progress in materials science · 2017. 9. 5. · inorganic nanomaterial assembly, wherein...

Progress in Materials Science 91 (2018) 1–23

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Progress in Materials Science

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Biotemplated synthesis of inorganic materials: An emergingparadigm for nanomaterial synthesis inspired by nature

http://dx.doi.org/10.1016/j.pmatsci.2017.08.0010079-6425/� 2017 Elsevier Ltd. All rights reserved.

⇑ Corresponding author at: Department of Materials Science and Engineering, Stanford University, 476 Lomita Mall, McCullough Room 246, Sta94305-4045, United States.

E-mail address: [email protected] (S.C. Heilshorn).

Brad A. Krajina c, Amy C. Proctor c, Alia P. Schoen a, Andrew J. Spakowitz a,b,c,Sarah C. Heilshorn a,b,⇑aMaterials Science and Engineering, Stanford University, Stanford, CA 94305, United Statesb Stanford Institute for Materials and Energy Science, SLAC National Accelerator Laboratory, Menlo Park, CA 94025, United StatescChemical Engineering, Stanford University, Stanford, CA 94305, United States

a r t i c l e i n f o

Article history:Received 22 May 2016Received in revised form 10 July 2017Accepted 7 August 2017Available online 8 August 2017

a b s t r a c t

Biomineralization, the process by which biological systems direct the synthesis of inor-ganic structures from organic templates, is an exquisite example of nanomaterial self-assembly in nature. Its products include the shells of mollusks and the bones and teethof vertebrates. By comparison, conventional inorganic synthesis techniques provide limitedcontrol over inorganic nanomaterial architecture. Inspired by biomineralization in nature,over the last two decades, the field of biotemplating has emerged as a new paradigm forinorganic nanomaterial assembly, wherein researchers seek to design novel nano-structures in which inorganic nanomaterial synthesis is directed from an underlyingbiomolecular template. Here, we review the motivation, mechanistic understanding, pro-gress, and challenges for the field of biotemplating. We highlight the interdisciplinary nat-ure of this field, and survey a broad range of examples of bio-templated engineering:ranging from strategies that exploit the inherent capabilities of proteins in nature, togenetically-engineered systems that unlock new capabilities for self-assembly with biomo-lecules. We illustrate that the use of biological materials as templates for inorganic self-assembly holds tremendous potential for nanomaterial engineering, with applications thatrange from electronics and energy to medicine.

� 2017 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 22. Nanomaterials: prospects, applications, and challenges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

2.1. Nanomaterials market forecast. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42.2. Applications of nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5

2.2.1. Biomedical applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2.2. Semiconductor applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2.3. Catalysis and electrochemistry applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 52.2.4. Synthetic challenges in nanomaterial chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62.2.5. Biotemplating for ‘‘greener” nanomaterial synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

3. Biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6

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2 B.A. Krajina et al. / Progress in Materials Science 91 (2018) 1–23

3.1. Biomineralization in nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63.2. Mechanisms of biomineralization . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7

3.2.1. Classical nucleation theory . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.2. Controversy over classical mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73.2.3. Biomineralization mechanisms: insight for nanoscale engineering. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8

4. Natural and recombinant protein inorganic templating . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8
4.1. Organic scaffolds in abalone shells: inspiration from nature . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84.2. Fibrous proteins. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94.3. Cage proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 10
4.3.1. Viral capsids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 104.3.2. Ferritin cages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 124.3.3. Heat shock proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12

5. Peptide only systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12
5.1. Peptide evolution by phage display . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135.2. Mechanisms of biomineralization by peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135.3. Examples of peptide systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13
5.3.1. Soluble peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 135.3.2. Peptide arrays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 145.3.3. Self-assembling peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

6. Protein-peptide hybrid systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
6.1. Phage capsid-peptide systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 166.2. Ferritin-peptide systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 176.3. Clathrin-peptide systems . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
7. Challenges in nanomaterials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18
7.1. Reproducibility in nanomaterial synthesis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.2. Mechanical properties of organic-inorganic hybrid materials . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.3. Environmental concerns . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 187.4. Health concerns. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19
8. Conclusion and outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

1. Introduction

Biological systems are exquisite examples of self-assembly in nature. Examples of biological self-assembly can be foundthroughout a vast hierarchy of length-scales, from the assembly of proteins and nucleic acids that form nanoscale viruses, tothe development of complex anatomies in multi-cellular animals. By comparison, the ability of materials scientists to designself-assembling architectures with multi-scale precision remains quite limited. However, the possibility of engineering novelfunctional materials with structures and resultant functions that are precisely tailored at the nanoscale excites tremendousinterest that continues to grow. The field of biotemplating emerged from the union of these two pursuits: the study of bio-logical self-assembly in nature, and the development of nanostructured inorganic materials with properties exceeding theirbulk counterparts. In nature, through the process of biomineralization, a wide variety of biological systems direct the growthof complex hierarchical inorganic mineral structures whose crystal phases and multi-scale architectures are precisely dic-tated by an underlying template composed of biomolecules. Drawing inspiration from biomineralization in nature, in thepast two decades, biotemplated synthesis of inorganic nanostructures has burgeoned as a field, wherein researchers seekto engineer new inorganic materials whose structures are directed by the unique recognition and self-assembly propertiesof biomolecules.

In this review, we provide a broad overview of the motivation, mechanistic understanding, progress, and challenges forbiotemplated synthesis of inorganic materials. To this end, we begin in Section 2 with a brief overview of the nanomaterialsfield, together with a survey of its potential applications, and current challenges in nanomaterials synthesis. The remainderof the review focuses on biotemplating of inorganic materials, organized from least-engineered to most-engineered strate-gies (illustrated in Fig. 1). Thus, in Section 3, we consider biomineralization as it occurs in nature, assess current models forits underlying mechanism, and preview the potential for mechanistic understanding of biomineralization to be appliedtoward nanoscale control of inorganic material synthesis. In Section 4, we review recent progress in using proteins as tem-plates for exercising control over nanoparticle structure and organization. In Section 5, we review advances in peptide sys-tems as molecular recognition motifs for directing nucleation of nanoparticles and controlling their growth and assembly atvarious length-scales. In Section 6, we provide examples in the literature in which peptide molecular recognition and proteinself-assembly are combined into protein-peptide fusion systems that direct inorganic material synthesis. Finally, in Section 7,we offer a perspective on the current challenges facing the field of biotemplating, including practical synthetic challenges, aswell as environmental and human health concerns.

Fig. 1. Overview of strategies for biotemplating of inorganic materials: from inspiration by nature to engineered biotemplating systems. (a)Biomineralization in nature provides inspiration for engineered biotemplating strategies. SEM images of the cell wall of Stephanopyxis turris are shownat two levels of magnification, which illustrates the elegant multi-scale patterning of silica in diatoms. This multi-scale self-assembly is directed by organicmolecules that serve as templates for silification. (b) Natural and recombinant protein strategies offer the opportunity to harness the intrinsic self-assemblyand biomineralization capabilities of naturally-occurring proteins. In the schematic shown, a recombinant protein cage from Pyrococcus furiosusencapsulates ferritin nanoparticles (blue sphere). This ferritin cage is combined with gold nanoparticles bearing amphiphilic ligands, which self-assembleinto binary nanoparticle superlattices composed of ferritin cages that are surface decorated with gold nanoparticles. (c) Synthetic peptide-based systemsenable the tunable design of novel biotemplates and broaden the scope of biotemplating beyond the functionality found in naturally occurringbiomolecules. In the schematic shown, amphiphilic peptides are designed to self-assemble into higher-order structures that can serve as 3D templates forinorganic materials. (d) Protein-peptide hybrid systems further expand biotemplate engineering by combining the intrinsic self-assembly capabilities ofnatural proteins with the broad tunability of synthetic peptides. In the schematic shown, self-assembled clathrin cages are decorated with bifunctionalpeptides that contain clathrin-binding and inorganic-binding domains. The modular design enables peptides to be used that direct the chemo-selectivesynthesis of inorganic materials within the clathrin cage, including titanium dioxide (red), cobalt oxide (green), and gold nanoparticles (blue). Scale bars are25 nm. Images from (a) were reproduced from Ref. [161] (copyright Springer-Verlag 2009) with permission of Springer. Images from (b) were adapted bypermission from Macmillan Publishers Ltd: Nature Nanotechnology (Ref. [162]), copyright 2012. Images from (c) were adapter from Ref. [125]. Reprintedwith permission from AAAS. Images from (d) were reproduced with permission from Ref. [142]. Copyright (2011) American Chemical Society.

B.A. Krajina et al. / Progress in Materials Science 91 (2018) 1–23 3

As we intend to illustrate in this review, the global market for nanomaterials is broad and growing as nanomaterialsbecome more prevalent in the biomedical, semiconductor, and energy fields. The precise structure of nanomaterials dictatestheir properties, and nanoscale materials have shown promise in applications where bulk material properties are insufficientfor the task. The development of scaffolds capable of reproducibly templating inorganic nanomaterials will be essential tothe development of next generation materials, because the unique properties of nanomaterials are highly dependent on theirsize and precise structures. Drawing inspiration from biomineralization in nature, researchers are increasingly investigatingthe use of proteins and peptides in inorganic nanotemplating. Protein and peptide based systems enable architectural con-trol over nanomaterial structure that is difficult to obtain using conventional synthetic techniques. The multi-faceted poten-tial for biotemplating includes shape and size control over nanoparticles, chemo-selection of specific inorganic species,spatially directed localization of nanomaterials, and exquisite 3D self-assembly capabilities (Fig. 2). In addition, the mild

Fig. 2. Biotemplates offer multifaceted control through four primary means: shape and size of the inorganic material; chemo-selectivity of inorganicsynthesis; inorganic localization; and higher-order assembly. (a) Organic templates can provide control over the size, crystal orientation, and morphology ofinorganic nanoparticles. In the schematic shown, two different peptides serve as capping agents to enable differential size selection for palladiumnanoparticles produced by reduction of palladium (II) with NaBH4 [106]. (b) Organic templates can function as chemo-selective inorganic binding agents.The image provided represents a typical conformation predicted by molecular dynamics simulation for a silver binding peptide interacting with the surfaceof gold and silver, illustrating its molecular recognition capabilities [105]. (c) Organic templates can be designed to direct the localization of inorganicmaterials to specific sites in relation to a protein. The schematic illustrates the capability of viral protein capsids to either encapsulate inorganicnanoparticles (red sphere), bind inorganic nanoparticles to specific sites within the interior (blue spheres), or to bind nanoparticles to sites on the exterior ofthe capsid (green spheres) [69]. (d) Organic templates can self-assemble into 3D structures, enabling bottom-up patterning of inorganic nanomaterials. Inthe example shown, a recombinant protein cage from Pyrococcus furiosus self-assembles to encage ferritin nanoparticles (blue spheres). These protein cagescan be combined with gold nanoparticles that are coated with amphiphiles, yielding ferritin cages decorated with gold (yellow spheres). These gold-decorated ferritin cages in turn self-assemble into binary super-lattices, as shown in the TEM images in the top panel. Scale bar is 50 nm. The schematic onthe middle-right illustrates the unit-cell of the super-lattice [162]. Image from (a) was reproduced with permission from Ref. [106]. Copyright (2012)American Chemical Society. Images from (b) were adapted with permission from Ref. [105]. Copyright (2014) American Chemical Society. Images from c)were reproduced from Ref. [69]. Reprinted with permission from AAAS. Images from (b) were adapted by permission fromMacmillan Publishers Ltd: NatureNanotechnology (Ref. [162]), copyright 2012.


conditions associated with protein and peptide based templating is highly advantageous, especially in light of the currentuncertainty about the environmental impacts of nanomaterials. While peptide-based templating of inorganic nanomaterialsis still an immature field, interest has risen tremendously in recent years and should continue to grow in tandem with therapidly developing market for novel nanomaterials.

2. Nanomaterials: prospects, applications, and challenges

2.1. Nanomaterials market forecast

As a result of their size, nanomaterials frequently possess unusual physical (structural, electronic, magnetic, and optical)and chemical (catalytic) properties. The global market for nanomaterials, valued in the billions of dollars, is massive and far-reaching with applications ranging from medicine to battery production [1,2]. The pharmaceutical, cosmetic, food, textile,automotive, and construction industries all sell a number of consumer products with incorporated nanomaterials, and aca-demic and industrial research in the area of nanomaterials has exploded over the past two decades [2–4]. Nanomaterialsdesign and synthesis represents an inherently interdisciplinary field of research, bringing together theoreticians and exper-imental researchers from diverse communities including organometallics, colloids, catalysis, soft matter, biology, polymerchemistry, and electrical engineering. The intersection between diverse scientific fields has led to some surprising hybridtechnologies, such as lithium batteries templated by viruses [5], and many manufacturing challenges in electrical engineer-ing may ultimately be solved using protein-based systems.


2.2. Applications of nanomaterials

2.2.1. Biomedical applicationsNanomaterials have enjoyed widespread popularity in the biomedical industry. Nanomaterials can be used in a variety of

biomedical contexts, including drug delivery, biosensing, and imaging [6–12]. Cancer diagnostics and targeted drug deliveryto tumors are among the most well studied applications of nanomaterials in healthcare [7,12]. Networks of gold nanoparti-cles and bacteriophage have shown promise as biological sensors and cell-targeting agents [9], and composite organic-inorganic nanoparticles have shown utility as Raman labels for tissue analysis [10]. Due to their small size, versatile surfacefunctionalization, and useful imaging properties, gold and silver nanoparticles have been widely studied for targeted treat-ment of tumors, including pancreatic tumors [12]. However, the biocompatibility of inorganic nanoparticles is shape, size,surface chemistry, and cell-type dependent [13,14]. Thus, the physical and chemical properties of nanoparticles must becarefully considered and controlled for biomedical applications.

2.2.2. Semiconductor applicationsSince the precise arrangement of metals and metal oxides into nanostructures influences the resulting optical and elec-

tronic properties, the role of nano-architecture has been widely studied in the semiconductor industry [15]. For example, theelectronic properties of nanoparticles for field emission applications has been a topic of extensive research [16,17]. Peptideswith semiconductor binding specificity for directed nanocrystal assembly have been identified to facilitate the productionand study of complex inorganic nanostructures [18].

2.2.3. Catalysis and electrochemistry applicationsNanomaterials have also been rapidly adopted in the electrocatalysis field. Catalysts can be highly clustered on the sur-

face of nanomaterials, leading to an electrocatalytically active interface that can be exploited in fuel cells and otherelectrocatalysis-based devices. Moreover, the size- and geometry-dependent electronic properties of nanoparticles presentthe opportunity for developing catalysts with superior properties. Nanodevices currently used in electrocatalysis include car-bon nanotubes [19–21], platinum nanoparticles [19], palladium nanoparticle-cored dendrimers [22], and colloidal goldnanoparticles [23]. Given the importance of materials development to the success of next generation energy technologies,

a)

b)

c)

d)

1 µm 1 µm10 µm

2 µm2 µm 200 nm

Fig. 3. Biomineralization in nature produces diverse hierarchical structures of inorganic materials. (a) SEM images of cell walls from diatoms. Diatomsexhibit a wide-range of multi-scale structures containing biomineralized silica, such as the cell walls from Thalassiosira pseudonana (left) and Stephanopyxisturris (right). (b) SEM images of the microstructure of silica cell walls in Gyrosigma balticum (left) and Ditylum brightwellii (right). (c) SEM image of thenacreous layer in the mollusk shell of Atrina rigida. The nacreous layer forms the inner structure of the mollusk shell and consists of layered tablets ofmineralized aragonite. (d) SEM image of the prismatic layer of the mollusk shell from Atrina rigida after partially dissolving inorganic minerals. Theprismatic layer contains a fibrous matrix of proteins that direct mineralization of calcite. Images from (a and b) adapted with permission from Ref. [163].Copyright 2008 American Chemical Society. Images (c and d) were reproduced with permission from Ref. [39] with permission of The Royal Society ofChemistry.


the range of nanostructures used for electrocatalysis will likely expand as more complicated architectures are produced andthe relationship between nanostructure and activity becomes better understood.

2.2.4. Synthetic challenges in nanomaterial chemistryAlthough nanomaterials excite broad interest in the scientific community, their synthesis presents both practical chal-

lenges and environmental concerns. Standard inorganic syntheses used to produce inorganic nanomaterials generallyinvolve the use of harsh conditions and solvents. In many cases, organic solvents and ionic liquids are used to control thecomposition, crystal structure, and assembly properties of nanomaterials, often under elevated temperature and pressureconditions [24–26]. The disposal of synthesis byproducts and used solvent, in addition to the energy required to maintainelevated temperature for large-scale reactions, represents an environmental burden that should be addressed as commercialscale production of inorganic nanoparticles becomes increasingly common [27,28]. Moreover, the ability to design nanoma-terials with complex architectures using standard inorganic synthesis techniques is currently limited, so alternative methodsare needed to meet the demand for new nanomaterials with novel, structure-dependent properties [29].

2.2.5. Biotemplating for ‘‘greener” nanomaterial synthesisWhile material synthesis routes that utilize milder conditions are increasingly being pursued and utilized [28,30,31],

many researchers have taken inspiration from nature for greener inorganic materials synthesis [32–34]. Natural biominer-alization produces complicated, tunable nanostructures under environmentally friendly conditions. By utilizing naturallyoccurring or naturally inspired templates, nanomaterials may be produced without the use of harsh solvents or high tem-peratures. In addition, natural inorganic nanocomposites possess a wide variety of complex 3D architectures not currentlyaccessible through conventional chemical techniques, making their use appealing from both an environmental and a designperspective.

3. Biomineralization

3.1. Biomineralization in nature

Biomineralization is a widely observed process whereby biological organisms direct the assembly of inorganic mineralstructures [35]. In contrast to the growth of inorganic materials typically achieved in laboratories, this directed assemblyin biological systems often gives rise to elaborate architectures with hierarchical levels of complex spatial organization,spanning nanoscale control of crystal patterning and growth up to meso- and macroscale morphological patterns [36,37].Products of biomineralization can be observed, for instance, in the intracellular micro-skeletons of unicellular radiolarian,

Fig. 4. Mechanisms of crystal nucleation and growth. (a) Illustration of crystal nucleation and growth in terms of classical nucleation theory. In the top leftpanel, red circles represent a series of nanocrystals that form spontaneously in solutions, but are thermodynamically unstable due to high interfacial freeenergy, and undergo spontaneous dissolution. Above a critical size, the nanocrystals that form are thermodynamically stable, and further crystal growth is athermodynamically favored process (top right panel). The bottom plot illustrates the dependence of the free energy of crystal formationDG on crystal size r,as predicted by classical nucleation theory. Crystals must reach a critical size rc in order for spontaneous crystal growth to occur, which coincides with theglobal maximum in the free energy curve DGc. The red curve represents a hypothetical free energy landscape in the absence of interface stabilizing agents(homogeneous nucleation). Crystal growth is facilitated by the presence of agents, such as biomolecules, that reduce the interfacial energy of nano-crystalsand lead to heterogeneous nucleation (blue curve). (b) Alternative pathways to crystal growth. Crystals may also form directly or indirectly through avariety of pathways and intermediates, including direct nucleation of the crystal from atoms or molecules, the intermediate formation of metastableclusters, and the formation and subsequent direct or indirect crystallization of an amorphous bulk phase. (c) A hypothetical non-classical pathway to crystalformation involving the formation of metastable clusters, rather than direct nucleation from primary particles. In the hypothetical pathway, the energeticlandscape contains a series of local minima that enable the formation of metastable intermediates, thereby bypassing direct nucleation of crystals fromions. Figure (b) was reprinted by permission from Macmillan Publishers Ltd: Nature Materials from Ref. [51], copyright (2013). Figure (c) was reproducedwith permission from Ref. [43], published by the Royal Society of Chemistry.


the morphologically diverse frustules that enclose diatoms, the shells of mollusks, and the bones and teeth of vertebrates[37–39] (Fig. 3). Although widely varying in form and biological origin, biomineralization products are unified by their emer-gence from organic templates, wherein macromolecular scaffolds and binding motifs are hypothesized to orchestrate thepatterned nucleation and controlled subsequent growth of inorganic minerals into higher-order assemblies [40,41]. Beyondits obvious biological significance, the elegant mutli-scale control of inorganic structures achieved in biomineralization,which is unrivaled by conventional synthetic materials chemistry, provides inspiration for novel pathways to materialsdesign and synthesis through biomimetic templates.

3.2. Mechanisms of biomineralization

Although biomineralization in nature proceeds through a complex interplay of physiochemical and biological processes ata hierarchy of length-scales, at the most basic level, biomineralization is initiated by organic scaffolds capable of exertingprecise control over spatial localization of mineral nucleation and growth [40]. Thus, a natural starting point for the devel-opment of a rational framework for design of inorganic nanostructures via biomimetic templates is understanding the fun-damental principles of crystal nucleation and growth. A deep understanding of the fundamental physio-chemical processesthat govern biomineralization may provide key insights into the design of nanostructured materials that are assembled fromthe bottom-up.

3.2.1. Classical nucleation theoryThe most commonly consulted theoretical framework for understanding growth of crystals from supersaturated solutions

is provided by classical nucleation theory [42,43] (Fig. 4). Within classical nucleation theory, crystal growth is viewed as athermodynamically-driven process governed by the balance of free energies between the ionic or molecular precursors insupersaturated solution, the species in the bulk of the crystal, and the excess free energy at the crystal/solution interface(the interfacial energy). Crystallization of atoms or molecules from the supersaturated solution is driven by the lower freeenergy in the bulk crystal, but growth of small crystals is kinetically hindered by the excess interfacial energy relative tothe free energy in solution. Since the surface area grows with the crystal radius r as r2, whereas the crystal volume growsas r3, the interfacial energy dominates for small particles, and the bulk free energy prevails for sufficiently large particles.This gives rise to a dependence of the crystal free energy on the radius that exhibits a local maximum at a correspondingcritical nucleation radius. Spontaneous density fluctuations in the supersaturated solution due to thermal fluctuations yieldcrystal aggregates with varying sizes. Those with sizes below the critical radius are thermodynamically unstable with respectto growth, and those exceeding the critical radius overcome the nucleation barrier and proceed to grow bulk crystals throughaddition of atoms or molecules. During the course of subsequent crystal growth through the creation of atomic steps, theformation of new crystal step edges also presents a free energy barrier, which gives rise to a critical step size for crystalgrowth propagation from the existing crystal surface [36].

3.2.2. Controversy over classical mechanismsAlthough appealing in their simplicity, the principles of classical nucleation theory have been vigorously challenged in the

last two decades by experimental observations of mineral crystallization, general theoretical considerations, and computersimulations [42]. The phenomenology predicted by the classical theory relies on the assumptions that aggregates of ions ormolecules form nuclei with internal energies comparable to the bulk crystal, and that the notion of a phase interface in suchaggregates is well-defined in terms of classical thermodynamics. Theoretically, such assumptions may be regarded as ques-tionable at nanoscopic length-scales, and recent experimental observations suggest a more complex reality. For instance,multiple studies of crystallization mechanisms in calcium carbonate reported the existence of (meta)stable aggregates,‘‘pre-nucleation clusters,” that serve as precursors to crystal nucleation, rather than nucleation and growth by direct additionof ions [44–46]. Pre-nucleation clusters also have been observed in calcium phosphate systems [47]. Moreover, it has beenproposed that in calcium carbonate systems, nucleation of an amorphous precursor phase represents an alternative pathwayto crystallization in addition to direct nucleation of the crystal phase [48,49]. Such observations contrast with the classicaltheory, wherein crystal nucleation proceeds through the accretion of individual ions or molecules, and the crystal structureresembling the bulk is nucleated directly, rather than through maturation of metastable intermediates.

However, mounting evidence suggests that crystallization cannot be described by a single universal pathway. Other stud-ies of organic template directed crystallization of calcium carbonate [43] and glucose isomerase [50] have demonstratedcrystallization in accordance with classical nucleation theory, challenging the notion that pre-nucleation clusters and meta-stable intermediates represent a universal pathway to mineralization. Indeed, in a recent study on crystallization of mag-netite, it was proposed that direct nucleation from ionic precursors, pre-nucleation clustering, and nucleation of meta-stable intermediates together form a system of possible crystallizationmechanisms that are available to supersaturated min-eral solutions (middle panel of Fig. 4). The selection of these is dictated by the mixture phase diagram and the energetic land-scapes of aggregates, metastable phases, and the bulk crystal [51,52]. Moreover, nucleation can be bypassed duringmineralization without invoking pre-nucleation clusters or metastable intermediates under solutions conditions near thecritical point for liquid-liquid spinodal decomposition. In the vicinity of the critical point, the liquid-cluster interfacial energyvanishes, leading to unstable density fluctuations at all length scales, and broad distributions of cluster sizes that form with-out an apparent nucleation energy barrier [53]. Similarly, a recent molecular dynamics study predicted that amorphous pre-


cursors will arise in calcium carbonate mixtures when the degree of supersaturation coincides with the liquid-liquid coex-istence regime [53]. These studies indicate that the crystallization pathway can modified by the regime of the thermody-namic phase diagram within which the mixture resides. Together, these findings paint a picture of crystal nucleation andgrowth that is richer and more complex than that provided by classical theory, with a network of possible pathways replac-ing a single universal nucleation mechanism.

Despite controversy over crystallizationmechanisms, it is becoming increasingly accepted that the formation of precursoraggregates and intermediate metastable states represent important possible mechanisms for biomineralization [42,54].However, many mechanistic studies have emphasized mineralization from homogeneous saturated solutions, where the roleof organic interfaces in heterogeneous nucleation, such as that which occurs in biological systems, cannot be probed. From aclassical perspective, organic interfaces may facilitate and direct mineralization by reduction of interfacial energy of nucle-ated crystals, thereby reducing the critical nucleus size. Such a model for mineralization at organic interfaces has been val-idated in studies involving calcium carbonate crystallization on alkyl thiol self-assembled monolayers, where directmanipulation of interfacial energies by modulation of the self-assembled monolayer leads to corresponding control over cal-cium carbonate crystallization consistent with classical nucleation theory [43]. Recently, experiments have also revealed thecapacity of biomimetic organic matrices with negatively charged acid groups to localize the nucleation of metastable amor-phous calcium carbonate through enhanced concentration of calcium ions in the vicinity of acid-bearing sites [55]. This workdemonstrates that biomimetic matrices are capable of organizing the spatial localization of crystal precursors in a supersat-urated solution, suggesting that organic interfaces are capable of mediating controlled mineralization through non-classicalmechanisms. These studies provide emerging evidence that organic interfaces can direct both spatial organization and mech-anistic pathways of inorganic mineralization.

3.2.3. Biomineralization mechanisms: insight for nanoscale engineeringA detailed mechanistic understanding of nucleation and growth processes in biomineralization is not only of fundamental

interest, but also may provide a predictive framework for the rational design of novel synthetic nanomaterials. Recent workprovides indications of the potential for a mechanistically-informed biomimetic approach toward engineering nanostruc-tures with structural control that surpasses conventional materials chemistry. For instance, in a simple model system fororganic templating involving self-assembled organic thiol monolayers, cooperative reorganization of the underlying organictemplate was strongly coupled to the ability of the template to direct the growth of crystals with preferred crystal orienta-tion [56]. Thus, the flexibility of organic templates to reorganize may provide an important design parameter for controllingthe crystal orientation of nanostructures. Observations of biomineralization mechanisms in vivo also may provide directinsights into design of templating systems in vitro. For example, inspired by the observation of ferrihydrite precursors in bac-terial magnetite mineralization, an in vitro peptide-based scheme was developed for crystallizing magnetite with superiorcontrol over particle shape and size compared to traditional coprecipitation of iron(II) and iron(III) [57]. Similarly, followingidentification of a matrix protein implicated in the directed mineralization of calcium oxalate needles in Musa spp. (banana),a highly conserved peptide sequence in the matrix protein was utilized to direct the growth of similarly elongated crystalfibers in vitro [58]. Although our understanding of underlying mechanisms for organic template directed mineralizationremains in a nascent stage, these findings reflect a growing potential to harness mechanistic understanding of biomineral-ization in the development of nanostructures with unprecedented bottom-up architectural control.

4. Natural and recombinant protein inorganic templating

Proteins in nature offer a multitude of compelling examples of their ability to direct the synthesis of complex, hierarchicalstructures through biomineralization. Moreover, outside of biomineralization, proteins, themselves, are well-known for theirability to self-assemble into a variety of organizations not easily achieved by synthetic organic materials. However, with thedevelopment and increasing sophistication of recombinant techniques, proteins are no longer limited purely to those foundin nature, and a vast design space exists for developing protein technologies with self-assembly capabilities not accessiblesynthetically or in nature. Consequently, inspiration from natural biomineralization scaffolds combined with opportunitiesthrough recombinant protein technology inspires researchers to pursue a variety of novel protein systems as templates innanoparticle engineering.

4.1. Organic scaffolds in abalone shells: inspiration from nature

Some of the earliest evidence of the capacity of proteins to self-assemble nanostructures with properties exceeding thoseof synthetic systems arose from biomineralization studies on abalone shells. Abalone shells comprise a multi-lamellar hier-archy of highly-oriented, interdigitating calcium carbonate crystals that are separated by thin organic lamellae [59]. Theirnano- and micro-structure confers a tensile fracture toughness that is �3000 times that of synthetically produced calciumcarbonate crystals [60]. The unique properties and multi-scale structure are intimately connected to the proteins that con-stitute the organic phase. Even in solution, polyanionic proteins isolated from either the calcite or aragonite phases of aba-lone shells were shown to direct mineralization of calcium carbonatecrystals with distinct phases (calcite or aragonite) andmorphologies, depending on the chemical identity of the protein templates, suggesting that protein expression provides a

Fig. 5. Collagen scaffolds direct the hierarchical synthesis of alumina nanofibers. (a) Schematic of the collagen scaffold used to direct alumina assembly. Thecollagen triple-helix self-assembles into fibrils, which further organize into higher-order fibers, forming a hierarchically structured template. The collagen isfunctionalized with black wattle tannin, which enables chelation of alumina ions. After immobilization of the alumina within the collagen scaffold, thecollagen scaffold is removed by heat treatment. (b) SEM image showing the network of alumina fibers formed after removal of the collagen scaffold. Scalebar is 10 lm. (c) Higher-magnification SEM image of the alumina fiber network, illustrating the hierarchical organization of alumina fibers, which arecomposed of smaller alumina fibrils. Scale bar is 500 nm. Images were reprinted with permission from Ref. [66]. Copyright (2008) American ChemicalSociety.


switchable mechanism for controlling phase and orientation of crystal growth in vivo [61]. At a larger scale, atomic forcemicroscopy (AFM) and scanning electron microscopy (SEM) studies of shell formation in mollusks suggest that the 2D geom-etry and pore structure of the protein-based organic lamellae guides the growth of more complex 3D mineral structures withmineral bridges that interconnect hierarchically stacked pearls [59]. These studies highlight two promising features ofprotein-based inorganic templating: precise molecular recognition for control of crystal location, phase, and orientation,and self-assembling higher-order structures that orchestrate the controlled formation of elaborate 3D geometries.

4.2. Fibrous proteins

Due to inspiration from biomineralization in nature, fibrous proteins and biopolymers have attracted interest in the mate-rials science community as scaffolds for templated synthesis of inorganic materials [62–68]. Collagen and chitin are twoprominent examples of fiber-forming biopolymers that serve as organic templates for mineralization in vivo. Their tendencyto form fibers at multiple length-scales (nano-fibers, micro-fibers, and macro-fibers) results in hierarchically organized scaf-folds that function both to guide the morphology of deposited minerals as well as to localize mineral forming precursorswithin the matrix, leading to unique morphologies and properties [64]. Fibrous proteins have been demonstrated to providesimilar functionality in vitro. Collagen scaffolds were shown to direct mineralization of alumina mesoporous materials [66](Fig. 5), as well as titanium dioxide nanofibers with unusual catalytic properties for degradation of organic toxins [68]. Sim-ilarly, native fish scales, which comprise a chitin scaffold that recruits a number of small proteins, proved to be a template forproducing porous carbon materials with promising performance as electrochemical capacitors [67]. On a smaller length-scale, fibers formed from lysozyme were found to electrostatically direct the assembly of gold nanoparticles along the fibers

5 nm 15 nm 25 nm

10 s 30 s 10 min 30 min 60 mina)

b)

100 nm 100 nm 100 nm

Fig. 6. Lysozyme amyloid nanofibers direct the deposition of gold nanoparticles into 1D arrays. (a) Atomic force microscopy images of amyloid nanofiberson glass during the time-course of deposition with gold. Total elapsed deposition time is indicated in the upper right of each panel. (b) Atomic forcemicroscopy images of amyloid nanofibers decorated with gold nanoparticles with indicated diameters (upper right of each panel). The spacing betweennanoparticles deposited on the nanofibers can be tuned based on the nanoparticle diameter. Lower-right panel shows the scale-bar for the z-height inpanels (a and b). Images were adapted with permission from Ref. [65]. Copyright (2014) American Chemical Society.


into arrays with tunable particle spacing [65] (Fig. 6). These works, among others, suggest that fibrous proteins represent apromising route toward templated synthesis of complex 3D inorganic architectures.

4.3. Cage proteins

4.3.1. Viral capsidsIt is often desirable to control size and organization of inorganic nanoparticles on scales much smaller than the size scales

of organization in fibrous proteins. Viral capsids are a simple and elegant example of the ability of proteins to self-assembleinto precise, responsive nano-structures (see Fig. 7 for examples). In naturally occurring viruses, the viral capsid self-assembles from protein subunits encoded by the viral genome to form a protective coat that serves to package and protectnucleic acids and functions as a responsive delivery system for infecting a target host with the enclosed genetic cargo [69–72]. The biological challenges encountered by viruses impose stringent demands on the ability of the capsid to robustly andstably self-assemble in its target host, and to remodel in response to environmental cues. In addition, viral capsids typicallypossess a high degree of structural regularity and symmetry, and are found to exist in a variety of self-assembled architec-tures spanning a range of length-scales, including helical, icosahedral, and more complex geometries [69,71]. In vitro studiesreveal that even a particular protein coat composition can give way to a rich self-assembly phase diagram with diverse mor-phologies governed by ionic strength and pH [73]. These appealing properties of naturally occurring capsids, and theimmense potential for engineering new protein capsids through recombinant approaches, excites considerable interest inthe nanoscience and nanotechnology communities to develop virus-like particles with new functional properties, eitheras delivery systems or templates for self-assembly.

A number of groups have investigated the potential use of viral capsids as templates for engineering inorganic nanopar-ticle assemblies with constrained sizes and/or spatial organization. For example, multiple variants of cowpea chlorotic mot-tle virus (CCMV) capsids are capable of directing reduction of gold(III) precursors to form gold nanoparticle-decoratedcapsids through tyrosine residues, with high selectively for reduction of gold(III) over several other metal precursors [74].Alternatively, the endogenous histidine residues on the CCMV capsid can serve as highly specific nucleation sites for goldin the presence of a non-reducible precursor, yielding gold nanoparticle-patterned capsids with spatial organization dictatedby the highly symmetric, repeating organization of native histidine residues on the icosahedral capsid [74]. Capitalizing onthe cylindrical geometry of tobacco mosaic viral capsids, cylindrical nano-arrays of gold, platinum, and palladium nanopar-ticles were formed through reduction of cationic precursors by acidic residues [75]. It was further demonstrated that intro-ducing mutations in the viral capsid to alter the density of negative charges produced distinct changes in the spatial

Symmetry-directed self-assembly

Geometry-directed self-assembly

Size-constrained nanoparticle synthesis

Nanoparticle-templated virus-like-particle

self-assembly

a) b) c) d)

Fig. 7. Diverse strategies for organic-inorganic hybrid material self-assembly using protein cages. Nanoparticles (represented schematically as silver,yellow, and copper colored spheres) can be patterned using self-assembling protein cages for spatially directed synthesis and nanoparticle shape and sizecontrol. (a) Nanoparticles can be synthesized or selectively bound on the surface of viral capsids with spatial organization dictated by the underlyingsymmetry of the capsid. In the schematic illustration provided, the viral capsid presents inorganic binding motifs on the purple colored domains of the self-assembled capsid, resulting in selective patterning of the nanoparticles (silver spheres) onto the surface that mirrors the underlying symmetry of thetemplate. (b) Nanoparticles can be assembled in arrays whose geometries are directed by the protein cage, such as the cylindrical Tobacco Mosaic Viruscage. In this schematic, gold nanoparticles are synthesized within the interior of a cylindrically shaped viral capsid due to presentation of gold bindingresidues on the surface. (c) The well-defined size and porous structure of protein cages can be exploited for the size-constrained synthesis of nanoparticleswithin the interior. In this schematic, a porous protein cage permits diffusion of precursors into the interior of the self-assembled cage and promotes thesynthesis of a nanoparticle (copper colored sphere) within the interior whose size is dictated by the size of the enclosing capsid. (d) Inorganic nanoparticlescan be functionalized to template the self-assembly of virus-like-particles from protein cage subunits. In this schematic, a nanoparticle (yellow sphere) isdecorated with amphiphilic molecules possessing an inorganic binding domain (red tails) and a charged domain (green spheres) that interact with proteinmonomers of a self-assembling capsid. The protein monomers self-assemble around the functionalized inorganic nanoparticle, forming a virus-like particlewith an inorganic nanoparticle in the interior. Viral capsid illustrations were generated using the UCSF Chimera package from the Computer GraphicsLaboratory [85] using files contributed to the Protein Data Bank from Refs. [164–167].


organization of nucleated nanoparticles. In addition, the well-defined sizes of viral cages and their porous structures, whichallow diffusion of reaction precursors, enable them to behave as size-constrained nano-reactors that direct the synthesis ofinorganic nanoparticles with controlled size distributions within the interior of the capsid [76]. These studies demonstratethe potential of viral capsids to produce nano-arrays of metal nanoparticles with geometry and dimensionality dictated bythe underlying geometry of the self-assembled capsid, and spatial distributions that can be tuned by engineering the presen-tation of nucleation-directing amino acid residues. Viral capsids thus provide a versatile platform for nano-particle miner-alization and assembly.

Alternatively, inspired by the ability of many wild-type viral capsids to self-assemble around their electrostaticallycharged genetic cargo, other researchers explored inorganic-organic hybrid materials in which the viral protein cage encap-sulates functionalized nanoparticle templates. The potential to enclose nanoparticles in protein coats similar to nativeviruses offers the possibility of producing nanoparticles equipped with proteins that are engineered for specific responsive-ness or other functionality. For example, gold nanoparticles functionalized with carboxylate-terminated thiolalkylated tet-raethylene glycol chains can serve as templates for self-assembling protein cages from brome mosaic virus proteins [77].Under suitable conditions, a protein coat efficiently assembled around the gold nanoparticles to form caged structures withprotein stoichiometry, symmetry, and low polydispersity reminiscent of the icosahedral capsids that form around the RNAtemplate in the native brome mosaic virus. In order to develop a deeper understanding of the mechanisms that underlie theprotein cage assembly around charged nanoparticles, a combinatorial study was designed to decouple geometric and elec-trostatic effects. Below a critical charge density essentially no protein cages are formed, irrespective of the total nanoparticle


charge [78]. This furnishes evidence of the possibility to rationally design protein cage assembly based on physical principlesand independently tunable parameters.

4.3.2. Ferritin cagesProtein cage structures for biotemplated inorganic nanoparticle synthesis are not limited to viral capsids. One of the most

common non-viral proteins used for synthesizing inorganic nanomaterials is ferritin. Ferritin is a class of spherical proteincages made up of 24 identical self-assembling subunits. It performs a crucial role in maintaining iron homeostasis by seques-tering iron as a nanoparticle of iron oxide (ferrihydrite) in the interior cavity of the cage [79]. This demonstration of naturallyoccurring nanoparticle synthesis has made ferritin a popular target for developing alternative syntheses. In addition to ironoxide [80], ferritin has been used to generate particles of silver [81], cobalt oxide [82], nickel [83], chromium [83], and cal-cium carbonate [84], among others.

4.3.3. Heat shock proteinsHeat shock proteins represent yet another example of naturally occurring proteins that can self-assemble into symmetric

cage structures, which can serve as nano-reactors that are amenable to chemical or genetic engineering. The self-assembledstructures possess pores that enable exchange of reactants and products between the interior and the environment, and pre-sent amino acid residues that function as spatially defined reactive sites. For example, the heat shock protein cage fromMethanococus jannaschii can direct the size-constrained synthesis of ferrihydrite nanoparticles from iron precursors viaacidic amino acid residues in the interior of the protein cage [85]. Moreover, both endogenous lysine groups and geneticallyengineered thiols served as reactive sites for conjugation to fluorophores with well-defined spatial locations. Capitalizing onthe ability of the same heat shock protein to nucleate controlled synthesis of platinum nanoparticles, a biomimetic hydro-genase was developed to efficiently catalyze hydrogen gas production [86] by platinum catalyzed reduction of protons. Dueto their ability to crystallize into well-defined arrays, protein cages formed from heat shock proteins also can direct the self-assembly of micron-sized nanoparticle arrays, such as quantum dot arrays for electronic or photonic applications [87]. Sucharrays are amenable to genetic modification to design non-native pore sizes for quantum dot incorporation [87].

5. Peptide only systems

A powerful feature of proteins that direct biomineralization in living organisms is the existence of specific peptide bindingmotifs that enable spatial localization of inorganic precursors. The vast diversity of peptide sequences available for explo-ration, together with the existence of high throughput combinatorial techniques for their selection [88], renders the designof novel self-assembling peptides with high affinities for crystal precursors a promising route toward synthesis of nanostruc-tures with refined control over composition, morphology, and spatial distribution. The ability of peptides to provide molec-ular recognition functionalities has been demonstrated not only to promote nanoparticle nucleation in solution conditions

Fig. 8. Phage display enables high-throughput evolution of molecular-recognition peptides. (a) A large combinatorial library of peptide-encoding DNAsequences (green, yellow, and red colored segments) is generated and integrated into the genome of the desired phage (black circles). (b) Phages areamplified in a bacterial host, generating phages that present peptides (green, yellow, and red colored ellipsoids) from the combinatorial library on thesurface of their protein coat. (c). Phage capsids that present peptides with high affinities for a desired material (e.g. a nanocrystal, represented here by a redsphere) are purified by affinity selection. (d) Sequences of the affinity-selected, molecular-recognition peptides are directly determined by sequencing thegenomes of the purified phage capsids. Phage capsid illustrations were generated using the UCSF Chimera package from the Computer Graphics Laboratory[85] using files contributed to the Electron Microscopy Databank from Refs. [168,169].


under which it would otherwise not occur, but also to provide morphological control of crystal growth, such as by high speci-ficity of binding to specific crystal faces [89–91].

5.1. Peptide evolution by phage display

Naturally occurring peptides are highly evolved to serve specific functionality in living systems, but they are capable ofnucleating only a limited range of crystal morphologies and compositions. A broad range of potential synthetic nanomate-rials with important technological applications, such as semiconductors, are not naturally synthesized in biological systems.Thus, the development of peptide-based organic templating systems for nanoparticle synthesis hinges crucially on the abilityto design novel peptide sequences with functionalities not currently found in nature. To this end, evolution of peptides byphage display is a powerful combinatorial technique for screening vast arrays of peptide sequences to identify molecularrecognition peptides with high binding affinities for nanocrystals or crystal precursors [88] (Fig. 8).

Phage display is an in vitro technique in which a large library (�109) of peptides are fused into the coat protein genes ofphage virions, thereby producing an array of phages that display distinct peptide sequences on their capsids. These virionscan subsequently be separated by binding affinity purification with a desired binding ligand, thereby isolating peptides withhigh binding specificity. The physical linkage between the phage phenotype and the corresponding genome encoding for thepeptide allows the specific peptide sequences to be directly identified. Although this technique is predicated on evolutionbased on affinity to the final crystal, and not nucleation kinetics, it was recently demonstrated that binding affinity to modelself-assembled monolayers is inversely related to the free energy barrier to nucleation, consistent with classical nucleationtheory [92]. Thus, binding affinity to the desired crystal product may serve as a proxy for nucleation efficiency for some sys-tems. Phage display has been successfully utilized to evolve novel peptide sequences with high affinities for a variety of sub-strates, including Al [93], carbon nanotubes [94], graphene [95], and semiconductors [18], and also for discovery of peptideswith catalytic activity for inorganic nanocrystal synthesis [96]. Moreover, the incorporation of non-natural amino acids fur-ther expands the available peptide library, and was implemented to develop peptides with high metal ion binding affinities[97]. Thus, the high-throughput nature of phage display and the possibility of incorporating a variety of non-natural aminoacids enables the development of peptide sequences for assembly of nano-architectures that are not only biomimetic, butthat yield products that extend beyond those found in the biological realm. The ability to directly evolve peptide sequencesbased on their desired functional properties also circumvents challenges associated with predicting peptide properties in denovo peptide design.

5.2. Mechanisms of biomineralization by peptides

Phage display technology enables high throughput selection of peptide sequences with desired binding properties with-out a priori knowledge, but it does not provide direct understanding of the underlying molecular mechanisms that confermolecular recognition properties of peptides [98]. Moreover, peptide design based purely on combinatorial techniquesmay overlook peptides with desirable functional properties due to biased and incomplete libraries, [98] or desired function-ality that does not directly translate from binding affinity alone. For example, a peptide sequence selected by affinity-basedphage display may direct growth of widely varying morphologies under different reaction conditions [99]. Thus, knowledgeof the fundamental principles that mediate peptide molecular recognition may offer powerful input for further refinementand discovery of peptide structures.

Although recent studies on calcite nucleation by model self-assembled monolayers demonstrate crystallization behavioradherent to classical nucleation theory [92], where crystal binding energy directly correlates with nucleation efficiency, anumber of studies in peptide-mediated nucleation point to the addition of more subtle and complex factors at work.Non-classical effects aside, peptide binding itself depends on a multitude of factors, including electrostatic and hydrophobicproperties of the substrate [100], as well as flexibility and conformational entropy of the peptide adsorbed to the target sur-face [101–103]. Even within a particular amino acid primary sequence, constraints that impose changes in secondary struc-ture can have profound changes on binding energies, as demonstrated in a series of gold adsorption studies involving linearand cyclized forms of peptides with identical primary sequences [104]. The situation is further complicated by non-classicalnucleation mechanisms. For instance, a recent study combining experiments and molecular dynamics simulations onpeptide-mediated binding and nucleation of silver revealed pronounced differences in growth of silver nanoparticlesbetween peptides with comparable binding energies [105], but different simulated 3D conformations. Overall, the currentbody of work reveals a complex structure-function landscape for peptide recognition systems involving classical and non-classical mechanisms, where a variety of approaches, including combinatorial screening, post-screening site-specific muta-tions [99,102,106], molecular simulations [101–105], and computational bioinformatics [107], are required to elucidate themolecular underpinnings of technologically-relevant functionality.

5.3. Examples of peptide systems

5.3.1. Soluble peptidesA number of specific peptide template systems for biomineralization have been developed, which exhibit varying levels of

structural complexity, ranging from single peptides to hierarchical assemblies. The simplest technologically relevant peptide

Fig. 9. Soluble peptides direct the synthesis of palladium and platinum nanoparticles with tunable morphology. (a) Schematic of the peptide templatesystem. The peptides consist of a hydrophobic domain (red), a hydrophilic domain (blue), and the R5 inorganic template sequence, which was isolated fromthe diatom Cylindrothica fusiformis. The peptides self-assemble into micelles with a hydrophobic core and a hydrophilic exterior that presents the R5 peptidesequence, which directs inorganic nanoparticle growth (left). The resultant micelles can be used to template the synthesis of a variety of inorganicnanoparticles, including palladium and platinum (shown here). Depending on the chemical identity and the precursor concentration, different nanoparticlemorphologies arise. In the case of palladium, increasing the precursor concentration results in a transition from spherical nanoparticles to nanoribbons, andfinally to branched nanoparticle networks (NPNs). In the case of platinum, only spherical nanoparticles are produced regardless of the precursorconcentration. (b) TEM images illustrating the influence of increasing the molar ratio of palladium (II) to peptide (PdX indicates X moles of palladium (II) topeptide) on nanoparticle morphology. Scale bars are 20 nm. Images were reprinted with permission from Ref. [109]. Copyright (2013) American ChemicalSociety.


systems are soluble peptides, which have been successfully implemented to direct growth of metal nanoparticles with a vari-ety of sizes, morphologies, and consequent optical and or catalytic properties. Such peptides often contain a-helical domainsthat direct spatial localization of metal ions, with subsequent growth that is morphologically tunable through a range of fac-tors. Even a single peptide sequence can orchestrate the synthesis of distinct morphological properties under different envi-ronmental conditions. This has been demonstrated, for instance, in work where a single peptide sequence can form an a-helical secondary structure at mildly alkaline conditions, but random coil structure under strongly alkaline conditions. Con-sequently, this peptide directs the assembly of fluorescent silver nano-particles with distinctly different sizes and emissionspectra at different pH [108]. The ability of metal-binding peptides to strongly localize metal ions also leads to a delicatedependence of particle morphology and functionality on peptide to metal ratios, as shown in a study of peptide-mediatedsynthesis of catalytic palladium nanoparticles [109] (Fig. 9). Tertiary structure also provides an important tuning parameterfor nanoparticle synthesis. For example, a-helical peptides that spontaneously assemble into supercoiled trimers, tetramers,and hexamers promote nucleation of fluorescent silver nanoparticles with varying optical properties that are directly relatedto the expected number of metal ions sequestered by the coiled-coil multimers [110]. Similarly, it was found that the num-ber of tandem a-helical repeats in homologous repeat proteins could control the morphology and optical properties of goldnanoparticles in template-directed synthesis [111]. Therefore, even comparatively simple soluble peptide systems yieldopportunities for template-directed synthesis of nanoparticles with a variety of technologically-relevant properties, whichmay be tuned at the level of primary, secondary, and tertiary structures.

5.3.2. Peptide arraysSoluble peptide systems enable growth of nanoparticles with some degree of tunability, but they provide limited control

over hierarchical spatial organization of nanoparticle assemblies. A promising strategy for exerting more elaborate self-assembly of superstructures is the use of 2D peptide arrays as programmable templates that direct nucleation of nanopar-

Fig. 10. Self-assembling collagen-like peptides serve as templates for metallic nanowire synthesis. (a) Schematic of the collagen-like peptide nanofibertemplate system. In step 1, collagen-like peptides equipped with lysine residues can be functionalized with amine-reactive gold nanoparticles, which serveas nucleation sites for electroless plating by reduction of gold or silver. In step 2, the gold-decorated collagen-like peptides self-assemble into nanofibers.Finally, in step 3, electroless plating by reduction of silver produces metallic nanowires whose morphology is dictated by the underlying peptide nanofibertemplate. (b) TEM images of nanofibers following 1, 2, and 3 electroless silver deposition cycles (left to right). Scale bars are 100 nm. Images werereproduced from Ref. [124] with permission of the Royal Society of Chemistry.


ticles into pre-determined spatial distributions. Inspiration for such peptide arrays can be found in nature, such as in the S-shells that enclose many prokaryotic organisms, which are composed of proteins that self-assemble into 2D lattices of a vari-ety of forms [112]. S-shells containing regularly ordered functional groups can be exploited as templates to guide the growthof nanoparticle superlattices with a variety of pre-programmed symmetries and compositions, including metallic and semi-conducting arrays [113–117]. Furthermore, S-shells isolated from living organisms can be further chemically modified toincorporate novel functionality and specificity not found in naturally occurring protein lattices, opening broader possibilitiesfor bottom-up assembly of supramolecular architectures [113]. Potentially even greater flexibility may be afforded by the


design of bio-inspired synthetic peptide array systems, such as the peptide-functionalized graphene templates developed topattern nanoparticle arrays with electrocatalytic functionality [118]. In direct contrast with conventional lithographic pat-terning techniques, which involve expensive, top-down sequential processing [115], these peptide array approaches enablebottom-up, in situ self-assembly of superstructures with architectures that are dictated by the spatial distribution of phys-iochemical properties of the underlying template.

5.3.3. Self-assembling peptidesIn addition, peptides can be designed to spontaneously self-assemble into other 2D or 3D architectures through inter-

molecular associations, such as nanofibers and nanotubes. Self-assembled scaffolds comprise individual components thatspontaneously self-assemble into stable supramolecular structures under specific conditions. Spontaenous self-assemblyoften occurs in response to shifts in pH, ionic strength, and concentration, [119] and the non-covalent bonds that stabilizethe resulting structures typically include hydrogen bonds, ionic bonds, hydrophobic interactions, and van der Waals inter-actions [120]. Self-assembly of peptide-amphiphiles is often driven by the tendency of the aliphatic tails to aggregate inaqueous solution at high concentrations. Several peptide-based self-assembling systems have been developed in recent years[121], including some materials applicable to inorganic nanotemplating. For example, single tail peptide-amphiphile mole-cules that self-assemble into nanofibers of varying morphology, surface chemistry [119,122–129], and bioactivity have beendesigned to template minerals such as hydroxyapatite [125].

Through judicious amino acid selection and alkyl tail modification, peptide-amphiphiles have been developed to undergopH-induced self-assembly into nanofibrous structures that mimic the architecture of the native extracellular matrix [125].Drawing inspiration from the natural tendency of collagen fibrils to promote oriented hydroxyapatite crystal growth, it wasdiscovered that incorporation of acidic phosphoserine residues into the peptide sequence of a known self-assembling pep-tide could promote the formation of calcium phosphate minerals along the fibrillar surface post-assembly. Acidic phospho-rylated moieties are known to play an essential role in biomineralization processes, and the designed peptide sequenceresulted in a surface display of phosphate similar to the repetitive organization of phosphate groups found in natural phos-phophoryn proteins.

Moreover, peptide sequences may be modified to produce scaffolds with less common natural functions, such as conduct-ing nanowires. For example, metal-binding peptide sequences may be introduced into self-assembling peptide sequences topromote the association of inorganic precursors with the peptide surface. The diversity of peptide sequences available andtheir inherent responsiveness to solution conditions offers the possibility for bottom-up self-assembly of functionalizednanowires with morphologies tuned by peptide composition or solution conditions. In recent years, peptide nanofiber-directed synthesis has been used to robustly produce a variety of inorganic nanomaterials with controllable morphologies,including 1D silver nanowires [124] (Fig. 10), 1D platinum nanostructures [129], lipophilic inorganic nanoparticles [126], sil-ica nanotubes [127], and coaxial metal tubes [122].

Peptide-directed inorganic templating may be used to produce more complex inorganic nano-assemblies. While 1D and2D peptide scaffolds have served as the primary biologically inspired templates to date, 3D hierarchical nanomaterial archi-tectures are becoming increasingly prevalent. For example, doughnut-shaped peptide nano-assemblies have been utilized tocreate nanoreactors [130,131]. In another exemplary case, alternative 3D architectures were used to produce palladiumnanoparticle networks and ferroelectric barium titanate nanoparticles under mild conditions [132]. As peptide self-assembly becomes better understood and more predictable, peptide templating will likely become an increasingly facileand effective method of synthesizing complex inorganic nano-assemblies not accessible via more conventional assemblytechniques.

6. Protein-peptide hybrid systems

While a wide range of inorganic structures have been produced using exclusively protein or exclusively peptide tem-plates, a number of innovative templating systems were recently developed that utilize both peptides and proteins to createnovel and broadly applicable scaffolds. Such systems combine the high specificity of molecular recognition peptides and the3D architecture conferred by protein self-assembly, opening the potential for engineering precisely spatially organized bind-ing motifs for nucleation of inorganic materials along with other functionalities.

6.1. Phage capsid-peptide systems

Viral capsids are a natural platform for functionalization with peptides. For example, a highly versatile protein-peptideplatform was developed using genetically engineered M13 bacteriophage that recognizes specific inorganic molecules andselectively directs their growth into higher-ordered structures. The engineered M13 system has been used produce nanoma-terials with a diverse range of functions, including nanocrystals applicable in the semiconductor industry [18,133], cathodesfor lithium-oxygen batteries [134], and ordered arrays of quantum dots. Other applications for the M13 bacteriophage sys-tem include the production of dye-sensitized solar cells [135,136], templating of magnetic nanoparticles for targeted in vivoimaging of prostate cancer [137], and stabilization of single-walled carbon nanotubes for use in noninvasive imaging andsurgical guidance of submillimeter tumors [138]. Like many other protein-peptide fusion systems, the M13 phage system

clathrin-binding inorganic-bindingLinker

KDVSLLDLDDFN GGGG RKLPDAPGMHTWEEEEAHHAHHAAD

KDVSLLDLDDFNKDVSLLDLDDFN

GGGGGGGG

TP1TP2TP3

a)

b)

c)

Fig. 11. Template engineering through epitope recognition (TEThER): Clathrin-peptide hybrid systems provide tunable inorganic nanoparticle synthesisthrough modular design. (a) Schematic of a clathrin cage. The clathrin cage (left) is formed from repeat units of triskelion monomers (highlighted in lightblue and shown on the right) that self-assemble into a protein cage. (b) Design of TEThER peptides. Heterobifunctional peptides are synthesized that consistof a clathrin binding domain and an inorganic binding domain separated by a glycine spacer. The inorganic binding domain can be designed in a modularfashion to produce peptides with affinity for the inorganic material of interest. Here, TP1, TP2, and TP3 represent peptides whose inorganic binding domainsexhibit affinity for titanium dioxide, cobalt oxide, and gold, respectively. (c) Schematic of the TEThER templating process. Clathrin triskelion monomers self-assemble into clathrin cages in solution (left). The desired TEThER peptide can be bound to the clathrin cage through eptitope recognition at the clathrinbinding domain, yielding a decorated clathrin cage that presents inorganic binding domains on its surface (middle). Incorporation of the appropriateinorganic precursors yields directed synthesis of inorganic nanoparticles within or on the surface of the clathrin cage (right). In the schematic provided, theclathrin template is functionalized with either titanium oxide (red), cobalt oxide (green), or gold (blue) nanoparticles, depending on the choice of TEThERpeptide and inorganic precursors. Images from a) were reproduced from Ref. [170] under a CC-BY-4.0 license agreement. Images from b) and c) wereadapted with permission from Ref. [142]. Copyright (2011) American Chemical Society.


combines the ability of the capsid proteins to self-assemble into a well-defined architecture with the precise recognition fea-tures of engineered peptides.

6.2. Ferritin-peptide systems

Similar to phage capsids, the self-assembled ferritin protein cage offers a variety of opportunities for molecular engineer-ing through genetic or post-translational incorporation of molecular recognition peptides. For example, ferritin protein cageswere genetically engineered to incorporate either titanium or carbon nanotube binding peptide aptamers with selectiveaffinity to their target substrates while retaining the ability of endogenous ferritin subunits to self-assemble into cage struc-tures and mineralize iron [139]. A similar strategy was pursued to achieve the selective growth of silver nanoparticles in theinterior of a ferritin cage genetically modified to display a peptide known to reduce silver(I) ions to silver [140]. Even broaderopportunities exist for producing inorganic-ferritin hybrid materials functionalized with molecular recognition peptides fora variety of applications. For instance, ferritin cages were engineered to present surface peptides that block protein receptorsimplicated in asthma, and the symmetric organization of peptide ‘‘bunches” mediated significantly improved affinity of theprotein-peptide chimera to the target receptor compared to the soluble peptide alone [141].


6.3. Clathrin-peptide systems

Clathrin protein cages are similarly amenable to augmentation with molecular recognition peptides. To move away fromthe need for chemical and genetic modifications which may disrupt protein integrity or assembly properties, a strategy wasdeveloped to mimic the naturally occurring phenomenon of adaptor proteins that bind to a central protein complex at speci-fic sites to modify the overall function of the complex (Fig. 11). Peptides were designed to functionalize clathrin cage scaf-folds through site-specific molecular recognition using short binding sequences from clathrin-binding proteins. Thesepeptides also contained inorganic-nucleating peptide sequences similar to those discussed above, rendering them bifunc-tional. The use of non-covalent, site-specific functionalization allowed the components to be treated in a mix-and-matchmanner, making the self-assembled clathrin platform versatile for a range of modifications. Clathrin cages, complexed witha family of engineered peptides, was used to separately synthesize nanoparticles of titania, cobalt oxide, and gold [142]. Thisstrategy, termed TEThER (Template Engineering Through Epitope Recognition), was extended to the use of multiple peptidesaimed at distinct binding sites on the clathrin inner and outer surfaces to generate multi-material nanoparticles containingsilver and gold in different arrangements. Particles were synthesized with mixed domains or core-shell arrangements of sil-ver and gold [143]. Furthermore, careful control of the ratio of inorganic precursor, bi-functional peptide, and clathrin cagescaffold was shown to lead to tunable arrangements of synthesized gold particles into solutions of dispersed or clusteredparticles, illustrating the potential of this non-covalent functionalization strategy for predictable and controlled hierarchicalassembly [144].

7. Challenges in nanomaterials

7.1. Reproducibility in nanomaterial synthesis

While biomaterial templates are a promising and innovative platform for the production of nanomaterials with complexarchitecture, lower costs, high-throughput production methods, and increased reproducibility will be essential to the devel-opment of next-generation commercial optical, electronic, and magnetic materials and devices. Many complicated newnanomaterials will rely on robust connections between multiple components, and defects in any of the 2D and 3D nanos-tructures used in these systems could impair function and reduce or eliminate their industrial utility [145]. Theoretical guid-ance has been successfully used to guide experimental design and reduce defect formation in some conventional templatingsystems, including one scaffold for the growth of zinc oxide nanowire arrays [146]. However, similar approaches are notcommonly used in peptide-based templating. Moreover, techniques to quickly and cost-effectively characterize nanomate-rials to screen for defects and enhanced properties are still in the early developmental stage. Benchtop production methods,which are commonly used to produce nanomaterials in academic settings, rarely include advanced control systems. Whilenanomaterials created using benchtop production methods are useful in many industrial applications in spite of the rela-tively low level of automation and reaction control involved in their synthesis, cost effective reaction scale-up presents abarrier to industrial adoption. More fundamentally, the use of nanomaterials is also limited by the current level of under-standing of what nanoarchitectures are most useful in a given application [147]. Future research into nanostructure-property relationships will be necessary to fully exploit the complex inorganic nanoarchitecutres that may be produced fromengineered biological templates.

7.2. Mechanical properties of organic-inorganic hybrid materials

Although protein-templated nanomaterials are highly versatile and could be used in diverse industrial applications, thelow mechanical strength [148,149] of most protein-templated nanomaterials has limited their adoption outside the biomed-ical industry. Even in nonstructural applications, such as optical and electronic devices, certain mechanical properties arerequired to ensure functionality. However, natural inorganic-protein nanocomposites such as bone, teeth, and nacre exhibitmechanical toughness and strength that notably exceed their individual components [150]. Increased understanding of theviscoelastic properties of strong naturally occurring nanocomposites will likely inform future design strategies, leading totougher bioinorganic nanocomposites that can be exploited in a wider range of commercial products [151,152].

7.3. Environmental concerns

The influence of nanomaterials, including protein-templated inorganic nanocomposites, on the environment and humansafety is still poorly understood. While the market for nanomaterials is rapidly expanding, research into their environmentalimpact is in its infancy. Reports have discussed environmental hazards; however, the cause and extent of the environmentalburden is currently unknown [153–155]. This is due in part to the vast range of nanomaterials that have been developed, andtheir rapid ongoing discovery. The small size of nanomaterials increases the risk of their unintentional release into the envi-ronment through atmospheric emissions and waste streams at production facilities. Unintentional release of nanomaterialsinto the atmosphere could ultimately result in soil and water contamination as the airborne nanomaterials deposit on avail-able surfaces. While low-level contamination may not be harmful to the environment, significant levels of environmental


accumulation due to spillage or other production or disposal methods could prove to be toxic to surrounding wild life and,indirectly, to humans.

7.4. Health concerns

In addition to environmental considerations, human safety must be considered when designing nanomaterials, especiallynanomaterials intended for clinical use [14,156–159]. Off target cytotoxicity is one of the primary safety issues encounteredduring clinical nanomaterial administration. Nanomaterials often acquire protein coronas and interact with secondary sitesin vivo [158,160]. Nanomaterial shape and exposure route often dictate material toxicity by affecting site-specific accumu-lation of nanoparticles and subsequent local toxicity. Shape in particular plays a large role in determining biodistribution, asrod shaped particles are more easily internalized by cells and more frequently exocytosed than spherical nanoparticles [14].Moreover, shape and administration route can affect the interaction between nanomaterials and phagocytic cells. Ingestionof nanomaterials by phagocyctic cells can lead to undesired removal and accumulation of particles in the organs such as theliver, spleen, and lungs, which may reduce the potency of nanomaterial-based treatments while increasing the risk ofadverse immune response following recognition of the nanomaterials by phagocytic cells. Intelligent materials design couldpotentially mitigate many off-target effects, facilitating the use of nanomaterials in the treatment of diseases other than can-cer, where cytotoxic side effects are the norm.

Other safety issues to consider include unintentional exposure to nanomaterials, including those not intended for clinicaluse, through inhalation and skin exposure [159]. Inhaled nanoparticles, especially very small nanoparticles (20 nm particles),have very high deposition efficiencies in the lungs, where their accumulation may lead to severe immune responses. Theeffects of nanoparticles on the skin are unclear since it is not yet known how many nanomaterials can penetrate the skinand how nanomaterial properties change under different exposure conditions. However, introduction of nanomaterials intothe bloodstream via initial skin contact could have detrimental health consequences, especially in the case of toxic metalnanoparticles. Inhalation and skin exposure are especially important concerns in the context of manufacturing, since workersafety may be compromised if nanomaterial toxicology is not well understood.

8. Conclusion and outlook

In spite of the potential challenges associated with their design, manufacture, and use, nanomaterials will become moreprevalent in the biomedical, semiconductor, and energy fields. The unique properties derived from the nanoscale structure ofthese materials will enable new and useful products to be produced on increasingly large scales as the field develops. Theability to precisely template inorganic nanomaterials will be key to developing next-generation nanomaterials, since com-plex nanostructures are likely to produce different effects than the simple 1D and 2D architectures used at present. Whileconventional synthetic techniques are increasingly being adapted to meet nanomaterial design challenges, proteins and pep-tides enable facile control over nanomaterial structure under mild conditions. Biotemplates, inspired from biomineralizationin nature, promise to provide multifaceted control over inorganic nanomaterial synthesis. As we describe in this review, evi-dence already is emerging for their potential to control the shape and size of nanoparticles, their molecular recognition capa-bilities for selective binding to specific inorganic species, their ability to spatially localize inorganic materials to specificunderlying patterns, and their 3D self-assembly capabilities. The integration of these control strategies in next-generationbiotemplates may yield unprecedented control in synthesizing hierarchical nanostructures. The reduced environmentaland health burden associated with the production of nanomaterials at ambient temperature in aqueous solvent using pep-tide or protein-based templates makes them an attractive platform for next generation materials design. However, the chal-lenges described above associated with bio-templated nanomaterials will need to be addressed before the platform’spromise can be fulfilled. While a great deal of progress has been made in the past decade to elucidate design principlesand explore potential applications, bioengineered templating of inorganic nanomaterials is still an immature field with con-siderable untapped potential.

Acknowledgements

The authors gratefully acknowledge helpful discussions with Sebastian Doniach and Nick Melosh. This work was sup-ported by the Department of Energy, Office of Basic Energy Sciences, Division of Materials Sciences and Engineering, undercontract DE-AC02-76SF00515. B.A.K. was supported by the Stanford BioX Fellowship Program. ACP was supported by theDepartment of Defense (DoD) through the National Defense Science & Engineering Graduate Fellowship (NDSEG) Program.Molecular graphics images (protein cages and phage illustrations) were produced using the UCSF Chimera package from theComputer Graphics Laboratory, University of California, San Francisco (supported by NIH P41 RR-01081).

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