assembly of bacteriophage into functional materials · optical phage-based sensors, which combine...

SUNG HO YANG,1,2 WOO-JAE CHUNG,1,2 SEAN MCFARLAND,1,2 ANDSEUNG-WUK LEE1,2*1Department of Bioengineering, University of California, Berkeley, California 94720, USA2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California94720, USAE-mail: [email protected]

Received: April 30, 2012Published online: December 20, 2012

ABSTRACT: For the last decade, the fabrication of ordered structures of phage has been of greatinterest as a means of utilizing the outstanding biochemical properties of phage in developinguseful materials. Combined with other organic/inorganic substances, it has been demonstrated thatphage is a superior building block for fabricating various functional devices, such as the electrodein lithium-ion batteries, photovoltaic cells, sensors, and cell-culture supports. Although previousresearch has expanded the utility of phage when combined with genetic engineering, mostimprovements in device functionality have relied upon increases in efficiency owing to thecompact, more densely packable unit size of phage rather than on the unique properties of theordered nanostructures themselves. Recently, self-templating methods, which control both ther-modynamic and kinetic factors during the deposition process, have opened up new routes toexploiting the ordered structural properties of hierarchically organized phage architectures. Inaddition, ordered phage films have exhibited unexpected functional properties, such as structuralcolor and optical filtering. Structural colors or optical filtering from phage films can be used foroptical phage-based sensors, which combine the structural properties of phage with target-specificbinding motifs on the phage-coat proteins. This self-templating method may contribute notonly to practical applications, but also provide insight into the fundamental study of biomacro-molecule assembly in in vivo systems under complicated and dynamic conditions. DOI10.1002/tcr.201200012

Keywords: bacteriophage, functional materials, liquid crystals, self-assembly, template synthesis

1. Introduction

The controlled assembly of basic structural building blocksinto long-range, ordered, periodic structures is an importantchallenge in many science and engineering fields, includingphysics, chemistry, biology, materials science, and electricalengineering. For the development of functional nanosystems,bottom-up material-assembly approaches have attempted to

integrate various components, including proteins, blockcopolymers, and nanoparticles.[1–4] It is postulated that thewell-defined or well-ordered structures that are formed by self-assembly have great promise for fabricating devices on a largescale. Ordered structures on the nano- or micrometer scalepossess several advantages for improving the performance of

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Assembly of Bacteriophage intoFunctional Materials

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integrated devices. 1) Their close-packed structures, whichresult from highly ordered materials, like crystals, are essentialfor improving the efficiency of devices such as solar cells, sec-ondary batteries, and memory-storage devices.[5,6] Further-more, the number of building-block units per volume is criticalin keeping costs low whilst maintaining high efficiency. 2)Ordered structures frequently enhance the performance of adevice, thereby augmenting the properties of its functionalbuilding blocks.[7,8] For example, directionally ordered buildingblocks can maximize functional performance by summing unitproperties such as magnetic strength, electric field, and dipolemoment.[9] 3) Ordered structures can provide unexpected anduseful functional properties beyond that offered by each build-ing block individually. Regardless of the inherent properties ofa building block, ordered structures can exhibit collective char-acteristics, such as structural color,[10] surface plasmon reso-nance,[11] and optical filtering.[12] These emergent functions,which are provided by ordered structures, can be synergisticallycombined with the native functionalities that are provided bythe underlying building blocks to yield devices that exceed thescope of what would otherwise be possible.

Although methods for the self-assembly of functionalmaterials have born witness to a number of significant advance-ments over the past decade, the generation of highly complexand hierarchically organized structures remains challenging.The fields of nanotechnology and materials science have con-currently been inspired by self-assembly processes in biologicalsystems, such as glass sponges (optical fibers),[13] brittle stars(optical lens array),[14] diatoms (sophisticated periodic struc-tures),[15] abalone shells (fracture-resistance materials),[16] andbones (support structure for vertebrates).[17] In biological

systems, the assembly of hierarchically organized, functionalstructures is directed by the mutual orchestration of proteinsand genes, which simultaneously regulate the synthesis oforganic and inorganic precursors. Although mimicking theself-assembly processes of biological systems is a promisingroute for the creation of new bio-nanomaterials, such processesremain obscured by the highly complex interplay betweengenes and proteins within an organismal context.

As a tool for overcoming this hurdle, the genetic engineer-ing of viruses offers new opportunities to integrate variousnanoscale materials into hierarchically ordered structuresthrough biologically inspired material-assembly processes.Phage, which is composed of relatively few proteins and genescompared to prokaryotic and eukaryotic cells, represents asimplified starting point for embarking upon such work. Bymimicking natural evolutionary processes, phage can beselected that specifically recognizes desired targets on themolecular level, thereby adding to their versatility as a basicbuilding block.[18] Through phage display and geneticengineering, phage has been used as a specific template forproducing functional nanomaterials, such as conductive/semiconductive nanowires.[19–21] For a decade, many research-ers have exploited virus particles for assembling orderednanostructures and for fabricating useful materials and devicesfor energy,[22–24] batteries,[25,26] biosensors,[27] and tissue-regenerating materials.[28] In this Personal Account, we intro-duce recent accomplishments in the assembly of bacteriophagefor fabricating functional devices and review the potentialfuture applications of this emerging technology.

2. Structure and Characteristics of Bacteriophage

Bacteriophage (phage) is a virus that replicates itself and propa-gates its genetic information by infecting bacterial host cells.[29]

Phage may take many different shapes, from linear (M13, Fd,F1),[30] to spherical (MS2),[31] to sophisticated head-tail struc-tures (T4, T7).[32] Among these shapes, the rod-like, filamen-tous shape of M13 phage constitutes an excellent biologicalbuilding block for the assembly of new materials. There areseveral reasons for this viewpoint (Figure 1):

First, normal, wild-type M13 bacteriophage exists as afilamentous nanofiber with a diameter of 6.6 nm and a lengthof 880 nm. This highly anisotropic shape, along with themonodispersity that results from its high-fidelity biologicalreproduction, endows this phage with the ability to exhibitliquid-crystalline properties. In suspension, this propertyallows for the artificial formation of ornate ordered structures.

Second, the surface of the phage can be modified bothgenetically and chemically to introduce specific peptides. TheM13 phage is composed of several major- and minor coatproteins that encapsulate its single-stranded DNA genome.

Dr. Seung-Wuk Lee earned his PhD inChemistry and Biochemistry from theUniversity of Texas at Austin in 2003under the supervision of ProfessorAngela Belcher. He completed post-doctoral work with Professor CarolynBertozzi in the Molecular Foundryat Lawrence Berkeley National Labora-tory. He is a currently a Professor in the Department ofBioengineering, University of California, Berkeley. Hiscurrent research interests include phage-based biomimeticmaterials, bionanotechnology, stimuli-responsive biopoly-mers, and regenerative medicine. In addition, based onphage-based techniques, he is developing a general conceptthat is termed “Virotronics”, which exploit the unique prop-erties of genetically engineered phage to carry informationto build functional medical- and electronic materials anddevices.

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The viral capsid is composed of about 2700 copies of thehelically organized pVIII major coat protein and 5–7 copies ofminor coat proteins pIII, pVI, pIX, and pVII.[33,34] The pVIIImajor coat proteins make up the side-walls of the sheath, whilstcomplexes of the pIII/pVI and pIX/pVII minor coat proteinsare located at either end. Thanks to advances in biotechnologyand phage engineering over the last two decades, biochemicalmodifications on each of the coat proteins have been achievedthrough genetic manipulation[35–39] and site-specific organic-synthesis approaches.[23,40–47]

Third, phage that bind specifically to a particular targetmaterial can be isolated through the use of phage display (seebelow).[48–49] These specific interactions between phage andtarget materials provide the capability to formulate phage-based hybrid materials.

3. Liquid-Crystalline Properties of Phage

Unlike spherical particles, it is well-known that rod-shapedmolecules or colloidal particles exhibit liquid-crystallineordered phases in suspension. Owing to their responses toelectromagnetism and polarized light, liquid crystals are usedfor many applications, including liquid-crystal displays[50] andoptical switches.[51] It is known that there are two types ofliquid-crystalline phases: thermotropic and lyotropic liquid-crystalline phases. Thermotropic phases are determined withina certain temperature range by the degree of each particle’sthermal movement. Lyotropic phases are influenced not onlyby temperature but also by the concentration of the particles.Hence, lyotropic liquid-crystals exhibit more-varied phasesthan thermotropic liquid crystals. In biological systems, thelyotropic liquid crystal is a driving force for the self-assembly ofproteins and cell membranes.

Because of their long, rod-like shape and monodispersity,filamentous viruses also show liquid-crystalline behavior in

suspension.[52] Thus, phage has drawn great attention as anexperimental model system for probing the structure anddynamics of liquid crystals on the molecular level, not least, inpart, owing to the ease with which organized phage structurescan be imaged by techniques as simple as optical microscopy(Figure 2). The liquid-crystalline behavior of the virus isdependent on various factors, such as phage concentration,ionic strength, and external fields.[52–55] Among them, the con-centration of phage is the most-significant factor that affectsthe lyotropic liquid-crystal phase. At low concentrations(below 5 mg ml-1), phage are randomly distributed withoutany order and are referred to as being in an isotropic phase.Within the concentration range 10–20 mg ml-1, at which thedistance between neighboring phage becomes smaller than itslength, phage particles align into orientationally ordered con-figurations, called nematic-phase liquid crystals, owing to stericforces between the phage particles. At this concentration, nem-atically ordered phage begin to intertwine with each otheralong the perpendicular axis of the layers, which results in ahelicity of the phage particles. Within the concentration range20–80 mg ml-1, the helicity of the layers becomes more dis-tinct and leads to the emergence of cholesteric-phase liquidcrystals.[53,56] These phage exhibit unique optical characteristics,such as a fingerprint texture and the capacity to reflect circu-larly polarized light.[53,56,57] At very high concentrations (above100 mg ml-1), phage are ordered both positionally and orien-tationally along one direction within well-defined layers, calledsmectic-phase liquid crystals.[58] According to the degrees ofpositional and orientational order, there are many differentsmectic phases. For example, in the Smectic A phase, phage areoriented along the layer normal, whilst, in the Smectic C phase,

Fig. 1. a) Schematic structure of M13 bacteriophage and b) a genomic dia-grams which shows each protein that is expressed on the M13-phage surface.

Fig. 2. Schematic structures of the liquid-crystalline phases of M13 bacte-riophages. Top: typical liquid-crystalline phages; bottom: hierarchicallyordered liquid-crystalline phages.


A s s e m b l y o f B a c t e r i o p h a g e i n t o F u n c t i o n a l M a t e r i a l s

they are tilted at an angle from the layer normal. After com-plete dehydration, the phage mostly arrange into their thermo-dynamically more-stable phases, such as the smectic phases.[54]

Recently, a method that can hierarchically order typicalcrystalline phases was developed by kinetically tuning thedehydration reaction. As a function of concentration andevaporation speed, nematic orthogonal twist, cholesteric helicalribbon, and smectic helicoidal filaments have been formed(Figure 2). These hierarchically ordered structures will befurther discussed in Section 9. On the other hand, Fraden andco-workers observed many other interesting microstructuresfrom mixed suspensions of rod-shaped phage and sphericalcolloids. In their study, they elucidated the fundamental rela-tionships between molecular properties, such as length andcharge, and macroscopic properties, such as phase behav-ior.[50,52,55,56,58] This binary phase behavior has also been used tofabricate highly ordered functional hybrid composites.[59]

4. Modification of the Phage Surface

Bacteriophage has served as an ideal template for the realizationof various functional materials and devices because of the pro-cessability of its surface proteins, as well as its well-definednanostructure. The capsids of bacteriophage can be modifiedby genetic engineering or by chemoselctive organic synthesis tointroduce specific amino acids or specific functional groupsonto desired coat proteins. To understand the genetic engineer-ing of the phage, it is helpful to understand the typical “lifecycle” of phage: Briefly, contact with a bacterial host cell isinitiated by pIII, which recognizes and binds to the sex pilus ofbacteria that carry the F plasmid. Subsequently, the phagegenome is injected into the host cell. The foreign DNA repro-duces their corresponding genes and proteins by hijacking themetabolism of the host cell. After the assembly of gene and coatproteins, the reproduced phage are released from the host cell.

By manipulating the infection process in an artificialmanner, such as through heat or electric shock,[60] recombinantgenes can also be delivered into the bacterial host. Engineeredphage can then be produced as normal by the hijacked metabo-lism of the host cell. This process allows specific amino acids tobe expressed on target sites in coat proteins by delivering thecorresponding nucleotide sequence in a recombinant gene.

On the other hand, chemoselective synthetic approacheshave been developed to introduce various functionalities ontothe surface of phage. In general, amino acids in coat proteins,such as lysine, aspartic/glutamic acid, cysteine, and tyrosine,are conjugated with functional derivatives through relativelymild reactions, such as EDC/NHS activation, maleimide-thiolcoupling, diazonium reagents, and click chemistry.[23,40–47] Byintroducing functional derivatives onto the surface of phagethrough such reactions, the phage can be employed in drug-

delivery systems,[61] as MRI-contrast agents,[62,63] cell-imagingagents,[44] and biosensors,[27] or in photosyntesis.[23] Organicsynthetic approaches and their applications have been reviewedin detail elsewhere by Francis and co-workers and by Wanget al.[42,45,47]

5. Phage Display

Phage display is a high-throughput, combinatorial screeningprocess that identifies, by selecting from random peptide librar-ies, functional peptides that are expressed as fusion proteinson the phage coat surface. This method is considered to bean accelerated evolutionary screening process for selectingpeptides that can specifically bind to target materials(Figure 3).[48,49] For phage display, a random phage library mustfirst be established. This library is accomplished by splicingrandom sequences into the appropriate site of a chosen coatprotein gene. The expressed recombinant protein will thendisplay a highly diverse library of peptides (up to 1011 randomsequences) on the surface of the phage capsid.[48,49,64] Theselibraries can then be screened against the target material.

Among the library population, some phage will bind tothe target with greater affinity, owing to interactions betweentheir displayed peptide and the target material. After washingaway weakly bound phage, the remaining strongly boundphage are eluted separately and are then amplified by infectinghost bacteria. This process is repeated over several rounds toisolate the most-strongly binding phage. Finally, the specificbinding peptides can be identified through DNA analysis ofthe selected phage’s genome.

The phage-display method was originally invented toidentify epitope-like short peptides sequences that can replace

Fig. 3. Schematic diagram of the phage-display processes for identifying aspecific peptide against the target substrates.



antibodies.[37] The specific sequences that are identifiedthrough phage display against specific target materials canfunction as epitopes through protein folding. Various epitope-like peptides have been isolated through phage display againstproteins,[65–67] DNA,[68] receptors,[69] and tissues.[70] Recently,this method was further advanced to identify the short peptidemotifs that specifically bind to inorganic materials, such asGaAs, GaN, Ag, Pt, Au, Pd, Ge, Ti, SiO2, quartz, CaCO3,ZnS, CdS, Co, TiO2, ZnO, CoPt, FePt, BaTiO3, CaMoO4,and hydroxyapatite, including carbon nanomaterials, such asC60, carbon nanotubes, and graphene.[71–75] It has been foundthat these specific peptides are highly effective at nucleatingand mineralizing various inorganic materials.[76–78] Further-more, phage that display specific peptides have also been usedas a template for various new functional nanomaterials,such as conductive/semi-conductive nanowires.[19–21] For moreinformation about target-specific peptides, inorganic-material-binding motifs have previously been summarized in variousreviews and books.[71,77,79]

6. Assembly of Phage with Organic Materials

M13 phage possesses advantageous structural features as abuilding block for 1D fibers because of its anisotropic shapeand monodisperse size. By exploiting rod-shaped phage struc-tures, Lee and Belcher fabricated micro- and nanosized-diameter fibers through wet-spinning and electrospinning,respectively.[80] Suspensions of M13 phage were extrudedthrough the capillary tubes into a cross-linking solution ofglutaraldehyde to generate fibers with a diameter of 10–20 mm.The suspension of the smectic ordered phage was transformedinto nematic structures that ran parallel to the fiber long axis,driven by flowing forces. In addition, fibers with a diameter of100–200 nm were fabricated by electrospinning. After blend-ing with polyvinyl pyrrolidone (PVP) to improve their process-ability, the PVP-blended phage was then electrospun intocontinuous fibers.

The authors confirmed that this method was biocompat-ible by showing that resuspended phage fibers in buffer solu-tion maintained their infectivity against bacterial hosts. Chianget al. developed two strategies that could introduce function-alities onto the M13 phage fibers, that is, before or afterwet-spinning the phage into fibers.[81] In the former case, tet-raglutamate phage, named E4-phage, were chemically conju-gated with amine-terminated cadmium selenide quantum dots(QDs). Microfibers were then fabricated through a wet-spinning process. The resulting QD-modified fibers emittedred light under exposure to UV light. In the latter case, geneti-cally modified M13 phage that displayed a gold-specificpeptide sequence (Val-Ser-Gly-Se-Ser-Pro-Asp-Ser) were firstspun into microfibers. Gold nanoparticles were then closelyassembled along microfibers through the specific interactions.

Polymeric templates have also been utilized in the 2Dassembly of M13 phage. Yoo et al. found that M13 phagecould be closely ordered through preferential electrostaticinteractions between phage particles and two oppositelycharged weak polyelectrolytes, such as linear polyethylenimine(LPEI) and poly(acrylic acid) (PAA).[82] The spontaneous 2Dassembly of phage was driven by a combination of competitiveelectrostatic interactions between phage particles and polyelec-trolyte, the inter-diffusion of the polyelectrolyte, and the aniso-tropic shape of the M13 phage. The phage were assembledaccording to the following procedure (Figure 4a): First, nega-tively charged phage were randomly deposited on the positivelycharged top surface of LPEI/PAA multilayers (step 1). Next,the adsorption of PAA on the top phage layer of the thinphage/LPEI/PAA multilayers led to a separation process thatforced the phage to the surface because of stronger electrostaticinteractions between LPEI and PAA, rather than with phage(step 2). Finally, the floating phage spontaneously ordered intoa monolayer structure owing to mutually repulsive interactionsbetween neighboring phage particles (step 3). The repeateddeposition of LPEI and PAA led to a free-standing film thatpresented more closely packed structures. They showed thatthe resulting phage films could be utilized as scaffolds fornucleating cobalt nanowires or for aligning gallium-nitridenanoparticle over multiple-length scales by expressing a specificpeptide sequence on pVIII major coat proteins of the M13phage. Furthermore, Yoo et al. spatio-selectively assembledphage onto patterned, multilayer polyelectrolyte that was gen-erated by solvent-assisted capillary-molding methods.[83] Thesize of the patterns could be adjusted by the swelling propertiesof the underlying patterned polyelectrolyte multilayer. Toexamine the possibility of biocompatible application, thesurface of patterned phage was modified with biotin-conjugated anti-Fd bacteriophage that specifically bould topVIII major coat proteins of the M13 phage. The biotin-modified pattern was visualized by exposing the substrate toAlexa-Fluor-conjugated streptavidin.

On the other hand, Solis et al. also developed phage-patterning methods by applying a subtractive contact-printingtechnique for patterning phage antibodies.[84] They immobi-lized individual phage into single-element arrays by optimizingboth the concentration of the phage suspension and the featuresize of the antibody.

These techniques for the ordered patterning and close-packing of phage are important for utilizing phage as abiosensor and energy-storage/-conversion device because suchcontrollability is directly related to the efficiency and/or sensi-tivity of such devices. Nam et al. utilized polymer-inducedclose-packed M13-phage structures to realize high-voltagelithium-ion batteries (Figure 4b).[25] The phage was first engi-neered to express both tetraglutamate (4E) and gold-bindingpeptide motifs on the pVIII major coat proteins for nucleating



gold and cobalt-oxide nanoparticles, respectively. By using theengineered phage as a scaffold, gold-nanoparticle-incorporatedcobalt-oxide nanowires (Au-Co3O4 nanowires) were producedwith a high degree of crystallinity under aqueous conditions atroom temperature. By applying these phage-based Au-Co3O4

nanowires to the anodic electrode of a lithium-ion battery, theenergy-storage capacity of the battery was improved by morethan 30% compared to that with pure Co3O4 nanowires. Namet al. proposed that this improvement in electrochemical prop-erties could be explained by the participation of gold nanopar-ticles in the redox reaction, which may enhance the electronic

conductivity of the Co3O4 nanoparticles, or may result in acatalytic effect.

To organize phage-based electrodes over large lengthscales, engineered M13 phage was assembled in 2D on the topof LPEI/PAA multilayers through competitive electrostaticinteractions (Figure 4c, d). After that, close-packed Au-Co3O4

nanostructures were formed on the assembled phage films onthe centimeter-length scale. The cell that was constructed withphage-based electrodes reached 94% of its theoretical capacity,with a high cycling rate. The resulting virus-based lithium-ionbattery exhibited lightweight, flexible, and transparent proper-

Figure 4. a) Schematic diagram of the strategy for the monolayer assembly of M13 phages on the polyelectrolytemultilayer of LPEI/PAA. b) The virus-enabled synthesis and assembly of nanowires as negative-electrode materials forLi-ion batteries. c, d) Phase-mode AFM image of 2D-assembled Co3O4 nanowires, driven by liquid-crystallineordering of the engineered M13 phage, and its magnified image. Z range is 30o.



ties, as well as superior electrochemical performance comparedto conventional batteries. This enhanced performance ismainly caused by the monodispersity of the Au-Co3O4 nanow-ires, the close-packed assembly of the layered nanostructures,and a high surface-area-to-volume ratio. Nam et al. furtherdeveloped phage-based electrodes for the fabrication of anarray of micro-electrodes, based on the hypothesis that micro-electrodes that are ordered in multidimensional architecturescould be beneficial for improving electrochemical performanceand for designing new types of batteries.[85] Co3O4 nanowireswere spatio-selectively assembled on the micropatternedPDMS template through the combination of phage engineer-ing, LPEI/PAA layer-by-layer, and microcontact-printing. Apatterned array of micro-electrodes was fabricated by stampinga self-assembled layer of phage-templated Co3O4 onto aplatinum-microband current collector. The resulting micro-electrode array was electrochemically functional as a compo-nent of a lithium-ion battery.

Although the phage was not assembled in an orderedmanner, Lee et al. developed a phage-based cathodic electrodefor lithium-ion batteries.[26] The major coat protein of the M13phage was engineered to express both tetraglutamate (4E) fornucleating amorphous anhydrous FePO4 (cathode material)and peptide motifs (Asp-Met-Pro-Arg-Thr-Thr-Met-Ser-Pro-Pro-Pro-Arg) for capturing conductive single-walled carbonnanotubes (SWNTs). They found that the incorporation ofSWNTs improved electrochemical performance by increasingthe local electronic conductivity in the cathode. The phage-based cathode showed a greatly enhanced capacity of134 mAh g-1 at a high discharge rate of 3 C, compared to thebest reported capacity (80 mAh g-1 at 3 C). In addition, almostno fading of the capacity was observed, even after up to 50cycles.

7. Self-Assembly of Phage withInorganic Materials

Increasingly, phage has been utilized to assemble nanomateri-als, driven by liquid-crystalline ordering of the engineeredphage particles. The ordered hybridization of phage and inor-ganic materials is mainly based upon the specific interactionsbetween them, which can be engineered through phage-displaymethods. Lee et al. reported the multi-length-scale ordering ofquantum dot (QD)-hybrid biocomposite structures by usinggenetically engineered M13 phage (Figure 5).[59] After selectinga zinc sulfide (ZnS)-binding peptide sequence (Cys-Asn-Asn-Pro-Met-His-Gln-Asn-Cys) through the screening of phage-display libraries, the peptide was expressed on the pIII minorcoat protein. The engineered phage was suspended in solutionsof the ZnS precursor to form a phage/ZnS hybrid liquid-crystalline suspension. Liquid-crystalline suspensions varied in

structure as a function of phage concentration and the smecticphase (at 127 mg ml-1), cholesteric phase (at 76–28 mg ml-1),and nematic phase (at 22 mg ml-1) were all observed. Inparticular, in the cholesteric phase, cholesteric pitchesexponentially decayed with respect to an increase of phageconcentration.

In addition, the structure could become nematic under amagnetic field, presumably because of the diamagnetic prop-erties of the major coat protein of the M13 phage. 3D-ordered,self-supporting phage/ZnS hybrid films were induced by slowevaporation; the viruses and nanocrystals gradually progressedthrough isotropic, nematic, cholesteric, and smectic phases inthe suspension media, while they were still mobile, and com-plete dehydration finally afforded free-standing films that werecomposed of a smectic-like lamellar structure. Interestingly, thesurface viral morphology was smectic O and the interior mor-phologies were smectic A and C. The resulting films weretransparent and continuous over many centimeters. Moreover,the films exhibited an ordered pattern of 1 mm fluorescentlines, which implied that the ZnS-QDs were confined at theend of the M13 phage, owing to the specific binding. Lee andBelcher also developed a nanoparticle-alignment method by

Fig. 5. a) Photograph of the free-standing ZnS-phage film. b) POM (x20)image, which shows birefringent dark- and bright-band patterns (periodiclength: 72.8 mm). These band patterns were optically active and their patternsreversed depending on the angle between the polarizer and the analyzer. c)Photoluminescent image, with an excitation wavelength of 350 nm and withfiltering below 400 nm, which shows a striped pattern (width: about 1 mm,x50). d) AFM image of the ZnS-phage film. The phage forms parallel alignedherringbone patterns that are almost at right angles to the adjacent direction(arrows).



using anti-streptavidin phage.[53] After isolating a streptavidin-binding motif (His-Pro-Gln) through the screening of a phage-display library, the anti-streptavidin phage was assembled withstreptavidin-conjugated gold nanoparticles. The mixed suspen-sion showed liquid-crystalline character and the structurestransformed from the nematic phase (14 mg ml-1), to the cho-lesteric phase (about 76–20 mg ml-1), to the smectic phase(about 83 mg ml-1) as a function of phage concentration.Smectic-ordered self-supporting phage/Au hybrid films wereformed by slowly drying mixed suspensions on gold films.They further applied this method for ordering organic dyes orbiological molecules by assembling anti-streptavidin phagewith streptavidin-conjugated fluorescein or phycoerythrin. Thepatterned fluorescence in the hybrid films confirmed that fluo-rescein or phycoerythrin were localized between the smecticlayers.

Multiple binding sites have been introduced on the surfaceof phage, with each site being used to align different nanopar-ticles. Huang et al. reported the assembly of nanoarchitecturesof various geometries and materials, such as arrays of goldnanoparticles or CdSe-QDs, by using highly engineered M13phage as a template.[86] M13 phage was rationally engineered toexpress gold-binding motifs (Val-Ser-Gly-Ser-Ser-Pro-Asp-Ser)on pVIII major coat proteins and streptavidin-binding motifs(Ser-Trp-Asp-Pro-Tyr-Ser-His-Leu-Leu-Gln-His-Pro-Gln) onpIII minor coat proteins. The engineered phage directed theformation of heterostructures on which the gold nanoparticles(5 nm) arranged into an ordered 1D array on major coat pro-teins along the viral axis, whilst the streptavidin-conjugatedgold particles (15 nm) bound to one end of the phage. Byreplacing the streptavidin-coated gold nanoparticles withstreptavidin-conjugated CdSe-QDs, they demonstrated thatnanoarchitectures that were composed of hetero-nanoparticlearrays could be rationally designed. In addition, they showedthat gold-nanoparticle arrays on pVIII proteins could subse-quently function as templates for highly conductive continu-ous nanowires by interconnecting gold-nanoparticle arrayswith the electro-less deposition of gold.

On the other hand, metal ions have also been used tomediate the assembly of phage to achieve nanostructured tem-plates for inorganic alloys or biocomposites. Lee et al. usedgenetically engineered phage to form the mechanical frame-work for nanophase Co-Pt alloys, which is an extensivelystudied magnetic material because of its high magnetocrystal-line anisotropy.[87] Biopanning against Co2+ ions specified Co2+-binding peptide sequences on the pVIII major coat protein.The isolated sequence (Glu-Pro-Gly-His-Asp-Ala-Val-Pro)contained a well-known metal-binding motif (Glu-X-X-His)that is frequently found in active sites of metalloproteins. In thepresence of Co2+ ions, the engineered M13 phage becamedirectionally ordered and assembled into fibrous structureswith a sponge-like morphology at low phage concentrations

(about 108 pfu mL-1), which was much lower than that requiredfor liquid-crystalline formation (about 1012 pfu mL-1).[53] Itseems that Co2+ ions increase the local concentration of phageparticles by stimulating the aggregation of phage bundles. Thephage bundle acts as a template for nucleating Co-Pt nanopar-ticles, which are uniformly deposited on the fibrous structuresand show both a crystalline nature and a narrow size distribu-tion. They also demonstrated that Co-Pt alloys have typicalsuperparamagnetic properties with high anisotropy.

Wang et al. reported that Ca2+ ions triggered the self-assembly of wild-type Fd phage, which could act as both ascaffold and as a Ca2+ source for the oriented nucleation ofbone minerals.[88] The formation of phage bundles could beexplained by the fact that Ca2+ ions served as a cross-linkingbridge between negatively charged wild-type Fd phage, whichcontained two negatively charged amino acids (1 Glu and 3Asp) on the surface of the pVIII major coat protein. By reactingthe Ca2+-ion/phage bundles with phosphate ions, hydroxyapa-tite (HAP) nanocrystals were grown along the long axis ofphage bundles. The oriented nucleation and growth of HAPnanocrystals resembled the natural bone-formation mecha-nism; bone minerals crystallized parallel to the long axis ofthe collagen fibrils. In addition, the Ca2+-ion/phage bundlescompleted the formation of HAP nanocrystals within 12 h,whereas most previously reported substrates needed muchlonger time periods, ranging from several days to weeks.

By using a similar method, He et al. also produced phage/HAP biocomposites by using M13 phage that was geneticallyengineered to display the negatively charged peptide octa-glutamate (8E) on the pVIII major coat protein.[89] Afterassembling the 8E-phage into a nanofibrous bundle with Ca2+

cations, precursors for HAP accumulated within the bundle,thereby making the local environment supersaturated, whichresulted in the nucleation of HAP nanocrystals parallel to thelong axis of the phage bundle. They also found that theco-assembly of phage and collagen into nanofibers improvedthe mineralization process, in terms of a stronger directionalpreference along the long axis and more-compact mineralizedstructures. Such improvements could be explained by thehypothesis that glutamates on the side-wall of the phage couldenhance the hydrogen-bonding interactions with collagenfibers, thus resulting in their hierarchical co-assembly. Theseresults indicated that rationally designed phage structures canplay a similar role to that of real tissues in biological systems.

Other practical applications of phage-inorganic hybridmaterials have also emerged. Liu et al. have developedhumidity-tunable surface plasmon resonance (SPR) sensorswith virus-based nanocomposite films.[90] They fabricated uni-formly distributed gold-nanoparticles/phage nanocompositefilms by alternately depositing negatively charged gold nano-particles and positively charged M13 phage that display tet-raarginine (4R) on the pVIII major coat proteins. The



nanoarchitecture of the films could be tuned by using layer-by-layer assembly; the gold nanoparticles could be more-closelypacked, thereby leading to a dense accumulation of nanopar-ticles, as a function of increasing the number of depositedlayers. They found that the SPR absorbance of the nanocom-posite at lmax, or lmax itself, could be systematically tuned byincreasing the number of deposited gold nanoparticles. Thisphenomenon was likely caused by changes in the amount ofdeposited nanoparticles or changes in the dielectric constant.In addition, the nanocomposite exhibited decreased SPRabsorbance and a blue-shift of the lmax value as a function ofincreased humidity. The SPR response showed a linear rela-tionship with the relative humidity in the range 19–86%,which implied that it was possible to apply the phage-basednanocomposite as a humidity sensor. The humidity depen-dence can be explained by the fact that the SPR spectra of theAuNPs are highly dependent upon the interparticle spacing.Dehydration may flatten the phage-based nanocomposite,which leads to a corresponding decrease in the interparticlespacing.

8. Self-Assembled Phage to ControlCellular Morphology

A filamentous bio-polymer structure is one of the most-common features in biological materials that make up extra-cellular matrices, such as collagen, carbohydrate, and elastin.[91]

As an approach to mimic such structures, the fabrication ofmacroscopically ordered architectures based on fibrillar tem-plates of M13 phage has been of great interest. As discussedabove, M13 phage demonstrates lyotropic liquid-crystallinebehavior; thus, the direction of the assemblies can be affectedby the shear flow that is exerted on them. In addition, filamen-tous colloidal particles tend to align in a flow field, owing to thetorque that the flowing solvent exerts on the particle’s cores.[92]

Merzlyak et al. reported an approach for the fabrication ofanisotropic structured materials from genetically engineeredM13 phage by using a flow-induced injection method.[93] Theyfabricated aligned phage-nanofiber matrices to examine theircapability for controlling the macroscopic behavior of neuralcells. Liquid-crystalline suspensions of RGD- and IKVAV-modified phage were mixed with neural progenitor cells andmanually injected into liquid agarose (Figure 6a). The struc-ture of the spun phage fibers persisted in the agarose aftercooling and solidification. The phage fibers that wereentrapped in the agarose possessed nematic-ordered liquid-crystalline structures with uniaxial orientation that wasinduced by flow-injection. This fact was verified by polarizedoptical microscopy and by SEM, which showed liquid-crystalline alignment of the fiber, which was over a centimeterin length, parallel to the long axis (Figure 6b, c). Neural pro-

genitor cells that were cultured in the phage matrices modifiedwith signaling motifs (RGD, IKVAV) were observed to remainviable, proliferate, and differentiate into neural cells. In addi-tion, the liquid-crystalline phage matrix could control thedirection of neuronal growth along the length of the fibers(Figure 6d). Such structurally aligned functional phage matri-ces may offer promising opportunities for therapies thataddress challenging biomedical problems, such as the regenera-tion of nerve tissue within the central nervous system.

The same approach was employed by Yoo et al. to fabri-cate liquid-crystalline fiber matrices for bone-tissue engineer-ing.[94] By using genetic engineering, they created phage thatdisplayed the DGEA peptide (a known signaling motif thatinduces bone-cell differentiation) on every copy of the capsid’spVIII major coat protein. MC-3T3 bone-progenitor cellswithin the DGEA-phage fiber matrix in agarose were stimu-lated to express actin filaments, osteogenic proteins, such ascollagen type I, osteopontin, alkaline phosphatase, osteocalcin,and matrix acidic phosphoprotein I, even at an early stage(12–24 h).

Kuncicky et al. first reported the flow-assembly methodfor aligning rod-shaped viruses into large-scale ordered films byusing the receding meniscus of a virus-containing suspen-sion.[95] Virus fibers were aligned in a linear manner, normal tothe liquid/air/solid three-phase contact line, when droplets of asuspension of the virus on a surface receded by using an appa-ratus for controlled deposition (Figure 7a). The scale of the

Fig. 6. Nematic aligned M13 phage fibers for neural-tissue engineering. a)Schematic diagram of the fabrication of the nematically ordered phage fiber inagarose gel through flow-induced alignment, in which an oriented nanofibrousphage matrix directs the neural cells. b) Photograph of the phage fibers (length:about 1 cm) spun in agarose. c) Polarized optical microscopy (POM) image ofthe nematic liquid-crystalline structure of phage fiber (scale bar: 50 mm).d)POM images of the differentiated neural cells within the RGD-phage matrixafter 6 days, which show that the neuritis extended parallel to the long axis ofthe phage fiber. The red arrow indicates the direction of phage alignment; theblue arrows indicate the oriented outgrowth of neurites.



deposition process is only limited by the size of the substrateand it is scalable beyond centimeter-length scales because thealignment process only occurs at the three-phase-contact line,regardless of the volume of the suspension of the virus. Theresulting aligned virus films served as a template for metallizingwires. This directional virus-assembly method inspired manyother research groups to investigate the behavior of orientedassemblies of M13 phage and the potential of the assembledmatrix for tissue engineering.[96–98]

Rong et al. chemically modified M13 phage with RGDpeptides to enhance the binding between the cells and the M13matrices.[98] The incorporation of RGD peptides onto thesurface of the phage was carried out in two steps: Alkyne-derivatized N-hydroxysuccinimidyl ester was coupled to theamino group on the surface of phage. A CuI-catalyzed azide-alkyne 1,3-dipolar cycloaddition reaction was performed toconjugate the azide-derivatized RGD peptides onto the alkynylfunctional group on the surface. The conjugation was verifiedby MS (MALDI-TOF) analysis of the protein subunits. Thechemically modified phage were aligned by using the samemethod as that reported by Kuncicky et al.[95] The phage solu-tion (20 mg ml-1) was first deposited onto the positivelycharged silane-coated cover glass and the meniscus wasdragged, thereby resulting in the formation of a thin-film

matrix that was composed of ordered phage. AFM analysisshowed that the phage (wildtype/RGD-phage = 3:1) aligneduniformly parallel to the dragging direction. NIH-3T3 andChinese hamster ovarian (CHO) cells that were cultured onthe aligned phage matrices both elongated their cytoskeletonand nuclei in a single direction along the long axis of the fibrilsin the matrices.

On the other hand, genetically engineered M13 phage hasbeen employed to make aligned biomimetic extracellularmatrices by using a shearing method.[96,97] Merzlyak et al. engi-neered M13 phage to display RGD peptides or RGE peptides(as control peptides) on its major coat protein (pVIII) byusing a partial library-cloning approach.[99] On one edgeof 3-aminopropyltriethoxy-silane-treated glass substrates, adroplet of phage solution (10–30 mg ml-1) was placed anddragged (by using a glass slide) parallel to the long axis of thesubstrate. By applying a shear force in a single direction, theengineered phage were aligned in that same direction. Thesheared phage films exhibited anisotropic surface morphologythat was formed with aligned fibrous structures parallel to theshear direction (Figure 7b), whereas drop-cast phage filmsexhibited a surface morphology of randomly organizedbundles. The roughness and diameters of the bundled struc-tures in the sheared films were tunable by varying the initialphage concentration (rms = 3.1–22.4 nm, d = 20–900 nm,respectively; Figure 7c). When neural progenitor cells (NPCs)were seeded on the aligned phage films (RGD, RGE and wild-type), four hours later, only the cells on the RGD-phage filmextended their filamentous cytoskeleton. This result indicatesthat the integrin motifs on the matrix played a crucial role incell-adhesion and spreading.

In addition, the anisotropic nanogrooves that were formedin the RGD-phage matrix contributed to the orientation ofcellular growth (over 80% of the elongated cells were alignedwithin a 20° angle of the aligned phage fibers). The NPCs thatwere cultured on the RGD-phage in differentiation mediaexhibited an outgrowth of neurites in the direction parallel tothe aligned phage fibers, whereas few cells were attached toRGE- and wild-type-phage matrix and formed round shapesunder the same conditions. The aligned RGD-phage matrixprovided both biological and biochemical cues for the attach-ment and elongation of NPCs and the outgrowth of theneurites. The genetically engineered phage matrix showedversatility for the directional growth of other types of cells, suchas preosteoblasts and NIH-3T3 cells (Figure 7d).[97]

On the other hand, self-standing phage-based alignedfibers have been developed for cell-encapsulation and 3D cell-cultures. Chung et al. took advantage of the electrostatic inter-actions between abundant net negative charges on the surfaceof the phage building blocks and cationic polymers at theinterface where the membrane formation occurs to makeself-standing phage-based fibers.[97] First, the liquid-crystalline

Fig. 7. Directional assembly of virus particles by using shearing force. a)Schematic representation of the apparatus for depositing aligned virus-fibercoatings. b) Atomic force microscopy (AFM) images that show long-range-ordered liquid-crystalline viral films (20 mg ml-1, scale bar: 5 mm). c) ShearedM13-phage films, which show different surface roughness depending on theinitial concentration. d) Fluorescence image of NIH-3T3 fibroblasts that weregrown on the RGD-phage-aligned film in serum-free media for 12 h. Theactin filaments are stained with phalloidin (green) and the nuclei are counter-stained with DAPI (blue); scale bar: 100 mm.



phage suspension was mixed with cells of interests and injectedinto a solution of the cationic polymer (poly-l-lysine). Theorientation of the liquid-crystalline phage was flow-induced byinjection into the polymer solution through the syringe; thus,the structure of the liquid-crystalline phage matrix was pre-served by the resulting phage/polymer complex and its sur-roundings. NIH-3T3 fibroblasts that were encapsulated withRGD-phage/PLL migrated and elongated their bodies alongthe phage matrix (day 7), thus indicating that cells respondedto the chemical and physical cues from the directionally orga-nized phage-based fiber matrices. The directionally orderedphage fibers supported cell-viability and directional growth,thus demonstrating its potential use as a material for tissueregeneration.

The chemical micro-pattern has been employed to selec-tively attach and align phage for the directional guidance ofhuman fibroblasts (Figure 8).[100] Yoo, Chung et al. preparedtwo chemical striped patterns (1-octadecanethiol (ODT)/cysteamine on gold) with different spacing (cysteamine/ODT,5:10 mm and 10:20 mm) by using a microcontact-printingmethod. Next, the stripe pattern was immersed in a suspensionof RGD-phage and pulled vertically, thereby resulting in theselective deposition of phage onto the cysteamine regions.During the drying process at the contact line, two dominantmovements of the meniscus affected the deposition of phage onthe cysteamine regions: the downward movement of the menis-cus and the de-wetting from hydrophobic to hydrophilicregions. Vertical orientation of the phage fibers was observed

along the central axis of the vertical hydrophilic stripes,whereas diagonal orientation of phage deposition was observednear the side edges. The selectively deposited phage structures,which provided topographical cues to the cells, along withbiochemical signals, appeared critical to guiding humanfibroblasts to grow and elongate in a uniform direction. Thisresult shows the hybrid bottom-up and top-down approachescan be useful for the study of cell behavior in differingmicroenvironments.

9. Hierarchical Assembly of Phage

Although many organized structures of phage have beencreated with the help of cross-linking agents, polyelectrolytes,and inorganic nanomaterials, it still remains a challenge toassemble phage by itself into ordered structures. Along theselines, Lee et al. fabricated long-range-ordered virus-based filmsby using only M13 phage as a building block.[54] The films wereformed by the slow evaporation of a suspension of phage andthe thickness of the free-standing films was tuned by varyingthe phage concentration. The resulting films predominantlyexhibited chiral smectic structures. Although nematic orderedstructures were formed at very low concentrations, the struc-tural controllability of the phage films still lacked the complex-ity and hierarchy that is present in liquid-crystalline phases insuspension. In suspension, phage can assemble into variousstructures and sustain their conformation owing to the liquid-

Fig. 8. Schematic illustration of selective phage patterning for the directional growth of human fibroblast cells.Hydrophobic/hydrophilic striped patterns (1-octadecanethiol/cysteamine) were created by using a microcontact-printing method. Negatively charged RGD-phage was selectively aligned on the cysteamine-stripe patterns throughvertical pulling of the patterned substrate that was immersed in the phage suspension. The RGD-phage-patternedsubstrates were then used for the directional guidance of human fibroblast cells.



crystalline properties of anisotropic-shaped viruses. However,slow dehydration, which led to solid or free-standing films,mostly resulted in more-thermodynamically stable morpholo-gies, because the phage particles gradually lost their mobilitywhilst drying and ended up in a highly concentrated phase.Otherwise, the phage were deposited onto the substrates withdisordered structures, driven by fast evaporation.

Most recently, Chung et al. developed a versatile strategyfor the self-templated assembly of M13 phage into hierarchi-cally organized structures; this strategy was suitable for variousfunctions, including the generation of structural colors, thedirectional guidance of cell growth, and the templating oforganic-inorganic composites through biomineralization.[101]

Unlike previous methods, they introduced a kinetic elementinto phage assembly by using a simple and highly tunableinstrument that allowed for the fine control of both meniscusmovements on the substrate and of competing interfacial forcesat the meniscus line (Figure 9). Briefly, phage that displayedtetraglutamate (4E) on the pVIII major coat protein wereassembled into large-area films by pulling substrates verticallyfrom suspensions of phage at precisely controlled speeds. At theliquid/solid/air triple-phase interface, evaporation proceededfaster and resulted in the accumulation of phage. Thus, chiralliquid-crystalline phase transition at the meniscus eventuallyled to the deposition of phage particles on the substrate in ahighly ordered manner. At this moment, the liquid-crystallinedeposition process was affected by the geometric shape of the

meniscus and by the resulting meniscus forces that were exertedon the deposition front.

A range of self-templated structures were formed bytuning various parameters, such as phage concentration,pulling speed, ionic concentration, phage surface chemistry,and the surface properties of the substrate. For example, at lowconcentrations (0.1–0.2 mg ml-1), the phage formed nemati-cally ordered fibril bundles parallel to the pulling direction,with regularly spaced ridge (stick) regions and groove (slip)regions owing to the stick-slip motion of the meniscus. Athigher concentrations (0.2–1.5 mg ml-1), the phage assembledinto nematic orthogonal twist structures, which consisted ofalternating nematic grooves and cholesteric ridges that wereorthogonally aligned to each other during stick-slip motion.Phage in the grooves aligned parallel to the pulling direction,whereas those at the ridge tops aligned perpendicular to thepulling direction (Figure 10a–c). Within a specific range ofphage concentrations (0.25–0.5 mg ml-1) and pulling speeds(10–20 mm min-1), phage in the ridges assembled into right-handed cholesteric helical ribbons (CHRs) along the left side ofthe meniscus and left-handed CHRs along the right side of themeniscus. This phenomenon was influenced by forces thatoriginated from the curvature of the meniscus at the outeredges of the substrate (Figure 10d–f ). At much-higher concen-trations (4–6 mg ml-1), the deposited phage exhibited zig-zag-patterned periodic structures, termed smectic helicoidalnanofilaments (SHNs, Figure 10g–i). The phage are packed

Fig. 9. M13 phage are assembled into functional materials by using a self-templating process. The hierarchicalassembly of phage can be controlled by competing interfacial forces at the meniscus, where the liquid-crystalline-phase-transition occurs.



into tilted smectic C bundles, thus forming the length of theresulting helicoidal nanofilaments, which extend parallel to thedirection of pulling (Figure 10h). Owing to the influence ofphage assembly by opposite meniscus forces from the sideedges, each left and right side of the film exhibited oppositeoptical responses under polarized light (Figure 10i). Byorthogonally aligning the phage-microstructures with alternat-ing SHNs and nematic hierarchical structures, the films wereable to allow the selective reflection or transmission of visiblelight, depending on the direction of the incident light. TheSHN structure also exhibited iridescence under white light,which is an exquisite optical property that has been found tooccur naturally in blue-faced monkeys,[102] bird skins,[103] andbeetle exocuticles.[104]

Furthermore, they finely tuned the structural colors of thefilms by controlling the pulling rates. Because the nanofilamentstructures were less ordered, owing to to the faster pulling rates,the position of the primary reflection wavelength shifted fromred to blue. Inspired by hierarchically organized extracellularmatrices in biological systems, they examined the capability ofthe nanostructued phage as biomimetic tissues for cell-culturesor biomineralization. The phage was engineered to display theRGD peptide, which is a specific motif that promotes integrin-mediated cell-adhesion, on the pVIII major coat protein andwas pulled into films that exhibited alternating SHNs andnematic hierarchical regions. The ordered phage structuresguided the growth of preosteoblast cells, by aligning theirbodies and internal actin filament networks parallel to the longaxes of the phage fibers on the nematic regions, but perpen-dicular to them on the SHN regions. The SHN films (whichwere assembled with the RGD phage and the 4E phage) werealso used as a template for the biomineralization of calciumphosphate, thereby resulting in tooth-enamel-like organic-inorganic composites that exhibited an about 20-fold increasein Young’s modulus when compared to native phage films.

10. Perspective

Over the last decade, the fabrication of ordered structures ofphage has been of great interest as a means of utilizing theoutstanding biochemical properties of phage in developinguseful materials. Combined with other organic/inorganic sub-stances, it has been demonstrated that phage is a superiorbuilding block for the fabrication of various functional devices,such as the cathode/anode in lithium-ion batteries, photovol-taic cells, phage-based sensors, and cell-culture supports.Although previous research has expanded the utility of phagewhen combined with genetic engineering, most improvementsin device functionality have relied upon increases in efficiencythat were attributed to the compact, more-densely packableunit size of phage and not upon the unique properties of theordered nanostructures themselves.

Recently, self-templating methods that control boththermodynamic and kinetic factors during the depositionprocess have opened up new routes for exploiting the orderedstructural properties of hierarchically organized phagearchitectures.[101] For example, directionally ordered phage canmaximize their piezoelectric strength, which is a useful prop-erty for energy-generating devices, by summing the dipolemoment of each phage.[105] In addition, films of ordered phagehave exhibited unexpected functional properties, such as struc-tured color and optical filtering. Structural colors or opticalfiltering from phage films can be used in optical phage-basedsensors, by combining these structural properties with target-specific binding motifs on the phage coat proteins.

This self-templating method may not only contribute topractical applications, as described above, but it could also lendinsight into the fundamental study of biomacromolecularassembly in in vivo systems under complicated and dynamicconditions. The phage serves as an ideal model fibrillar build-ing block, in terms of its monodispersity and its ability todisplay functional peptides, so that the relationship betweenthe assembled structure and the resulting functions can beeasily elucidated. By investigating the kinetic and thermody-namic factors that play an important role in the formation ofmetastable structures, we may further develop a better under-standing of nature’s sophisticated self-assembly processes.Although this self-templating approach demonstrated the greattunability and versatility of phage structures in response toconcentration and pulling speed, many engineering parametersremain unexplored. For example, persistence length, differen-tially charged or hydrophobic proteins, and substrate compo-sition may all influence the assembly process.

The effective diameter and persistence length of thephage particle changes in response to different pH values andionic strength, owing to the polyelectrolytic properties ofphage. The effective diameter and persistence length stronglyinfluence the phase before drying, even at the meniscus,thereby resulting in the formation of bundles of different sizesand helical pitch. The surface properties of the substrateupon which phage-assembly takes place is also a parameterthat might be considered. The contact angle of the recedingmeniscus and the tendency of the phage to adhere to thesubstrate can be controlled by surface modification of thesubstrate and may affect the competing interfacial forces thatare exerted on the meniscus, thus creating various unprec-edented structures. In addition, if multiple components areused in the solution, we can expect the formation of even-higher-level-ordered structures. This phenomenon can beeasily observed in nature. For example, beneficial transpar-ency properties are generated in arrays of close-packed,narrow collagen-fiber bundles when potentially interferingproteoglycan components are not present to obscure visiblelight.[106]



Recently, a new way of controlling the interfacial tensionin membranes that are condensed from a dispersion of phageparticles in a non-adsorbing polymer has been developed,which acts through a depletion mechanism.[107] Typically,phage can only twist along the membrane edge, whereas thegeometry of the membrane prevents twisting in the interior.Then, the increased strength of chirality of the phage along themembrane increases the frustration of untwisted phage in thebulk, thereby lowering the interfacial tension to create macro-scopically twisted structures.

Now, there have been great advances in creating phage-based hierarchical structures by both minimizing the interac-tion energy between the phage particles under variousenvironmental conditions and by controlling the kineticfactors during the deposition process. In contrast to conven-tional liquid crystals, which would be unstable in the absenceof an external field, these newly fabricated architecturesimprint the liquid crystals into the biomimetic morphology,thereby affording many possible opportunities for the develop-ment of metamaterials, photonic crystals, organic photovolta-ics, sensor devices, and energy-generating devices.

The phage genome lacks mammalian promoters andsequences for genomic incorporation and, therefore, shouldnot have an effect, even when taken up into cells. Although thein vivo applicability of phage as a tissue-engineering materialremains to be confirmed, it remains an attractive material forin vitro studies on the effects of biochemical ligands, surfacetopography, and -alignment. Furthermore, genetic engineeringprovides additional control of the phage for biomedical engi-neering purposes. Although there are issues to be addressedbefore their use in future practical applications, includingstability and potential toxicity in biological applications, thedevelopment of phage-based structures has shown sufficientlygreat potential to be used as one of the next generation offunctional materials.

In summary, phage has been shown to possess a remark-able capacity for creating various new, functional propertieswithin the context of diverse, self-assembled structures. Kineticcontrol of these directed- or self-templated processes hasopened up many new self-assembly possibilities, with manyparameters remaining to be explored. Concurrently, the field of

phage engineering stands poised to generate exciting advances,in both fundamental science and in engineering applications,in the coming decades.

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