Assembly of Bacteriophage Into Functional Materials

THE CHEMICAL Assembly of Bacteriophage into RECORD Functional Materials

SUNG HO YANG,1,2 WOO-JAE CHUNG,1,2 SEAN MCFARLAND,1,2 AND SEUNG-WUK LEE1,2* 1Department of Bioengineering, University of California, Berkeley, California 94720, USA 2Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, California 94720, USA E-mail: [email protected]

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

ABSTRACT: For the last decade, the fabrication of ordered structures of phage has been of great interest as a means of utilizing the outstanding biochemical properties of phage in developing useful materials. Combined with other organic/inorganic substances, it has been demonstrated that phage is a superior building block for fabricating various functional devices, such as the electrode in lithium-ion batteries, photovoltaic cells, sensors, and cell-culture supports. Although previous research has expanded the utility of phage when combined with genetic engineering, most improvements in device functionality have relied upon increases in efficiency owing to the compact, more densely packable unit size of phage rather than on the unique properties of the ordered nanostructures themselves. Recently, self-templating methods, which control both thermodynamic and kinetic factors during the deposition process, have opened up new routes to exploiting the ordered structural properties of hierarchically organized phage architectures. In addition, ordered phage films have exhibited unexpected functional properties, such as structural color and optical filtering. Structural colors or optical filtering from phage films can be used for optical phage-based sensors, which combine the structural properties of phage with target-specific binding motifs on the phage-coat proteins. This self-templating method may contribute not only to practical applications, but also provide insight into the fundamental study of biomacro- molecule assembly in in vivo systems under complicated and dynamic conditions. DOI 10.1002/tcr.201200012

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

1. Introduction

The controlled assembly of basic structural building blocks integrate various components, including proteins, block into long-range, ordered, periodic structures is an important copolymers, and nanoparticles.[1–4] It is postulated that the challenge in many science and engineering ﬁelds, including well-deﬁned or well-ordered structures that are formed by self- physics, chemistry, biology, materials science, and electrical assembly have great promise for fabricating devices on a large engineering. For the development of functional nanosystems, scale. Ordered structures on the nano- or micrometer scale bottom-up material-assembly approaches have attempted to possess several advantages for improving the performance of

integrated devices. 1) Their close-packed structures, which systems, the assembly of hierarchically organized, functional result from highly ordered materials, like crystals, are essential structures is directed by the mutual orchestration of proteins for improving the efficiency of devices such as solar cells, sec- and genes, which simultaneously regulate the synthesis of ondary batteries, and memory-storage devices.[5,6] Further- organic and inorganic precursors. Although mimicking the more, the number of building-block units per volume is critical self-assembly processes of biological systems is a promising in keeping costs low whilst maintaining high efficiency. 2) route for the creation of new bio-nanomaterials, such processes Ordered structures frequently enhance the performance of a remain obscured by the highly complex interplay between device, thereby augmenting the properties of its functional genes and proteins within an organismal context. building blocks.[7,8] For example, directionally ordered building As a tool for overcoming this hurdle, the genetic engineer- blocks can maximize functional performance by summing unit ing of viruses offers new opportunities to integrate various properties such as magnetic strength, electric field, and dipole nanoscale materials into hierarchically ordered structures moment.[9] 3) Ordered structures can provide unexpected and through biologically inspired material-assembly processes. useful functional properties beyond that offered by each build- Phage, which is composed of relatively few proteins and genes ing block individually. Regardless of the inherent properties of compared to prokaryotic and eukaryotic cells, represents a a building block, ordered structures can exhibit collective char- simplified starting point for embarking upon such work. By acteristics, such as structural color,[10] surface plasmon reso- mimicking natural evolutionary processes, phage can be nance,[11] and optical filtering.[12] These emergent functions, selected that specifically recognizes desired targets on the which are provided by ordered structures, can be synergistically molecular level, thereby adding to their versatility as a basic combined with the native functionalities that are provided by building block.[18] Through phage display and genetic the underlying building blocks to yield devices that exceed the engineering, phage has been used as a specific template for scope of what would otherwise be possible. producing functional nanomaterials, such as conductive/ Although methods for the self-assembly of functional semiconductive nanowires.[19–21] For a decade, many research- materials have born witness to a number of significant advance- ers have exploited virus particles for assembling ordered ments over the past decade, the generation of highly complex nanostructures and for fabricating useful materials and devices and hierarchically organized structures remains challenging. for energy,[22–24] batteries,[25,26] biosensors,[27] and tissue- The fields of nanotechnology and materials science have con- regenerating materials.[28] In this Personal Account, we intro- currently been inspired by self-assembly processes in biological duce recent accomplishments in the assembly of bacteriophage systems, such as glass sponges (optical fibers),[13] brittle stars for fabricating functional devices and review the potential (optical lens array),[14] diatoms (sophisticated periodic struc- future applications of this emerging technology. tures),[15] abalone shells (fracture-resistance materials),[16] and bones (support structure for vertebrates).[17] In biological 2. Structure and Characteristics of Bacteriophage Bacteriophage (phage) is a virus that replicates itself and propa- Dr. Seung-Wuk Lee earned his PhD in gates its genetic information by infecting bacterial host cells.[29] Chemistry and Biochemistry from the Phage may take many different shapes, from linear (M13, Fd, University of Texas at Austin in 2003 F1),[30] to spherical (MS2),[31] to sophisticated head-tail struc- under the supervision of Professor tures (T4, T7).[32] Among these shapes, the rod-like, filamen- Angela Belcher. He completed post- tous shape of M13 phage constitutes an excellent biological doctoral work with Professor Carolyn building block for the assembly of new materials. There are Bertozzi in the Molecular Foundry several reasons for this viewpoint (Figure 1): at Lawrence Berkeley National Labora- First, normal, wild-type M13 bacteriophage exists as a tory. He is a currently a Professor in the Department of filamentous nanofiber with a diameter of 6.6 nm and a length Bioengineering, University of California, Berkeley. His of 880 nm. This highly anisotropic shape, along with the current research interests include phage-based biomimetic monodispersity that results from its high-fidelity biological materials, bionanotechnology, stimuli-responsive biopoly- reproduction, endows this phage with the ability to exhibit mers, and regenerative medicine. In addition, based on liquid-crystalline properties. In suspension, this property phage-based techniques, he is developing a general concept allows for the artificial formation of ornate ordered structures. that is termed “Virotronics”, which exploit the unique prop- Second, the surface of the phage can be modified both erties of genetically engineered phage to carry information genetically and chemically to introduce specific peptides. The to build functional medical- and electronic materials and M13 phage is composed of several major- and minor coat devices. proteins that encapsulate its single-stranded DNA genome.

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.

The viral capsid is composed of about 2700 copies of the helically organized pVIII major coat protein and 5–7 copies of Fig. 2. Schematic structures of the liquid-crystalline phases of M13 bacte- [33,34] riophages. Top: typical liquid-crystalline phages; bottom: hierarchically minor coat proteins pIII, pVI, pIX, and pVII. The pVIII ordered liquid-crystalline phages. major coat proteins make up the side-walls of the sheath, whilst complexes of the pIII/pVI and pIX/pVII minor coat proteins are located at either end. Thanks to advances in biotechnology suspension.[52] Thus, phage has drawn great attention as an and phage engineering over the last two decades, biochemical experimental model system for probing the structure and modifications on each of the coat proteins have been achieved dynamics of liquid crystals on the molecular level, not least, in through genetic manipulation[35–39] and site-specific organic- part, owing to the ease with which organized phage structures synthesis approaches.[23,40–47] can be imaged by techniques as simple as optical microscopy Third, phage that bind specifically to a particular target (Figure 2). The liquid-crystalline behavior of the virus is material can be isolated through the use of phage display (see dependent on various factors, such as phage concentration, below).[48–49] These specific interactions between phage and ionic strength, and external fields.[52–55] Among them, the con- target materials provide the capability to formulate phage- centration of phage is the most-significant factor that affects based hybrid materials. the lyotropic liquid-crystal phase. At low concentrations (below 5 mg ml-1), phage are randomly distributed without any order and are referred to as being in an isotropic phase. -1 3. Liquid-Crystalline Properties of Phage Within the concentration range 10–20 mg ml , at which the distance between neighboring phage becomes smaller than its Unlike spherical particles, it is well-known that rod-shaped length, phage particles align into orientationally ordered con- molecules or colloidal particles exhibit liquid-crystalline figurations, called nematic-phase liquid crystals, owing to steric ordered phases in suspension. Owing to their responses to forces between the phage particles. At this concentration, nem- electromagnetism and polarized light, liquid crystals are used atically ordered phage begin to intertwine with each other for many applications, including liquid-crystal displays[50] and along the perpendicular axis of the layers, which results in a optical switches.[51] It is known that there are two types of helicity of the phage particles. Within the concentration range liquid-crystalline phases: thermotropic and lyotropic liquid- 20–80 mg ml-1, the helicity of the layers becomes more dis- crystalline phases. Thermotropic phases are determined within tinct and leads to the emergence of cholesteric-phase liquid a certain temperature range by the degree of each particle’s crystals.[53,56] These phage exhibit unique optical characteristics, thermal movement. Lyotropic phases are influenced not only such as a fingerprint texture and the capacity to reflect circu- by temperature but also by the concentration of the particles. larly polarized light.[53,56,57] At very high concentrations (above Hence, lyotropic liquid-crystals exhibit more-varied phases 100 mg ml-1), phage are ordered both positionally and orien- than thermotropic liquid crystals. In biological systems, the tationally along one direction within well-defined layers, called lyotropic liquid crystal is a driving force for the self-assembly of smectic-phase liquid crystals.[58] According to the degrees of proteins and cell membranes. positional and orientational order, there are many different Because of their long, rod-like shape and monodispersity, smectic phases. For example, in the Smectic A phase, phage are filamentous viruses also show liquid-crystalline behavior in oriented along the layer normal, whilst, in the Smectic C phase,

they are tilted at an angle from the layer normal. After com- delivery systems,[61] as MRI-contrast agents,[62,63] cell-imaging plete dehydration, the phage mostly arrange into their thermo- agents,[44] and biosensors,[27] or in photosyntesis.[23] Organic dynamically more-stable phases, such as the smectic phases.[54] synthetic approaches and their applications have been reviewed Recently, a method that can hierarchically order typical in detail elsewhere by Francis and co-workers and by Wang crystalline phases was developed by kinetically tuning the et al.[42,45,47] dehydration reaction. As a function of concentration and evaporation speed, nematic orthogonal twist, cholesteric helical ribbon, and smectic helicoidal filaments have been formed 5. Phage Display (Figure 2). These hierarchically ordered structures will be further discussed in Section 9. On the other hand, Fraden and Phage display is a high-throughput, combinatorial screening co-workers observed many other interesting microstructures process that identifies, by selecting from random peptide librar- from mixed suspensions of rod-shaped phage and spherical ies, functional peptides that are expressed as fusion proteins colloids. In their study, they elucidated the fundamental rela- on the phage coat surface. This method is considered to be tionships between molecular properties, such as length and an accelerated evolutionary screening process for selecting charge, and macroscopic properties, such as phase behav- peptides that can specifically bind to target materials [48,49] ior.[50,52,55,56,58] This binary phase behavior has also been used to (Figure 3). For phage display, a random phage library must fabricate highly ordered functional hybrid composites.[59] first be established. This library is accomplished by splicing random sequences into the appropriate site of a chosen coat protein gene. The expressed recombinant protein will then display a highly diverse library of peptides (up to 1011 random 4. Modification of the Phage Surface sequences) on the surface of the phage capsid.[48,49,64] These Bacteriophage has served as an ideal template for the realization libraries can then be screened against the target material. of various functional materials and devices because of the pro- Among the library population, some phage will bind to cessability of its surface proteins, as well as its well-defined the target with greater affinity, owing to interactions between nanostructure. The capsids of bacteriophage can be modified their displayed peptide and the target material. After washing by genetic engineering or by chemoselctive organic synthesis to away weakly bound phage, the remaining strongly bound introduce specific amino acids or specific functional groups phage are eluted separately and are then amplified by infecting onto desired coat proteins. To understand the genetic engineer- host bacteria. This process is repeated over several rounds to ing of the phage, it is helpful to understand the typical “life isolate the most-strongly binding phage. Finally, the specific cycle” of phage: Briefly, contact with a bacterial host cell is binding peptides can be identified through DNA analysis of initiated by pIII, which recognizes and binds to the sex pilus of the selected phage’s genome. bacteria that carry the F plasmid. Subsequently, the phage The phage-display method was originally invented to genome is injected into the host cell. The foreign DNA repro- identify epitope-like short peptides sequences that can replace duces their corresponding genes and proteins by hijacking the metabolism of the host cell. After the assembly of gene and coat proteins, the reproduced phage are released from the host cell. By manipulating the infection process in an artificial manner, such as through heat or electric shock,[60] recombinant genes can also be delivered into the bacterial host. Engineered phage can then be produced as normal by the hijacked metabolism of the host cell. This process allows specific amino acids to be expressed on target sites in coat proteins by delivering the corresponding nucleotide sequence in a recombinant gene. On the other hand, chemoselective synthetic approaches have been developed to introduce various functionalities onto the 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 relatively mild reactions, such as EDC/NHS activation, maleimide-thiol coupling, diazonium reagents, and click chemistry.[23,40–47] By introducing functional derivatives onto the surface of phage Fig. 3. Schematic diagram of the phage-display processes for identifying a through such reactions, the phage can be employed in drug- specific peptide against the target substrates.

antibodies.[37] The specific sequences that are identified Polymeric templates have also been utilized in the 2D through phage display against specific target materials can assembly of M13 phage. Yoo et al. found that M13 phage function as epitopes through protein folding. Various epitope- could be closely ordered through preferential electrostatic like peptides have been isolated through phage display against interactions between phage particles and two oppositely proteins,[65–67] DNA,[68] receptors,[69] and tissues.[70] Recently, charged weak polyelectrolytes, such as linear polyethylenimine this method was further advanced to identify the short peptide (LPEI) and poly(acrylic acid) (PAA).[82] The spontaneous 2D motifs that specifically bind to inorganic materials, such as assembly of phage was driven by a combination of competitive GaAs, GaN, Ag, Pt, Au, Pd, Ge, Ti, SiO2, quartz, CaCO3, electrostatic interactions between phage particles and polyelec- ZnS, CdS, Co, TiO2, ZnO, CoPt, FePt, BaTiO3, CaMoO4, trolyte, the inter-diffusion of the polyelectrolyte, and the aniso- and hydroxyapatite, including carbon nanomaterials, such as tropic shape of the M13 phage. The phage were assembled [71–75] C60, carbon nanotubes, and graphene. It has been found according to the following procedure (Figure 4a): First, nega- that these specific peptides are highly effective at nucleating tively charged phage were randomly deposited on the positively and mineralizing various inorganic materials.[76–78] Further- charged top surface of LPEI/PAA multilayers (step 1). Next, more, phage that display specific peptides have also been used the adsorption of PAA on the top phage layer of the thin as a template for various new functional nanomaterials, phage/LPEI/PAA multilayers led to a separation process that such as conductive/semi-conductive nanowires.[19–21] For more forced the phage to the surface because of stronger electrostatic information about target-specific peptides, inorganic-material- interactions between LPEI and PAA, rather than with phage binding motifs have previously been summarized in various (step 2). Finally, the floating phage spontaneously ordered into reviews and books.[71,77,79] a monolayer structure owing to mutually repulsive interactions between neighboring phage particles (step 3). The repeated 6. Assembly of Phage with Organic Materials deposition of LPEI and PAA led to a free-standing film that presented more closely packed structures. They showed that M13 phage possesses advantageous structural features as a the resulting phage films could be utilized as scaffolds for building block for 1D fibers because of its anisotropic shape nucleating cobalt nanowires or for aligning gallium-nitride and monodisperse size. By exploiting rod-shaped phage struc- nanoparticle over multiple-length scales by expressing a specific tures, Lee and Belcher fabricated micro- and nanosized- peptide sequence on pVIII major coat proteins of the M13 diameter fibers through wet-spinning and electrospinning, phage. Furthermore, Yoo et al. spatio-selectively assembled respectively.[80] Suspensions of M13 phage were extruded phage onto patterned, multilayer polyelectrolyte that was gen- through the capillary tubes into a cross-linking solution of erated by solvent-assisted capillary-molding methods.[83] The glutaraldehyde to generate fibers with a diameter of 10–20 mm. size of the patterns could be adjusted by the swelling properties The suspension of the smectic ordered phage was transformed of the underlying patterned polyelectrolyte multilayer. To into nematic structures that ran parallel to the fiber long axis, examine the possibility of biocompatible application, the driven by flowing forces. In addition, fibers with a diameter of surface of patterned phage was modified with biotin- 100–200 nm were fabricated by electrospinning. After blend- conjugated anti-Fd bacteriophage that specifically bould to ing with polyvinyl pyrrolidone (PVP) to improve their process- pVIII major coat proteins of the M13 phage. The biotin- ability, the PVP-blended phage was then electrospun into modified pattern was visualized by exposing the substrate to continuous fibers. Alexa-Fluor-conjugated streptavidin. The authors confirmed that this method was biocompat- On the other hand, Solis et al. also developed phage- ible by showing that resuspended phage fibers in buffer solu- patterning methods by applying a subtractive contact-printing tion maintained their infectivity against bacterial hosts. Chiang technique for patterning phage antibodies.[84] They immobi- et al. developed two strategies that could introduce function- lized individual phage into single-element arrays by optimizing alities onto the M13 phage fibers, that is, before or after both the concentration of the phage suspension and the feature wet-spinning the phage into fibers.[81] In the former case, tet- size of the antibody. raglutamate phage, named E4-phage, were chemically conju- These techniques for the ordered patterning and close- gated with amine-terminated cadmium selenide quantum dots packing of phage are important for utilizing phage as a (QDs). Microfibers were then fabricated through a wet- biosensor and energy-storage/-conversion device because such spinning process. The resulting QD-modified fibers emitted controllability is directly related to the efficiency and/or sensi- red light under exposure to UV light. In the latter case, geneti- tivity of such devices. Nam et al. utilized polymer-induced cally modified M13 phage that displayed a gold-specific close-packed M13-phage structures to realize high-voltage peptide sequence (Val-Ser-Gly-Se-Ser-Pro-Asp-Ser) were first lithium-ion batteries (Figure 4b).[25] The phage was first engi- spun into microfibers. Gold nanoparticles were then closely neered to express both tetraglutamate (4E) and gold-binding assembled along microfibers through the specific interactions. peptide motifs on the pVIII major coat proteins for nucleating

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

gold and cobalt-oxide nanoparticles, respectively. By using the conductivity of the Co3O4 nanoparticles, or may result in a engineered phage as a scaffold, gold-nanoparticle-incorporated catalytic effect. cobalt-oxide nanowires (Au-Co3O4 nanowires) were produced To organize phage-based electrodes over large length with a high degree of crystallinity under aqueous conditions at scales, engineered M13 phage was assembled in 2D on the top room temperature. By applying these phage-based Au-Co3O4 of LPEI/PAA multilayers through competitive electrostatic nanowires to the anodic electrode of a lithium-ion battery, the interactions (Figure 4c, d). After that, close-packed Au-Co3O4 energy-storage capacity of the battery was improved by more nanostructures were formed on the assembled phage ﬁlms on than 30% compared to that with pure Co3O4 nanowires. Nam the centimeter-length scale. The cell that was constructed with et al. proposed that this improvement in electrochemical prop- phage-based electrodes reached 94% of its theoretical capacity, erties could be explained by the participation of gold nanopar- with a high cycling rate. The resulting virus-based lithium-ion ticles in the redox reaction, which may enhance the electronic battery exhibited lightweight, ﬂexible, and transparent proper-

ties, as well as superior electrochemical performance compared to conventional batteries. This enhanced performance is mainly caused by the monodispersity of the Au-Co3O4 nanowires, the close-packed assembly of the layered nanostructures, and a high surface-area-to-volume ratio. Nam et al. further developed phage-based electrodes for the fabrication of an array of micro-electrodes, based on the hypothesis that micro- electrodes that are ordered in multidimensional architectures could be beneficial for improving electrochemical performance [85] and for designing new types of batteries. Co3O4 nanowires were spatio-selectively assembled on the micropatterned PDMS template through the combination of phage engineering, LPEI/PAA layer-by-layer, and microcontact-printing. A patterned array of micro-electrodes was fabricated by stamping a self-assembled layer of phage-templated Co3O4 onto a platinum-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 ordered manner, Lee et al. developed a phage-based cathodic electrode for lithium-ion batteries.[26] The major coat protein of the M13 phage was engineered to express both tetraglutamate (4E) for Fig. 5. a) Photograph of the free-standing ZnS-phage film. b) POM (x20) image, which shows birefringent dark- and bright-band patterns (periodic nucleating amorphous anhydrous FePO4 (cathode material) length: 72.8 mm). These band patterns were optically active and their patterns and peptide motifs (Asp-Met-Pro-Arg-Thr-Thr-Met-Ser-Pro- reversed depending on the angle between the polarizer and the analyzer. c) Pro-Pro-Arg) for capturing conductive single-walled carbon Photoluminescent image, with an excitation wavelength of 350 nm and with nanotubes (SWNTs). They found that the incorporation of filtering below 400 nm, which shows a striped pattern (width: about 1 mm, SWNTs improved electrochemical performance by increasing x50). d) AFM image of the ZnS-phage film. The phage forms parallel aligned herringbone patterns that are almost at right angles to the adjacent direction the local electronic conductivity in the cathode. The phage- (arrows). based cathode showed a greatly enhanced capacity of 134 mAh g-1 at a high discharge rate of 3 C, compared to the best reported capacity (80 mAh g-1 at 3 C). In addition, almost structure as a function of phage concentration and the smectic no fading of the capacity was observed, even after up to 50 phase (at 127 mg ml-1), cholesteric phase (at 76–28 mg ml-1), cycles. and nematic phase (at 22 mg ml-1) were all observed. In particular, in the cholesteric phase, cholesteric pitches exponentially decayed with respect to an increase of phage 7. Self-Assembly of Phage with concentration. Inorganic Materials In addition, the structure could become nematic under a magnetic field, presumably because of the diamagnetic prop- Increasingly, phage has been utilized to assemble nanomateri- erties of the major coat protein of the M13 phage. 3D-ordered, als, driven by liquid-crystalline ordering of the engineered self-supporting phage/ZnS hybrid films were induced by slow phage particles. The ordered hybridization of phage and inor- evaporation; the viruses and nanocrystals gradually progressed ganic materials is mainly based upon the specific interactions through isotropic, nematic, cholesteric, and smectic phases in between them, which can be engineered through phage-display the suspension media, while they were still mobile, and com- methods. Lee et al. reported the multi-length-scale ordering of plete dehydration finally afforded free-standing films that were quantum dot (QD)-hybrid biocomposite structures by using composed of a smectic-like lamellar structure. Interestingly, the genetically engineered M13 phage (Figure 5).[59] After selecting surface viral morphology was smectic O and the interior mor- a zinc sulfide (ZnS)-binding peptide sequence (Cys-Asn-Asn- phologies were smectic A and C. The resulting films were Pro-Met-His-Gln-Asn-Cys) through the screening of phage- transparent and continuous over many centimeters. Moreover, display libraries, the peptide was expressed on the pIII minor the films exhibited an ordered pattern of 1 mm fluorescent coat protein. The engineered phage was suspended in solutions lines, which implied that the ZnS-QDs were confined at the of the ZnS precursor to form a phage/ZnS hybrid liquid- end of the M13 phage, owing to the specific binding. Lee and crystalline suspension. Liquid-crystalline suspensions varied in Belcher also developed a nanoparticle-alignment method by

using anti-streptavidin phage.[53] After isolating a streptavidin- (about 108 pfu mL-1), which was much lower than that required binding motif (His-Pro-Gln) through the screening of a phage- for liquid-crystalline formation (about 1012 pfu mL-1).[53] It display library, the anti-streptavidin phage was assembled with seems that Co2+ ions increase the local concentration of phage streptavidin-conjugated gold nanoparticles. The mixed suspen- particles by stimulating the aggregation of phage bundles. The sion showed liquid-crystalline character and the structures phage bundle acts as a template for nucleating Co-Pt nanopar- transformed from the nematic phase (14 mg ml-1), to the cho- ticles, which are uniformly deposited on the fibrous structures lesteric phase (about 76–20 mg ml-1), to the smectic phase and show both a crystalline nature and a narrow size distribu- (about 83 mg ml-1) as a function of phage concentration. tion. They also demonstrated that Co-Pt alloys have typical Smectic-ordered self-supporting phage/Au hybrid films were superparamagnetic properties with high anisotropy. formed by slowly drying mixed suspensions on gold films. Wang et al. reported that Ca2+ ions triggered the self- They further applied this method for ordering organic dyes or assembly of wild-type Fd phage, which could act as both a biological molecules by assembling anti-streptavidin phage scaffold and as a Ca2+ source for the oriented nucleation of with streptavidin-conjugated fluorescein or phycoerythrin. The bone minerals.[88] The formation of phage bundles could be patterned fluorescence in the hybrid films confirmed that fluo- explained by the fact that Ca2+ ions served as a cross-linking rescein or phycoerythrin were localized between the smectic bridge between negatively charged wild-type Fd phage, which layers. contained two negatively charged amino acids (1 Glu and 3 Multiple binding sites have been introduced on the surface Asp) on the surface of the pVIII major coat protein. By reacting of phage, with each site being used to align different nanopar- the Ca2+-ion/phage bundles with phosphate ions, hydroxyapa- ticles. Huang et al. reported the assembly of nanoarchitectures tite (HAP) nanocrystals were grown along the long axis of of various geometries and materials, such as arrays of gold phage bundles. The oriented nucleation and growth of HAP nanoparticles or CdSe-QDs, by using highly engineered M13 nanocrystals resembled the natural bone-formation mecha- phage as a template.[86] M13 phage was rationally engineered to nism; bone minerals crystallized parallel to the long axis of express gold-binding motifs (Val-Ser-Gly-Ser-Ser-Pro-Asp-Ser) the collagen fibrils. In addition, the Ca2+-ion/phage bundles on pVIII major coat proteins and streptavidin-binding motifs completed the formation of HAP nanocrystals within 12 h, (Ser-Trp-Asp-Pro-Tyr-Ser-His-Leu-Leu-Gln-His-Pro-Gln) on whereas most previously reported substrates needed much pIII minor coat proteins. The engineered phage directed the longer time periods, ranging from several days to weeks. formation of heterostructures on which the gold nanoparticles By using a similar method, He et al. also produced phage/ (5 nm) arranged into an ordered 1D array on major coat pro- HAP biocomposites by using M13 phage that was genetically teins along the viral axis, whilst the streptavidin-conjugated engineered to display the negatively charged peptide octa- gold particles (15 nm) bound to one end of the phage. By glutamate (8E) on the pVIII major coat protein.[89] After replacing the streptavidin-coated gold nanoparticles with assembling the 8E-phage into a nanofibrous bundle with Ca2+ streptavidin-conjugated CdSe-QDs, they demonstrated that cations, precursors for HAP accumulated within the bundle, nanoarchitectures that were composed of hetero-nanoparticle thereby making the local environment supersaturated, which arrays could be rationally designed. In addition, they showed resulted in the nucleation of HAP nanocrystals parallel to the that gold-nanoparticle arrays on pVIII proteins could subse- long axis of the phage bundle. They also found that the quently function as templates for highly conductive continu- co-assembly of phage and collagen into nanofibers improved ous nanowires by interconnecting gold-nanoparticle arrays the mineralization process, in terms of a stronger directional with the electro-less deposition of gold. preference along the long axis and more-compact mineralized On the other hand, metal ions have also been used to structures. Such improvements could be explained by the mediate the assembly of phage to achieve nanostructured tem- hypothesis that glutamates on the side-wall of the phage could plates for inorganic alloys or biocomposites. Lee et al. used enhance the hydrogen-bonding interactions with collagen genetically engineered phage to form the mechanical frame- fibers, thus resulting in their hierarchical co-assembly. These work for nanophase Co-Pt alloys, which is an extensively results indicated that rationally designed phage structures can studied magnetic material because of its high magnetocrystal- play a similar role to that of real tissues in biological systems. line anisotropy.[87] Biopanning against Co2+ ions specified Co2+- Other practical applications of phage-inorganic hybrid binding peptide sequences on the pVIII major coat protein. materials have also emerged. Liu et al. have developed The isolated sequence (Glu-Pro-Gly-His-Asp-Ala-Val-Pro) humidity-tunable surface plasmon resonance (SPR) sensors contained a well-known metal-binding motif (Glu-X-X-His) with virus-based nanocomposite films.[90] They fabricated uni- that is frequently found in active sites of metalloproteins. In the formly distributed gold-nanoparticles/phage nanocomposite presence of Co2+ ions, the engineered M13 phage became films by alternately depositing negatively charged gold nano- directionally ordered and assembled into fibrous structures particles and positively charged M13 phage that display tet- with a sponge-like morphology at low phage concentrations 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-closely packed, thereby leading to a dense accumulation of nanoparticles, as a function of increasing the number of deposited layers. They found that the SPR absorbance of the nanocomposite at lmax,orlmax itself, could be systematically tuned by increasing the number of deposited gold nanoparticles. This phenomenon was likely caused by changes in the amount of deposited nanoparticles or changes in the dielectric constant. In addition, the nanocomposite exhibited decreased SPR absorbance and a blue-shift of the lmax value as a function of increased humidity. The SPR response showed a linear relationship with the relative humidity in the range 19–86%, which implied that it was possible to apply the phage-based nanocomposite as a humidity sensor. The humidity depen- Fig. 6. Nematic aligned M13 phage fibers for neural-tissue engineering. a) dence can be explained by the fact that the SPR spectra of the Schematic diagram of the fabrication of the nematically ordered phage fiber in AuNPs are highly dependent upon the interparticle spacing. agarose gel through flow-induced alignment, in which an oriented nanofibrous Dehydration may flatten the phage-based nanocomposite, phage matrix directs the neural cells. b) Photograph of the phage fibers (length: which leads to a corresponding decrease in the interparticle about 1 cm) spun in agarose. c) Polarized optical microscopy (POM) image of the nematic liquid-crystalline structure of phage fiber (scale bar: 50 mm).d) spacing. POM images of the differentiated neural cells within the RGD-phage matrix after 6 days, which show that the neuritis extended parallel to the long axis of the phage fiber. The red arrow indicates the direction of phage alignment; the blue arrows indicate the oriented outgrowth of neurites. 8. Self-Assembled Phage to Control Cellular Morphology A filamentous bio-polymer structure is one of the most- genitor cells that were cultured in the phage matrices modified common features in biological materials that make up extra- with signaling motifs (RGD, IKVAV) were observed to remain cellular matrices, such as collagen, carbohydrate, and elastin.[91] viable, proliferate, and differentiate into neural cells. In addi- As an approach to mimic such structures, the fabrication of tion, the liquid-crystalline phage matrix could control the macroscopically ordered architectures based on fibrillar tem- direction of neuronal growth along the length of the fibers plates of M13 phage has been of great interest. As discussed (Figure 6d). Such structurally aligned functional phage matri- above, M13 phage demonstrates lyotropic liquid-crystalline ces may offer promising opportunities for therapies that behavior; thus, the direction of the assemblies can be affected address challenging biomedical problems, such as the regenera- by the shear flow that is exerted on them. In addition, filamen- tion of nerve tissue within the central nervous system. tous colloidal particles tend to align in a flow field, owing to the The same approach was employed by Yoo et al. to fabri- torque that the flowing solvent exerts on the particle’s cores.[92] cate liquid-crystalline fiber matrices for bone-tissue engineer- Merzlyak et al. reported an approach for the fabrication of ing.[94] By using genetic engineering, they created phage that anisotropic structured materials from genetically engineered displayed the DGEA peptide (a known signaling motif that M13 phage by using a flow-induced injection method.[93] They induces bone-cell differentiation) on every copy of the capsid’s fabricated aligned phage-nanofiber matrices to examine their pVIII major coat protein. MC-3T3 bone-progenitor cells capability for controlling the macroscopic behavior of neural within the DGEA-phage fiber matrix in agarose were stimu- cells. Liquid-crystalline suspensions of RGD- and IKVAV- lated to express actin filaments, osteogenic proteins, such as modified phage were mixed with neural progenitor cells and collagen type I, osteopontin, alkaline phosphatase, osteocalcin, manually injected into liquid agarose (Figure 6a). The struc- and matrix acidic phosphoprotein I, even at an early stage ture of the spun phage fibers persisted in the agarose after (12–24 h). cooling and solidification. The phage fibers that were Kuncicky et al. first reported the flow-assembly method entrapped in the agarose possessed nematic-ordered liquid- for aligning rod-shaped viruses into large-scale ordered films by crystalline structures with uniaxial orientation that was using the receding meniscus of a virus-containing suspen- induced by flow-injection. This fact was verified by polarized sion.[95] Virus fibers were aligned in a linear manner, normal to optical microscopy and by SEM, which showed liquid- the liquid/air/solid three-phase contact line, when droplets of a crystalline alignment of the fiber, which was over a centimeter suspension of the virus on a surface receded by using an appa- in length, parallel to the long axis (Figure 6b, c). Neural pro- ratus for controlled deposition (Figure 7a). The scale of the

matrix that was composed of ordered phage. AFM analysis showed that the phage (wildtype/RGD-phage = 3:1) aligned uniformly parallel to the dragging direction. NIH-3T3 and Chinese hamster ovarian (CHO) cells that were cultured on the aligned phage matrices both elongated their cytoskeleton and nuclei in a single direction along the long axis of the fibrils in the matrices. On the other hand, genetically engineered M13 phage has been employed to make aligned biomimetic extracellular matrices by using a shearing method.[96,97] Merzlyak et al. engineered M13 phage to display RGD peptides or RGE peptides (as control peptides) on its major coat protein (pVIII) by using a partial library-cloning approach.[99] On one edge of 3-aminopropyltriethoxy-silane-treated glass substrates, a droplet of phage solution (10–30 mg ml-1) was placed and dragged (by using a glass slide) parallel to the long axis of the substrate. By applying a shear force in a single direction, the engineered phage were aligned in that same direction. The sheared phage films exhibited anisotropic surface morphology Fig. 7. Directional assembly of virus particles by using shearing force. a) that was formed with aligned fibrous structures parallel to the Schematic representation of the apparatus for depositing aligned virus-fiber coatings. b) Atomic force microscopy (AFM) images that show long-range- shear direction (Figure 7b), whereas drop-cast phage films ordered liquid-crystalline viral films (20 mg ml-1, scale bar: 5 mm). c) Sheared exhibited a surface morphology of randomly organized M13-phage films, which show different surface roughness depending on the bundles. The roughness and diameters of the bundled struc- initial concentration. d) Fluorescence image of NIH-3T3 fibroblasts that were tures in the sheared films were tunable by varying the initial grown on the RGD-phage-aligned film in serum-free media for 12 h. The actin filaments are stained with phalloidin (green) and the nuclei are counter- phage concentration (rms = 3.1–22.4 nm, d = 20–900 nm, stained with DAPI (blue); scale bar: 100 mm. 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 film deposition process is only limited by the size of the substrate extended their filamentous cytoskeleton. This result indicates and it is scalable beyond centimeter-length scales because the that the integrin motifs on the matrix played a crucial role in alignment process only occurs at the three-phase-contact line, cell-adhesion and spreading. regardless of the volume of the suspension of the virus. The In addition, the anisotropic nanogrooves that were formed resulting aligned virus films served as a template for metallizing in the RGD-phage matrix contributed to the orientation of wires. This directional virus-assembly method inspired many cellular growth (over 80% of the elongated cells were aligned other research groups to investigate the behavior of oriented within a 20° angle of the aligned phage fibers). The NPCs that assemblies of M13 phage and the potential of the assembled were cultured on the RGD-phage in differentiation media matrix for tissue engineering.[96–98] exhibited an outgrowth of neurites in the direction parallel to Rong et al. chemically modified M13 phage with RGD the aligned phage fibers, whereas few cells were attached to peptides to enhance the binding between the cells and the M13 RGE- and wild-type-phage matrix and formed round shapes matrices.[98] The incorporation of RGD peptides onto the under the same conditions. The aligned RGD-phage matrix surface of the phage was carried out in two steps: Alkyne- provided both biological and biochemical cues for the attach- derivatized N-hydroxysuccinimidyl ester was coupled to the ment and elongation of NPCs and the outgrowth of the amino group on the surface of phage. A CuI-catalyzed azide- neurites. The genetically engineered phage matrix showed alkyne 1,3-dipolar cycloaddition reaction was performed to versatility for the directional growth of other types of cells, such conjugate the azide-derivatized RGD peptides onto the alkynyl as preosteoblasts and NIH-3T3 cells (Figure 7d).[97] functional group on the surface. The conjugation was verified On the other hand, self-standing phage-based aligned by MS (MALDI-TOF) analysis of the protein subunits. The fibers have been developed for cell-encapsulation and 3D cell- chemically modified phage were aligned by using the same cultures. Chung et al. took advantage of the electrostatic inter- method as that reported by Kuncicky et al.[95] The phage solu- actions between abundant net negative charges on the surface tion (20 mg ml-1) was first deposited onto the positively of the phage building blocks and cationic polymers at the charged silane-coated cover glass and the meniscus was interface where the membrane formation occurs to make dragged, thereby resulting in the formation of a thin-film self-standing phage-based fibers.[97] First, the liquid-crystalline

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 through vertical pulling of the patterned substrate that was immersed in the phage suspension. The RGD-phage-patterned substrates were then used for the directional guidance of human fibroblast cells. phage suspension was mixed with cells of interests and injected along the central axis of the vertical hydrophilic stripes, into a solution of the cationic polymer (poly-l-lysine). The whereas diagonal orientation of phage deposition was observed orientation of the liquid-crystalline phage was flow-induced by near the side edges. The selectively deposited phage structures, injection into the polymer solution through the syringe; thus, which provided topographical cues to the cells, along with the structure of the liquid-crystalline phage matrix was pre- biochemical signals, appeared critical to guiding human served by the resulting phage/polymer complex and its sur- fibroblasts to grow and elongate in a uniform direction. This roundings. NIH-3T3 fibroblasts that were encapsulated with result shows the hybrid bottom-up and top-down approaches RGD-phage/PLL migrated and elongated their bodies along can be useful for the study of cell behavior in differing the phage matrix (day 7), thus indicating that cells responded microenvironments. to the chemical and physical cues from the directionally organized phage-based fiber matrices. The directionally ordered phage fibers supported cell-viability and directional growth, 9. Hierarchical Assembly of Phage thus demonstrating its potential use as a material for tissue regeneration. Although many organized structures of phage have been The chemical micro-pattern has been employed to selec- created with the help of cross-linking agents, polyelectrolytes, tively attach and align phage for the directional guidance of and inorganic nanomaterials, it still remains a challenge to human fibroblasts (Figure 8).[100] Yoo, Chung et al. prepared assemble phage by itself into ordered structures. Along these two chemical striped patterns (1-octadecanethiol (ODT)/ lines, Lee et al. fabricated long-range-ordered virus-based films cysteamine on gold) with different spacing (cysteamine/ODT, by using only M13 phage as a building block.[54] The films were 5:10 mm and 10:20 mm) by using a microcontact-printing formed by the slow evaporation of a suspension of phage and method. Next, the stripe pattern was immersed in a suspension the thickness of the free-standing films was tuned by varying of RGD-phage and pulled vertically, thereby resulting in the the phage concentration. The resulting films predominantly selective deposition of phage onto the cysteamine regions. exhibited chiral smectic structures. Although nematic ordered During the drying process at the contact line, two dominant structures were formed at very low concentrations, the struc- movements of the meniscus affected the deposition of phage on tural controllability of the phage films still lacked the complex- the cysteamine regions: the downward movement of the menis- ity and hierarchy that is present in liquid-crystalline phases in cus and the de-wetting from hydrophobic to hydrophilic suspension. In suspension, phage can assemble into various regions. Vertical orientation of the phage fibers was observed structures and sustain their conformation owing to the liquid-

Fig. 9. M13 phage are assembled into functional materials by using a self-templating process. The hierarchical assembly of phage can be controlled by competing interfacial forces at the meniscus, where the liquid-crystalline- phase-transition occurs. crystalline properties of anisotropic-shaped viruses. However, meniscus and by the resulting meniscus forces that were exerted slow dehydration, which led to solid or free-standing films, on the deposition front. mostly resulted in more-thermodynamically stable morpholo- A range of self-templated structures were formed by gies, because the phage particles gradually lost their mobility tuning various parameters, such as phage concentration, whilst drying and ended up in a highly concentrated phase. pulling speed, ionic concentration, phage surface chemistry, Otherwise, the phage were deposited onto the substrates with and the surface properties of the substrate. For example, at low disordered structures, driven by fast evaporation. concentrations (0.1–0.2 mg ml-1), the phage formed nemati- Most recently, Chung et al. developed a versatile strategy cally ordered fibril bundles parallel to the pulling direction, for the self-templated assembly of M13 phage into hierarchi- with regularly spaced ridge (stick) regions and groove (slip) cally organized structures; this strategy was suitable for various regions owing to the stick-slip motion of the meniscus. At functions, including the generation of structural colors, the higher concentrations (0.2–1.5 mg ml-1), the phage assembled directional guidance of cell growth, and the templating of into nematic orthogonal twist structures, which consisted of organic-inorganic composites through biomineralization.[101] alternating nematic grooves and cholesteric ridges that were Unlike previous methods, they introduced a kinetic element orthogonally aligned to each other during stick-slip motion. into phage assembly by using a simple and highly tunable Phage in the grooves aligned parallel to the pulling direction, instrument that allowed for the fine control of both meniscus whereas those at the ridge tops aligned perpendicular to the movements on the substrate and of competing interfacial forces pulling direction (Figure 10a–c). Within a specific range of at the meniscus line (Figure 9). Briefly, phage that displayed phage concentrations (0.25–0.5 mg ml-1) and pulling speeds tetraglutamate (4E) on the pVIII major coat protein were (10–20 mm min-1), phage in the ridges assembled into right- assembled into large-area films by pulling substrates vertically handed cholesteric helical ribbons (CHRs) along the left side of from suspensions of phage at precisely controlled speeds. At the the meniscus and left-handed CHRs along the right side of the liquid/solid/air triple-phase interface, evaporation proceeded meniscus. This phenomenon was influenced by forces that faster and resulted in the accumulation of phage. Thus, chiral originated from the curvature of the meniscus at the outer liquid-crystalline phase transition at the meniscus eventually edges of the substrate (Figure 10d–f). At much-higher concen- led to the deposition of phage particles on the substrate in a trations (4–6 mg ml-1), the deposited phage exhibited zig- highly ordered manner. At this moment, the liquid-crystalline zag-patterned periodic structures, termed smectic helicoidal deposition process was affected by the geometric shape of the nanofilaments (SHNs, Figure 10g–i). The phage are packed

into tilted smectic C bundles, thus forming the length of the Recently, self-templating methods that control both resulting helicoidal nanofilaments, which extend parallel to the thermodynamic and kinetic factors during the deposition direction of pulling (Figure 10h). Owing to the influence of process have opened up new routes for exploiting the ordered phage assembly by opposite meniscus forces from the side structural properties of hierarchically organized phage edges, each left and right side of the film exhibited opposite architectures.[101] For example, directionally ordered phage can optical responses under polarized light (Figure 10i). By maximize their piezoelectric strength, which is a useful prop- orthogonally aligning the phage-microstructures with alternat- erty for energy-generating devices, by summing the dipole ing SHNs and nematic hierarchical structures, the films were moment of each phage.[105] In addition, films of ordered phage able to allow the selective reflection or transmission of visible have exhibited unexpected functional properties, such as struc- light, depending on the direction of the incident light. The tured color and optical filtering. Structural colors or optical SHN structure also exhibited iridescence under white light, filtering from phage films can be used in optical phage-based which is an exquisite optical property that has been found to sensors, by combining these structural properties with target- occur naturally in blue-faced monkeys,[102] bird skins,[103] and specific binding motifs on the phage coat proteins. beetle exocuticles.[104] This self-templating method may not only contribute to Furthermore, they finely tuned the structural colors of the practical applications, as described above, but it could also lend films by controlling the pulling rates. Because the nanofilament insight into the fundamental study of biomacromolecular structures were less ordered, owing to to the faster pulling rates, assembly in in vivo systems under complicated and dynamic the position of the primary reflection wavelength shifted from conditions. The phage serves as an ideal model fibrillar build- red to blue. Inspired by hierarchically organized extracellular ing block, in terms of its monodispersity and its ability to matrices in biological systems, they examined the capability of display functional peptides, so that the relationship between the nanostructued phage as biomimetic tissues for cell-cultures the assembled structure and the resulting functions can be or biomineralization. The phage was engineered to display the easily elucidated. By investigating the kinetic and thermody- RGD peptide, which is a specific motif that promotes integrin- namic factors that play an important role in the formation of mediated cell-adhesion, on the pVIII major coat protein and metastable structures, we may further develop a better under- was pulled into films that exhibited alternating SHNs and standing of nature’s sophisticated self-assembly processes. nematic hierarchical regions. The ordered phage structures Although this self-templating approach demonstrated the great guided the growth of preosteoblast cells, by aligning their tunability and versatility of phage structures in response to bodies and internal actin filament networks parallel to the long concentration and pulling speed, many engineering parameters axes of the phage fibers on the nematic regions, but perpen- remain unexplored. For example, persistence length, differen- dicular to them on the SHN regions. The SHN films (which tially charged or hydrophobic proteins, and substrate compo- were assembled with the RGD phage and the 4E phage) were sition may all influence the assembly process. also used as a template for the biomineralization of calcium The effective diameter and persistence length of the phosphate, thereby resulting in tooth-enamel-like organic- phage particle changes in response to different pH values and inorganic composites that exhibited an about 20-fold increase ionic strength, owing to the polyelectrolytic properties of in Young’s modulus when compared to native phage films. phage. The effective diameter and persistence length strongly influence the phase before drying, even at the meniscus, 10. Perspective thereby resulting in the formation of bundles of different sizes and helical pitch. The surface properties of the substrate Over the last decade, the fabrication of ordered structures of upon which phage-assembly takes place is also a parameter phage has been of great interest as a means of utilizing the that might be considered. The contact angle of the receding outstanding biochemical properties of phage in developing meniscus and the tendency of the phage to adhere to the useful materials. Combined with other organic/inorganic sub- substrate can be controlled by surface modification of the stances, it has been demonstrated that phage is a superior substrate and may affect the competing interfacial forces that building block for the fabrication of various functional devices, are exerted on the meniscus, thus creating various unprec- such as the cathode/anode in lithium-ion batteries, photovol- edented structures. In addition, if multiple components are taic cells, phage-based sensors, and cell-culture supports. used in the solution, we can expect the formation of even- Although previous research has expanded the utility of phage higher-level-ordered structures. This phenomenon can be when combined with genetic engineering, most improvements easily observed in nature. For example, beneficial transpar- in device functionality have relied upon increases in efficiency ency properties are generated in arrays of close-packed, that were attributed to the compact, more-densely packable narrow collagen-fiber bundles when potentially interfering unit size of phage and not upon the unique properties of the proteoglycan components are not present to obscure visible ordered nanostructures themselves. light.[106]

Fig. 10. Self-templated supramolecular structures. a) SEM image of the nematic orthogonal twist structure, which shows the periodic striped pattern. b) AFM image that shows left-handed rotation of phage-fiber bundles the accumulated from the groove to the ridge area. c) Schematic illustration of the alternating nematic groove and cholesteric ridge. d) Photograph of cholesteric helical ribbon (CHN) structures, which shows the curved meniscus (dashed red line) that induces the macroscopic twist in supramolecular phage structures. e) SEM images of the CHR structures on the left and right sides, which show opposite macroscopic handedness of the ribbons. f) AFM image, which shows that the twisted cholesteric phase (red dashed lines) is comprised of the CHR structures. g) AFM image of the Smectic helicoidal nanofilament (SHN) structure. h) Smectic C-phage bundles that constitute the length of the helicoidal filamentous matrix. i) POM image of the grain boundary (white dashed line) in the SHN structure, which shows opposite polarized optical responses from the left and right sides. Inset: iridescence on the film upon white-light illumination.

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