBP/AP), 6, 7, and 9. Here wepresent the bifunctional activity of 6R gold binding polypeptidecontaining GBP/AP construct by E. coli cultures. The constructswere produced by transforming the cells with the plasmid havingthe desired number of repeated unit of GBP-1 insert. Then,following the induction of the E. coli cultures with IPTG (iso-propyl-β-D-thiogalactopyranoside) for 6 h, the constructs in the

embled at strain centers on GaAs substrate via high-vacuum (molecular beamcles (12 nm diam.) self assembled on 7-repeat GBP-1 immobilized on polymervered with a polystyrene thin film, and the non-specific binder is gluteraldehyde.

Fig. 4. (a) Comparison of the catalytic activities of AP when fused to 6R-GBP-1 in the presence (squares) and absence (diamond) of gold nanoparticles. (b) The SDS–PAGE gel electrophoresis of WT and 6R-GBP-1/AP showing the relative position of wild type and GEPI-fused AP.

562 C. Tamerler et al. / Materials Science and Engineering C 27 (2007) 558–564

periplasmic fraction were isolated and the concentrated fractionwas purified via DEAE (diethylaminoethyl) column chromatog-raphy. Subsequently, the eluted fractions showing AP activity isconcentrated and then passed through gel filtration column. Aftereach step, we carried protein electrophoresis (SDS–PAGE) toassess the purity of the fraction. At each step of purification, wealso followed the enzymatic activity of AP through the change ofabsorbance in the presence of p-nitrophenylphosphate in an assaybuffer containing 10 mM Tris-Cl (pH:8,0), 1 mM MgCl2. Toobserve if the enzyme is also active in the presence of Au-NPs, wetested the enzymatic activity of the GBP/AP/Au-NPs construct.For this the hybrid construct was incubated overnight with Au-NPs at 37 °C. Similar AP activity profiles were obtained for boththe wild type and the recombinant AP with Au-NPs (Fig. 4(a)).The GBP/AP constructs were also tested for the stability of theirfused GBP-1 inserts in the main protein via SDS–PAGE gelelectrophoresis. The SDS–PAGE gel analysis in Fig. 4 (b) showsthe differences in the relative molecular weights of the wild typeAP and the recombinant 6R-GBP-1/AP construct compared withthe molecular weight marker.

Fig. 5. Aligned self-assembly of the quartz-binding phage mutants along specificmicroscopy images in (a) and (b) are amplitude and height, respectively.

Alkaline phosphatase is an essential enzyme for biomi-neralization of mammalian hard tissues being instrumental notonly in bone but also in cementum [32,33]. The GBP/AP bifunc-tional construct is promising, in particular in the area of peri-odontal regeneration. The enzyme is known to have amajor effectin controlling the extracellular phosphate concentration bycatalyzing the pyrophosphate degradation. The molecular con-structs developed in this work could provide excellent probingproperties to monitor biomineralization processes via goldbinding property and, therefore, could allow controlled spatialdelivery of AP to the desired locations, consequently promotingtissue repair or regeneration.

6. Biofabrication of silica using inorganic bindingpolypeptides

In nature, organisms exhibit diverse and complex silica shapesand structures which are used as skeletons, protection covers, orfunctional devices [4]. The proteins directing silica synthesis inbiological systems have been studied extensively. [9,10] For

crystallographic directions on single crystal quartz surface. The atomic force

Fig. 6. SEM images of biosilica formed in the presence of quartz-binding phage mutants. Samples for high magnification microscopy were prepared by (a) freezedrying and (b) air drying. The inset shows a TEM image of the air (slow) dried sample, revealing compact (dense) structure. A secondary electron image of the bulksample is in (c) and its energy dispersive X-ray spectrum in (d), both recorded with SEM.

563C. Tamerler et al. / Materials Science and Engineering C 27 (2007) 558–564

example, silaffins extracted from the cell wall of diatoms Cylin-drotheca or silicatein extracted from the spounge spicules ofTerhya aurantia [34,35]. Extracted, purified and engineered natu-ral proteins from biological hard tissues have been shown to retaintheir biomaterialization activity for silica formation. Silicabinding peptides were also identified using combinatorial selec-tion protocols, and some of these, in the presence of silica bindingphage clones, were used in the synthesis of the amorphous silicaprecipitates [36]. These engineered proteins, similar to naturalones, have been found to be rich in basic and hydroxyl groups,perhaps essential amino acids for silica formation.

Adapting phage display protocols, we also identified silica-binding polypeptides and used them for silica formation. [Un-published] Our approach, different than the previous selections,used oriented single crystal quartz substrates for this purpose. Ourlong-term goal in this research is to assess binding sequences thatare not only specific to crystalline silica (quartz) but also to a givencrystallographic surface (e.g., (100) versus (111) surfaces). In thiswork, selected sequences were categorized according to theirbinding activity as strong, medium, and weak binders, and thestrong binders were used for silica synthesis compared to thosefabricated using silicatein and silaffins in the literature [34,35].

We used 12-amino acid long M13 phage display library forthe selection. Five rounds of biopanning (see Fig. 1) were carriedout and 50 sequences were obtained presenting differentaffinities for silica substrate. Binding characterization of theselected phage samples were examined by atomic force micro-scopy (Fig. 5(a and b)) which displayed aligned mutant phagesalong specific crystallographic direction on quartz surface. Wealso performed immunofluorescence microscopy experiments toassess the degree of binding (data not shown). The selected highquartz-binding affinity phage clones were then tested for theirsilica biofabrication characteristics (one such binder, e.g., isDS202: RLNPPSQMDPPF). Samples containing 1013 phage

forming units were incubated in freshly prepared tetra methylortho silicate (TMOS) solution for 3–4 min at room temperatureand initial pH of 7.0. To test the presence of silica, first theTEOS/phage/buffer solutions were centrifuged and the samplesfor examination were taken (using a syringe) from the preci-pitated portion of the solution after removing the supernatant.The test samples were either freeze-dried or air-dried. Thesewere examined using scanning and transmission electron micro-scopy (SEM and TEM) and elemental compositional analyseswere accomplished using energy dispersive X-ray spectroscopy(EDXS) (Fig. 6). The samples under both drying conditionsdisplay highly porous three-dimensional structure as shown inFig. 6(a) and (b). From the SEM and TEM images (the latter inthe inset of Fig. 6(b)) the air-dried samples appear to be morecollapsed, therefore, denser, than the freeze-dried samples.When examined at low magnifications, such as shown in Fig.6(c), the air-dried sample appears as a hard bulk material withsmooth cleaved surfaces although the structure is amorphousunder these preparation conditions (as examined by XRD, datanot shown). The elemental composition of the bulk sampledetermined by EDXS shows Si and O (Fig. 6(d)), with aquantified value of the material close to SiO2 composition (thetiny peak shown above Si–K is from is from the thin Au layer onthe sample surface ensuring conduction during SEM observa-tion). Although studies are currently underway, the results so fardemonstrate the feasibility of using inorganic binding polypep-tides in the biofabrication of the inorganic material in biofriendlyenvironments, i.e., conditions similar to the metabolite produc-tions via biological pathways.

7. Concluding Remarks

Inorganic binding polypeptides generated via molecular bio-mimetics approach offers a great potential for synthesis, assembly

564 C. Tamerler et al. / Materials Science and Engineering C 27 (2007) 558–564

and creation of materials architectures at molecular or nanoscalelevels. Similar to the ways of Mother Nature, it is now feasible todesign hierarchical hybrid structures with robust features andstructural characteristics. The three examples given here demons-trate that the combinatorially selected inorganic specific peptides(GEPI) can be used to immobilize inorganic nanoparticles or theycan be part of a biologically active enzyme producing amultifunc-tional entity that can then be easily placed (i.e., directed immo-bilized) on a given substrate (nanoparticle or solid and flat bulksubstrate). Finally, a GEPI can be utilized for the synthesis ofinorganic materials that it was originally selected for. Theseresults could lead to new avenues in nanotechnology, biomimeticdisplay of enzymes and biotechnology areas with improvedfunctionality, all accomplished under mild conditions.

There are great opportunities in future materials science andengineering that are provided by the multifunctional properties ofthe engineered polypeptides. These can be categorized in twobasic pathways: On the one hand, taking advantage of the mole-cular binding properties the GEPI one can use it as a functionalprobe for target recognition in medical applications. And, on theother hand, as a catalyzer, bracer, template and assembler, a GEPIcould be utilized to fabricate addressable structures, clusters, andsystems with enhanced functional properties [7,23].

Acknowledgements

This research was supported by the US Army Research Office(PM: R. Campbell) through the DURINT (Defense UniversityInitiative on NanoTechnology) Program (Grant No: DAAD19-01-1-0499).

References

[1] C.M. Niemeyer, Angew. Chem., Int. Ed. Engl. 40 (2001) 4128.[2] P. Ball, Nature 409 (2001) 413.[3] M. Sarikaya, Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 14183.[4] H.A. Lowenstam, S. Weiner, On Biomineralization, Oxford Univ. Press,

Oxford, 1989.[5] S. Mann (Ed.), Biomimetic Materials Chemistry, VCH Publishers, New

York, 1996.[6] P. Calvert, S. Mann, J. Mater. Res. 23 (1988) 3801.

[7] M. Sarikaya, C. Tamerler, A.Y. Jen, K. Schulten, F. Baneyx, Nat. Mater. 2(2003) 577.

[8] M.A. Cariolou, D.E. Morse, J. Comp. Physiol., B 157 (1988) 717.[9] N. Kruger, R. Deutzmann, C. Bergsdork, M. Sumper, Proc. Natl. Acad.

Sci. U. S. A. 97 (2000) 14133.[10] J.N. Cha, K. Shimizu, Y. Zhou, S.C. Christiansen, B.F. Chmelka, et al.,

Proc. Natl. Acad. Sci. U. S. A. 96 (1999) 361.[11] M.L. Paine, M.L. Snead, J. Bone Miner. Res. 12 (1997) 221.[12] P.S. Stayton, G.P. Drobny, W.J. Shaw, J.R. Long, M. Gilbert, Crit. Rev.

Oral Biol. Med. 14 (5) (2003) 370.[13] R.H. Hoess, Chem. Rev. 101 (2001) 3205.[14] K.D. Wittrup, Curr. Opin. Biotechnol. 12 (2001) 395.[15] P. Samuelson, E. Gunneriusson, P.Å. Nygren, S. Ståhl, J. Biotechnol. 96

(2002) 129.[16] S. Brown, Nat. Biotechnol. 15 (1997) 269.[17] S. R. Whaley, D. S. English, E. L. Hu, P. F. Barbara, M. A. Belcher, Nature

405 (2000) 665.[18] R.R. Naik, S.J. Stringer, G. Agarwal, S.E. Jones, M.O. Stone, Nat. Mater. 1

(2002) 169.[19] Y.G. Sun, Y. Xia, Science 298 (2002) 2176.[20] S. Brown, M. Sarikaya, E. Johnson, J. Mol. Biol. 299 (2000) 725.[21] R.R. Naik, L. Brott, S.J. Carlson, M.O. Stone, J. Nanosci. Nanotechnol. 2

(2002) 95.[22] C.K. Thai, H. Dai, M.S.R. Sastry, M. Sarikaya, D.T. Schwartz, F. Baneyx,

Biotech. Bioeng. 87 (2004) 129.[23] M. Sarikaya, C. Tamerler, D.T. Schwartz, F. Baneyx, Annu. Rev. Mater.

Res. 34 (2004) 373.[24] C. Tamerler, M. Sarikaya, unpublished results (2004).[25] B. Ratner, F. Schoen, A. Hoffman, J. Lemons (Eds.), Biomaterials Science:

Introduction to Materials in Medicine, Academic Press, San Diego/USA,1996.

[26] M. Mrksich, Curr. Opin. Chem. Biol. 6 (2002) 794.[27] J. LaBaer, N. Ramachandran, Curr. Opin. Chem. Biol. 6 (2005) 14.[28] N.C. Seeman, A.M. Belcher, Proc. Natl. Acad. Sci. 99 (2002) 6452.[29] G. Schmid, Clusters and Colloids, VCH, Weinheim, 1994.[30] S. Narisawa, N. Frohlander, J.L. Millan, Dev. Dyn. 208 (1997) 432.[31] K.N. Fedde, L. Blair, J. Silverstein, S.P. Coburn, L.M. Ryan, R.S.

Weinstein, K. Waymire, S. Narisawa, J.L. Millan, G.R. Macgregor, et al.,J. Bone Miner. Res. 14 (1999) 2015.

[32] F.H. Nociti Jr., J.E. Berry, Foster, et al., J. Dent. Res. 81 (2002) 817.[33] M. Balcerzak, E. Hamade, L. Zhang, S. Pikula, G. Azzar, J. Radisson, J.

Bandorowicz-Pikula, R. Buchet, Acta Biochim. Pol. 223 (2003) 1019.[34] K. Shimizu, J. Cha, G.D. Stucky, D.E. Morse, Proc. Natl. Acad. Sci. U. S. A.

95 (2000) 6234.[35] N. Kröger, R. Deutzman, M. Sumper, Science 286 (1999) 1129.[36] R.R. Naik, P.W. Whitlock, F. Rodriguez, L.L. Brott, D.D. Glave, et al.,

Chem. Commun. 2 (2003) 238.