2.206.
Phages as Tools for Functional Nanomaterials Development
W-J Chung, M Sena, A Merzlyak, and S-W Lee, University of California, Berkeley, CA, USA
ã 2011 Elsevier Ltd. All rights reserved.
2.206.1.
2.206.1.1.
2.206.1.2.
2.206.1.3.
2.206.2.
2.206.2.1.
2.206.3.
2.206.3.1.
2.206.3.2.
2.206.4.
2.206.4.1.
2.206.4.2.
2.206.4.3.
2.206.4.4.
2.206.4.5.
2.206.5.
2.206.5.1.
2.206.5.2.
2.206.5.3.
2.206.6.
References
Introduction
Introduction of Bacteriophage
Structure of M13 Phage
Directed Evolution of Phage
Phages for Inorganic–Organic Hybrid Materials
Self-Assembly of Inorganic Materials using Phage
Phage for Energy Materials
Phage for Energy Storage Materials
Phage for Energy Producing Materials
Phage for Sensing Materials
Phage-Based Molecular Recognition
Phage for a Sensing Platform
Micro- and Nanomechanical Sensing
Electrochemical Sensing
Optical Sensing
Phage for Biomedical Application
Phage Therapy
Phage for Drug and Gene Delivery
Phage for Tissue-Engineering Materials
Summary and Future Perspectives
Glossary
Bacteriophage Bacteriophage (phage) is a prokaryotic
virus that can infect bacterial host cells. The name
‘bacteriophage’ can be translated as ‘bacteria eater’
in Greek.
Directed evolution Directed evolution is a method of
utilizing the power of natural selection at the molecular level
to evolve proteins or RNA that are customized to meet
desired specifications.
Molecular recognition element The molecular recognition
element is a specific amino acid sequence that binds to a
particular molecular target which can be discovered through
directed evolution processes.
Nanobot The nanobot is a new future machinery concept
proposed by Dr. Eric Drexler in his book Engines of Creation
in 1983. He coined the term ‘Nanobot (Nanoscale Robot)’
for systems that can self-assemble, self-evolve, and selfreplicate – serving as fabricators of the next generation of
improved future machine systems.
Abbreviations
AAV
DNT
EGF
ELISA
FACS
FDA
Adeno-associated virus
1,3-Dinitrotoluene
Epidermal growth factors
Enzyme-linked immunosorbent assay
Fluorescence-activated cell sorting
Food and drug administration
96
96
96
97
98
100
101
101
101
103
103
104
104
105
106
107
107
107
108
108
110
Phage display Phage display is an accelerated evolutionary
screening process that allows one to isolate peptides that
bind specifically to a particular target material.
Phage therapy Phage therapy is the therapeutic use of
bacteriophage to treat bacteria-related disease.
Phagemid The phagemid is a hybrid cloning vector from
the filamentous phage M13 and plasmids. The vector can
grow into a plasmid and also can be packaged as a
single-stranded DNA in viral capsids. In order to
enable single-stranded DNA replication and packaging of
the phagemid DNA into phage particles, bacterial host
containing phagemid is subjected to infection with a helper
phage to provide the viral components.
Virotronics The Virotronics is a novel virus-based material
design technology that can be used to create novel
functional materials with precise molecular-level structural
and functional control using unique biological advantages
of viruses, such as specific molecular recognition, evolution,
and self-replication.
FGF
HER2
HPQ
IKVAV
ITR
LAPS
LC
Fibroblast growth factors
Human epidermal growth factor receptor
Histidine-proline-glutamine tripeptide
Isolucine-lysine-valine-alanine-valine tetrapeptide
Inverted terminal repeat
Light-addressable potentiometric sensors
Liquid crystal
95
96
Biologically Inspired and Biomolecular Materials and Interfaces
ME
MEMS
MREs
PEG
PSMA
2.206.1.
Magnetoelastic
Microelectromechanical system
Molecular recognition elements
Polyethylene glycol
Prostate-specific membrane antigen
QCM
RES
RGD
SPR
TNT
Introduction
shape, viral particles can be used to catalyze self-assembly
of ordered nanostructures, which is useful in applications
including energy,16–18 biosensors,19 electronics,14 and tissue
regenerating materials.20,21 In this chapter, we introduce the
unique features of viruses, recent accomplishments in the
development of virus-based materials for use as tools to fabricate functional nanomaterials, and review the potential future
applications of this emerging technology.
Nanotechnology is an interdisciplinary science and engineering field that enables us to observe, measure, and control
matters at the molecular level. Its development has allowed
us to envision clean and efficient energy conversion devices,1,2
super-computers operated by light,3 and tissues and organs
regenerated by smart tissue scaffolding.4 Despite the existence
of various methodologies to manipulate atoms and molecules
into exquisite new functional materials, designing new materials with well-defined structures and desired functions is still
a challenge in materials science.5 Conventional materials
are developed through rational design and intensive characterization. Based on the results, new materials with improved
physical and chemical properties can be designed. Many new
materials are generated by this iterative design and performance characterization process. Nature, however, solves such
material design issues using an evolutionary approach. Nature
prepares a diversified set of candidates and tests them in a
given environment through mutation. The best candidates are
then selected and propagated through generations over the
course of millions of years. Examples of advanced biological
materials with well-defined structures and functions include
glass sponges (optical fibers),6 brittle stars (optical lens array),7
diatoms (sophisticated periodic structures), abalone shells
(fracture-resistance materials),8 bones (support structure for vertebrates),9 and cells (exquisite self-replicating biomachines).10
Efforts to mimic the unique biological structures in nature have
provided a variety of tools and resources that are useful to
scientists and engineers.11,12 However, whole organisms have
complex functions and self-templated hierarchical structures
that cannot be easily mimicked. These unique characteristics,
which have accumulated through evolution, are inherited
genetically. Genetic information is translated into proteins,
which work as molecular machines to control precisely programmed processes in biosystems. Although protein-based
‘bottom-up’ synthesis of nanoscale functional materials and
devices is one of the most promising areas in the newly
emerging field of nanotechnology,13 identifying active basic
building blocks from biological examples is still a challenge
because of their complex and encrypted sequence structure.
Genetic engineering of phage viruses provides opportunities
for building novel bio-nanomaterials by integrating biology,
chemistry, physics, materials science, electric engineering, and
other disciplines. By mimicking the evolutionary process
in nature, phages can be used as an information-mining tool
to identify functional peptide (or protein) sequences that
can specifically recognize desired materials at the molecular
level.14,15 These recognition elements can be used to guide the
design of unprecedented materials such as semiconductor and
metallic materials. Additionally, because of their well-defined
Quartz crystal microbalance
Reticuloendothelial system
Arginine-glycine-aspartate tripeptide
Surface plasmon resonance
1,3,5-Trinitrotoluene
2.206.1.1. Introduction of Bacteriophage
Bacteriophage (phage) is a prokaryotic virus that can infect
bacterial host cells.22 The name ‘bacteriophage’ can be translated as ‘bacteria eater.’ As its name implies, once a bacteriophage infects the bacterial host, the virus exploits the host’s
biosynthetic machinery in order to produce many identical
copies of itself. Phages are some of the most common organisms on earth.23 Phages are commonly composed of a protein
capsid, or shell, which encapsulates and protects the virus’s
genomic material (DNA or RNA) (Figure 1).24 There are
many types of phages, each having differing genomic materials,
replication processes, and shapes. Genomic materials can be
either DNA or RNA in a single-stranded or double-stranded
form.25 With regard to the replication processes, phages can
be lysogenic or lytic.25 Lysogenic phages infect the host cells by
injecting their genomic materials. The genetic materials uptake
the host cell metabolisms and reproduce the same genetic
materials and corresponding proteins. These protein products
are delivered to the host cell membranes, where new phages are
packaged and released without disruption of the host cell walls.
On the other hand, lytic phages are replicated and accumulate
inside the host cells rather than at the cell membrane. Following replication, the newly amplified phages destroy the host cell
wall and can infect other host cells. There are many different
shapes of phages such as linear (M13, Fd, F1)26 or spherical
(MS2)27 (Figure 1). Some shapes are quite sophisticated. For
example, T4 and T7 phages possess an icosahedral head and a
long tail connected through a cylindrical body. Regardless of
differences in shape, composition, and life cycle, all phages
share the capacity to make exact copies of themselves with
incredible structural precision. Because of this property,
phages are great candidates to aid the development of novel
bio-nanomaterials. Thanks to the commercially available genetic
tool kits, M13 phage, in particular, has been extensively used as
evolvable nanoscale material. Therefore, we discuss mainly M13
phage and introduce other phages whenever applicable.
2.206.1.2. Structure of M13 Phage
M13 phage is a bacterial virus that comprises a single-stranded
DNA encapsulated by several major and minor coat proteins.
Phages as Tools for Functional Nanomaterials Development
97
pIX
pVIII
6.6 nm
pIII
pVIII
M13 phage
genome
(b)
T4 bacteriophage
(c)
MS2 bacteriophage
880 nm
pIX
pIII
(a)
M13 bacteriophage
Figure 1 Schematic diagram of various distinct structures of various phages. (a) Long-rod structure of M13 bacteriophage with genomic schematic
diagrams to show the each protein expressed on the M13 phage surfaces. (b) Structure of T4 bacteriophage with icosahedral head and long tail
connected through cylindrical body. (c) Sphere structure of MS2 bacteriophage.
It has a long-rod filament shape that is approximately 880 nm
in length and 6.6 nm in diameter (Figure 1(a)).28,29 The viral
capsid is composed of 2700 copies of helically arranged major
coat protein, pVIII, and 5–7 copies of the minor coat proteins,
pIII, pVI, pIX, and pVII, located at either of its ends.28,29 M13
phage can only infect and propagate within bacteria displaying
F-pili, such as Escherichia coli.30 It is a nonlytic bacterial virus,
meaning that it does not break the bacterial cell membrane
upon exit, but instead is secreted through a protein pore channel in the bacterial membrane.28,31,32 Bacterial host growth is
slowed down because of the increased metabolic demands of
phage production, but continues after infection.33 These qualities allow for easy mass amplification of the bacteriophage in
bacterial culture. Over the past 2 decades, the biochemical
landscape of the phage structure has been greatly expanded
through genetic engineering of the phage21,34–36 and sitespecific organic synthesis approaches.37–40 Through genetic
engineering, many foreign or synthetic DNA have been integrated
into phage genome and expressed at various sites of the phage
body.36,41 Nonnatural amino acids have been expressed on
the phage surfaces using amber codon tRNA approaches.42,43
In addition, reactions have been developed that enable
site-specific modification of phage surfaces with chemicals such
as fluorescent dyes or chromophores for various applications
including biochemical imaging and energy harvesting.38–40
2.206.1.3. Directed Evolution of Phage
One of the most remarkable features of phage-based materials,
which distinguishes them from traditional engineering materials, is their ability to be chemically evolved in a directed
fashion. Evolution mainly consists of genetic diversification,
functional selection, and replication processes. In nature, mutation can occur during gene replication, resulting in diversified
species with various new functions. By mimicking this natural
process, phage can be used as a template to perform directed
evolution through a technique called phage display.36,41 Phage
display is an accelerated evolutionary screening process that
allows one to isolate peptides that bind specifically to a particular target material. All the phage coat proteins can be genetically modified to display relatively short (<8–20 amino acid)
peptide sequences. Insertion of randomized DNA sequences at
specific locations within the phage genome enables one to
generate a highly diverse library of peptides (up to 1011 random
sequences) expressed on the viral particle surface.36,44
To select the functional peptide sequence for a given target
material, the engineered phage library pool goes through several rounds of selection, which is typically based on affinity
for the target material (Figure 2). As for the affinity selection,
the phages are first allowed to bind to the target. The nonbound phages are then washed away, and the bound ones
are eluted separately. The eluted phages are then amplified
through E. coli bacterial host infection. This is repeated for
several rounds with increasingly stringent binding conditions
in order to enrich the phage pool with high-affinity peptides.
Finally, the dominant binding peptides emerge and are identified through DNA analysis of the phage genome. Phage
display on pIII is most often utilized, as it is the biggest
phage coat protein and allows for greater peptide sequence
variability. pIII is also conducive for display of constrained
libraries.36,45 The lower valency of the protein (only 5 copies
as opposed to 2700 for pVIII) allows for the selection of peptides with higher affinity toward the target, because of the lack
of avidity effects.35 Recently, major coat engineered libraries
have been developed and used for inorganic material syntheses
involving semiconductors, metallic materials, and conjugation
of phage with carbon nanotubes.14–16,18,46 By applying microelectromechanical system (MEMS) techniques, Liu et al. have
recently developed a microfluidic device that performs phage
display in an automated manner on a microliter scale.47
98
Biologically Inspired and Biomolecular Materials and Interfaces
Wash away
nonspecific
binders
Biopanning
Elution
Target
Combinatorial
phage library
Repeat:
use more difficult
binding conditions
Finding
functional
peptide
information
Phage display
No!
⫻106
Yes!
Bacterial
amplification
Consensus
sequence?
DNA analysis
Figure 2 Schematic diagram of the phage display processes to identify the functional recognition peptide motifs.
The phage display technique was originally developed for
small peptides and antibodies and for identifying epitope-like
ligands. The main advantage of this approach is that the use
of an amino acid library allows for identification of an epitope
sequence from a protein that is not necessarily in sequential
order, but could assemble and become functional through
protein folding.35 Further, presentation of such a library on a
phage protein allows for a direct connection between the peptide
identified and its encoding genetic sequence.34,35 Since the
inception of this method, a variety of peptide ligands have
been discovered that are involved in interactions including specific protein binding,48–50 DNA binding,51 receptor binding,52,53
and cell and tissue binding.53 Further, through the use of
phage-display, short peptide motifs can be further matured
for enhanced binding against the target of interest.54,55
For example, phage display was instrumental in determining the
best binding conformation for the well-known RGD motif, as
well as elucidating the different sequences of RGD flanking residues which confer binding specificity for certain classes of integrins.53 However, identified peptide sequences should be carefully
confirmed with appropriate binding assays. Results from nonspecific binding can mislead the interpretation, particularly when
panning phage libraries against cell surfaces, which include
many of their molecules as well as the target receptor.
2.206.2. Phages for Inorganic–Organic Hybrid
Materials
Phage display has also been utilized to identify affinity peptide
sequences for a variety of inorganic substances, such as semiconductor and magnetic and metallic materials.14,46,55–57 Specific binding peptide sequences identified through such
directed evolution screening methods have been summarized
GaAs
SiO2
Figure 3 Specific recognition of phage onto semiconductor surfaces.
Fluorescence image of the phage can bind to square patterned GaAs
semiconductor surfaces. Reproduced from Whaley, S. R.; English, D. S.;
Hu, E. L.; Barbara, P. F.; Belcher, A. M. Nature 2000, 405, 665–668,
with permission from Nature. © 2000 Macmillian Publishing Ltd.
in recent reviews.58–60 Phage display performed with inorganic
substrates can uncover novel information about the particular
binding interaction between inorganic crystals and organic
peptides. Belcher and coworkers have demonstrated that
a genetically engineered phage can specifically recognize inorganic semiconductor substrates. For example, Whaley et al.
demonstrated that the phage could be selected for binding to
GaAs (III–V semiconductor) (Figure 3).15 The resulting phages
and corresponding peptide sequences exhibit remarkable specificity in their binding to different crystallographic orientations, such as (100) and (111), and similar surface structures,
Phages as Tools for Functional Nanomaterials Development
such as (111)A (gallium-terminated) or (111)B (arsenicterminated) face of GaAs.15 Another study of phage display
screening using carbon nanotubes and graphite surfaces
showed that selective binding of sequences with multiple tryptophan residues could be attributed to aromatic ring interaction between the peptides and nanotubes.57,61 Recently,
similar display screening methods, such as bacterial cell surface
display60,62 and yeast display,63 have also been applied to
identify the specific binding peptide against the various inorganic seminconductor and metallic materials and uncover the
specific binding peptides and proteins.
By mimicking the biomineralization processes in nature,
the phages and their peptides discovered from phage display
can also be used for the synthesis of inorganic materials.
In nature, specific binding proteins against various inorganic
crystals, such as silica in diatoms,64,65 iron oxide in magnetotetic bacteria,66 calcium carbonate in abalone shells,67 or
calcium phosphate in bones and teeth,68,69 play a critical role
in the synthesis of hybrid organic–inorganic materials.70
Similarly, specific recognition peptides identified through
phage display for desired crystal surfaces can be exploited in
order to template the growth of various target inorganic crystals.
Flynn et al. identified specific binding peptide motifs with high
affinity for II–VI semiconductor materials (ZnS, CdS, PbS, etc.)
and used them to direct mineralization of different phases of
the inorganic crystals in controlled shape and morphology.55
99
The researchers showed, for example, that linear and constrained binding peptides selected for zinc sulfide crystal surfaces are capable of nucleating cubic (zinc blende) and
hexagonal (wurtzite) phases of ZnS nanocrystals, respectively.
Genetic engineering of functional peptides with crystalnucleating or recognition capabilities on multiple phage coat
proteins has allowed for construction of many interesting
functional nanomaterials and nanostructures. Single or multiple genetic modification schemes are depicted in Figure 4.
Single modification of pIII minor coat protein was used
to synthesize ZnS and CdS nanoparticles at the terminus
of the phage.14,55 Nam et al. modified both the end minor
coat proteins (pIII and pIX) with six histidines and HPQ
(His-Pro-Gln) streptavidin-binding peptide motifs. These
phages were then used to construct nanoscale viral ring
structures through specific binding of chemically conjugated
streptavidin and Ni-NTA linkers.71 By incorporating complementary leucine zipper sequences on both ends of the phage,
Sweeney et al. programmed phage to self-assemble into linear
structures.72 By expressing the semiconductor- and metalbinding peptides on the major coat proteins using phagemid
vectors, one-dimensional polycrystalline semiconductor and
magnetic and metallic nanowires can be synthesized on viral
templates.46 After high-temperature annealing processes, highquality single-crystal ZnS or CdS nanowires with uniform size
and shape could be fabricated on the templated phage
pIII engineering
pIIIV engineering
A
B
pIX
100 nm
(c)
200 nm
pIII + pIX engineering
6.6 nm
pVIII
5 nm
(b)
300 nm
(d)
a-FePO4
pVIII + pIII engineering
880 nm
SWNTs
pIII
(a)
100 nm
(e)
4 nm
(f)
Figure 4 Engineering of M13 phage for programmed functionalization from material synthesis to specific conjugation. (a) Schematic diagram of M13
phage. Single engineering: (i) pIII engineering: specific nanoparticle synthesis on minor coat proteins (b); (ii) pVIII engineering: major coat protein
engineering for the synthesis of ZnS nanowires which can be used for the semiconductor synthesis or energy producing nanoscale nanowired materials
(c). Multiple gene engineering: (i) pIII and pIX engineering to construct ring structures using fillamentous phage (d) and (ii) pIII and pVIII engineering
to synthesize energy storing phage for battery materials synthesis and wiring to SWNT (e, f). (b, c, e, f) Reproduced from Lee, S. W.; Mao, C. B.;
Flynn, C. E.; Belcher, A. M. Science 2002, 296, 892–895; Mao, C. B.; Solis, D. J.; Reiss, B. D.; et al. Science 2004, 303, 213–217; Lee, Y. J.; Yi, H.;
Kim, W.-J.; et al. Science 2009, 324, 1051–1055, with permission from Science. © 2002, 2004, and 2009 American Association for the Advancement of
Science. (d) Reproduced from Nam, K.; Beau, R. P.; Lee, S. W.; Belcher, A. M. Nano Lett. 2004, 4, 23–27, with permission from American Chemical
Society. © 2004 American Chemical Society.
100
Biologically Inspired and Biomolecular Materials and Interfaces
structures. These nanowires exhibited promising electrooptical
and magnetic properties.46 By inserting multiple major coat
protein genes in the phage genome, Nam et al. demonstrated
that the phage can nucleate gold and metal oxides simultaneously.17 A variety of architectures of phage-templated nanostructures have been successfully demonstrated using
concurrent modification of pIII and pVIII coat proteins.73
Both ends and major coat protein-engineered phage might
conduct multiple programmed operations. Such engineered
phages are expected to be able to serve as electronic components and wires, potentially resulting in self-assembled, selfinterconnected, self-fabricated electronic devices. As a proof of
concept, Huang et al. showed that exquisitely programmed
materials can perform electronic functions.73 They fabricated
two terminal electrodes, which were interconnected by virusbased conductive gold nanowire, and measured I–V behaviors,
which exhibited ohmic behaviors of the viral nanowires.
2.206.2.1. Self-Assembly of Inorganic Materials using
Phage
Because of their monodispersity and long-rod shape, many
filamentous viruses have been extensively studied as liquid
crystalline model systems.74,75 By changing variables such as
concentrations, ionic strength, and external fields, viruses are
able to form ordered liquid crystalline structures (Figure 5).
At a low concentration range (below 5 mg ml1), phages
are randomly ordered in an isotropic liquid crystalline phase.
With increasing concentration (10–20 mg ml1), the distance
between neighboring viral particles becomes smaller than
their length. The virus particles begin to sterically hinder each
other and align into orientationally ordered configurations
called nematic phase liquid crystals. In the nematic concentration range, the helicity exhibited by viral particles such as M13,
Isotropic
Nematic
F1, and Ff phages is conserved in the nematically ordered
phage layer. Therefore, each nematic layer becomes twisted
about the normal axis of the layer below it. In the concentration range between 20 and 80 mg ml1 (cholesteric phase), the
rotation of nematic layers generates optically distinguishable
periodic structures, which may resemble finger print textures in
the optical micrographs (Figure 5). Finally, above 100 mg ml1,
the viral particles acquire both positional and orientational
order (smectic phase).74 Some viral particles such as M13 viruses
have been studied at much higher concentration ranges as solid
phase two-dimensional thin films14,75 and one-dimensional
fibers.76 Two-dimensional viral liquid crystalline films grown
at the air–liquid–solid interfaces of a meniscus have been shown
to exhibit a chiral smectic C structure (Figure 5).75
The use of polymeric templates and surface patterning tools
has greatly expanded the self-assembly capabilities of phages.
Yoo et al. developed a novel M13 phage patterning methodology by combining M13 phages with polyelectrolytes such as
polyethyleneimine and poly acrylic acid.77 In this approach,
M13 phages were segregated from the bulk charged polymers
when deposited on the surface of a suitable substrate. The
resulting aligned films exhibited a high degree of positional
and orientational order and could be further modified with
inorganic nanomaterials such as cobalt and gallium nitride
thin films. Using solvent-assisted capillary molding methods,
Yoo et al. showed that phage can be densely packed atop
micropatterned surfaces. Surface feature size was dependent
on phage length as well as the swelling properties of the underlying electrolyte polymers.78 Solis et al. also showed that, by
combining soft-lithography techniques with phage antibodies,
they could first pattern phage antibodies on the gold substrates. By tuning the concentration of the phage solution,
they could immobilize phages on the gold substrates on a
large scale in a controllable manner.79 In the future, these
z
n
q
Smectic
Smectic C
Cholesteric phase
POM image
Cholesteric
f
Chiral smectic C
1 mm
Smectic C phase
AFM image
Figure 5 Schematic diagram of the M13 bacteriophage in a various liquid crystalline ordered structures and their microscopy image. Reproduced
from Lee, S. W.; Wood, B. M.; Belcher, A. M. Langmuir 2003, 19, 1592–1598, with permission from American Chemical Society. © 2003
American Chemical Society.
Phages as Tools for Functional Nanomaterials Development
surface patterning techniques may be useful for biosensor
development, semiconductor fabrication, and energy storage
and conversion devices.
Phage self-assembly combined with peptide display via
genetic modification has been exploited to organize a variety
of nanocomponents into periodically ordered films and
fibers.14,55,76 After the identification of ZnS-specific binding
peptides through phage display, ZnS nanoparticles were selfassembled into periodically ordered film structures. Similarly,
anti-streptavidin viruses were used to align gold nanoparticles, organic dyes, and biological molecules.80 Magnetic
nanoparticle–virus composite films (FePt, CoPt, etc.) were
constructed using both phagemid and pVIII engineering of
viruses.81 Finally, spontaneously ordered virus-based thin
films integrated with semiconductor (GaN) and metallic
(Au) nanoparticles have been fabricated on multilayered electrolyte polymer films using layer-by-layer deposition.17
Besides electronic and magnetic particles, viruses can also
be used for the assembly of biological tissues. Merzlyak et al.
engineered M13 phage with RGD (integrin-binding peptide)
and IKVAV (neural cell stimulating peptides) displayed on the
major coat proteins.21 Following formation of the liquid crystalline suspensions, these phages were spun along with target
cells using a micropipette. The resulting microfiber structures
contained aligned phages, which served as scaffolds for the
controlled growth of fibroblasts and neural stem cells.
Mechanically sheared thin films composed of liquid crystalline
phages can also exhibit directional organization and serve as
substrates that direct cell growth processes.20
2.206.3.
Phage for Energy Materials
In recent times, there has been a rising demand for novel
materials that can generate, store, and utilize energy with performance levels exceeding those of current technologies. In this
research area, the ability to nucleate nanocrystals and selfassemble long-range ordered phage structures has attracted
much attention.
2.206.3.1. Phage for Energy Storage Materials
One of the advantages of the use of engineered viruses as
molecular templates is the fact that they permit nanomaterial
synthesis and assembly in an energy-efficient and environmentally friendly manner. Nam et al. demonstrated that engineered
M13 phages can be used to synthesize and assemble nanowires
of cobalt oxide at room temperature, which can then be used
as materials in lithium-ion batteries.17 In this work, tetraglutamate (EEEE-, 4E) was first inserted into the N-terminus
of the pVIII major coat protein. This phage was further engineered to express gold-binding peptide motifs. Both gold and
cobalt oxide nanoparticles nucleated on the major coat protein, which resulted in hybrid nanowire structures composed
of cobalt oxide with spatially interspersed gold nanoparticles
(Figure 6(a)). These cobalt oxide nanowires mineralized by
the M13 virus in aqueous condition at room temperature
exhibited a high degree of crystallinity that was comparable
to that of cobalt oxide nanoparticles prepared using conventional high-temperature (>500 C) methods. When these
virus-based hybrid composite Au–Co3O4 nanowires were
101
applied to lithium battery electrode materials, the resulting
electrodes showed improved energy storage capacity compared
to the pure Co3O4 nanowires (>30% greater). This was mainly
because of the enhanced conductivity and catalytic activity of
the gold nanoparticles that participated in the redox reaction of
Li with cobalt oxide. In order to integrate these hybrid nanomaterials into a thin and flexible energy storage system, M13
viruses (4E) were assembled in a two-dimensional polyelectrolyte using layer-by-layer assembly (linear poly(ethyleneimine)/
poly(acrylic acid)). Spontaneously assembled viruses were
then transformed to cobalt oxide nanowires in a similar fashion, enabling fabrication of lightweight, flexible, and transparent anode electrode films. The resulting self-assembled
virus-based lithium-ion battery film exhibited enhanced battery performance in comparison to commercial batteries and
displayed a cycling rate remarkably close to the theoretical
limits. The main reason for the improved performance of
novel virus-based battery materials is that the monodisperse
nanowires templated by engineered viruses enable assembly
of close-packed layered structures with a high surface area to
volume ratio, and thus charge carrying capacity. The Belcher
group has also developed a method for spatial positioning of
virus-based electrodes using microcontact printing.18 After
coating a PDMS template (4-mm diameter circular patterns)
with alternating layers of positive and negative polyelectrolytes, the engineered virus (4E) solution was dropcasted on
the multilayers. The whole assembly was dipped into the
cobalt oxide precursor solution and cobalt oxide nanowires
formed on the micropatterned regions. Stamping the template onto a platinum microband current collector (cobalt
oxide side down) produced a patterned array that served
as the anode electrode component of an effective battery.
In order to fabricate cathode materials needed for a full
virus-based battery system, Lee et al. developed a novel multigene engineered virus.16 The new cathode-fabricating viruses
were engineered to nucleate iron phosphate on the pVIII
major coat protein and to bind to carbon nanotubes on pIII
minor coat proteins. The resulting viral particles successfully
mineralized iron phosphate and interconnected the conductive
carbon nanotubes, forming a conductive network cathode
material (Figure 6(b)). It was found that incorporating carbon
nanotubes increases the cathode’s conductivity without adding
much weight to the battery. By combining virus-based cathodes
and anodes (viral silver Ag nanowires), the Belcher group has
demonstrated complete virus-based 3-V batteries that can be
used to power light-emitting diodes (LEDs).
2.206.3.2. Phage for Energy Producing Materials
Inspired by the natural photosynthetic systems, the welldefined structure of viral protein shells has also been used to
develop biomimetic photosynthetic materials. In natural photosynthetic systems, several types of chromophores are spaced
precisely so that the resulting structures can transfer absorbed
solar energy in a highly efficient manner.81 Thus, in manmade
systems, the spacing between individual energy transferring
components should be regulated with nanometer precision.
In order to accomplish this, the well-defined structure of viral
particles such as MS2 phage has been taken advantage of for
precise spatial tuning of self-assembled structures that mimic
102
Biologically Inspired and Biomolecular Materials and Interfaces
Virus-based anode fabrication
Assembly
engineering
Virus biotemplating
Li ion battery
Macroscopic self-assembly of virus
M13 virus
Li+
Co3O4
or
Au-Co3O4
nanowire
(a)
Anode
Electrolyte
Cathode
Virus-based cathode fabrication
High-power lithium-ion battery
cathode
Cathode
a-FePO4
templated
virus nanowire
SWNT
Biomolecular
recognition
and attachment of
templated virus to SWNT
Electrolyte
Anode
(b)
Figure 6 Schematic diagram of the virus-enabled synthesis and assembly of nanowires as negative and positive electrode materials for Li-ion
batteries. (a) Rationally designed peptides were expressed on the major coat p8 proteins of M13 viruses to grow Co3O4 and Au–Co3O4 nanowires.
Macroscopic ordering of the viruses was used to fabricate an assembled monolayer of Co3O4 nanowires for flexible, lightweight Li-ion batteries.
Reproduced from Nam, K. T.; Kim, D.-W.; Yoo, P. J.; et al. Science 2006, 312, 885–888, with permission from Science. © 2006 American Association for
the Advancement of Science. (b) Fabrication of genetically engineered li-ion battery cathodes using multifunctional viruses and a photograph of the
battery used to power a green LED. Reproduced from Lee, Y. J.; Yi, H.; Kim, W.-J.; et al. Science 2009, 324, 1051–1055, with permission from Science.
© 2009 American Association for the Advancement of Science.
natural energy transfer systems.38,83 The protein structure of
viral particles can be modified by genetic engineering or chemoselective bioconjugation approaches for incorporating specific functional groups at desired locations.21,40 Using such
techniques, photocatalytic materials such as porphyrins can
be conjugated to genetically engineered M13 bacteriophage
for investigation of virus-based energy transfer reactions.84
Scolaro et al., for example, have genetically modified M13
phage to express inserted peptides containing tryptophan (Trp)
residues at the exposed N-terminal region of the pVIII major coat
protein. Cationic porphyrin derivatives were then electrostatically immobilized to the negatively charged M13 phage surface.
Fluorescence quantum yields of the resulting porphyrin–virus
hybrid structures were examined at three different wavelengths
(295, 400, and 438 nm) in order to observe energy transfer
events from the external tryptophan residues of M13 to the
contiguous porphyrins. In the presence of M13 with Trp residues, fluorescence quantum yields at 295 nm were found to be
approximately two times higher than those observed with
wild-type M13 phage. This only happened at 295 nm, which
indicated that Trp residues were actively involved in the
donor–acceptor coupling and energy transfer processes.
Site-specific chemical reactions have also been developed to
precisely control the protein shell structure for design of welldefined multifunctional viral particles used in photosynthetic
systems. Stephanopoulos et al. used viral capsids of sphereshaped MS2 viral particles, which can be assembled in a hollow 27-nm diameter shell, to arrange fluorescent dyes (Oregon
green) and zinc-porphyrin through chemoselective reactions
(Figure 7(a)).39 The maleimide-containing donor dyes were
targeted to the interior surface of MS2 phage, which was modified with cysteine residues. The dyes were chosen to have
emission spectra that overlapped with the porphyrin absorbance band. The exterior of the capsid was modified through
an oxidative coupling reaction on the aniline residue37
to introduce an aldehyde functional group, which was
further modified with the aminooxy-containing derivative of
Zn-porphyrin to form an oxime linkage. This arrangement
enables fluorescence resonance energy transfer from the dyes
inside to the porphyrin on the outside through the 2-nm-thick
protein shell. The electron-transfer ability of the complex
was monitored by photoreduction of methyl viologen by
excited-state Zn-porphyrins (Figure 7(b)). The addition of
2-mercaptoethanol completed the catalytic cycle by reducing
the porphyrin cations. Viral capsids with both fluorophore
donors and porphyrin acceptors showed a 3.5-fold increase
in the photoreduction of methyl viologen upon illumination
at 505 nm for 15 min in comparison to the capsids without
donor (Figure 7(c)). The results indicate that well-designed
nanostructures incorporating donor dyes and porphyrins at
Phages as Tools for Functional Nanomaterials Development
103
Nanoscale integration of sensitizing chromophores and porphyrins with bacteriophage MS2
Selfassembly
in E. coli
Cys 87
MS2-coat-protein dimer
SH
Zn-Por*
Zn-Por
e−
HO
S
.
+
Me – N
N – Me
e−
+
H+
20–120 porphyrins
installed outside
Up to 180 donor dyes
installed inside
(a)
HO
Acceptor attachment
by oxidative coupling
and oxime formation
Donor maleimide
attachment
Me – N
Zn-Por.+
+
N – Me
Relative [MV+] per mM
pAF 19
(b)
(c)
50
MS2 with 4 only
40
30
MS2 with 2 only
20
MS2 with 2 and 4
10
0
2: Oregon green 488
4: Porphyrin
Figure 7 Integration of sensitizing chromophores and porphyrins into subunits of bacteriophage MS2 and photoreduction of methyl viologen by a
sensitized porphyrin system. (a) Two mutations were introduced into subunits of the MS2 coat protein. After capsid formation in E. coli, the interior
and exterior surfaces were differentially modified in a multistep sequence. (b) Catalytic cycle of photoreduction. (c) Relative efficiency of the
photoreduction with different systems upon illumination at 505 nm for 15 min. Reproduced from Nicholas, S.; Carrico. Z. M. C.; Francis, M. B. Angew.
Chem. Int. Ed. 2009, 48, 9498–9502, with permission from Wiley-VCH Verlag GmbH & Co. KGaA © 2009 Wiley-VCH Verlag GmbH & Co. KGaA.
specific locations can perform energy transfer from the inside
to the outside of the capsids.
2.206.4.
Phage for Sensing Materials
Phages have been used extensively in sensing applications
over the course of the past 2 decades. Because of their
genetically tunable chemical surface properties, self-assembling
capabilities, and biological activity, phages have emerged as
valuable tools for detection of various targets including small
molecules,85–87 biomolecules,88–93 and whole cells.94–104
Phages have been applied to biological and chemical sensor
development in three fundamental ways19: First, phage display
techniques have been used for rapid evolutionary screening of
peptide molecular recognition elements (MREs) capable of
binding to a particular target of interest. Once their sequence
is identified, these MREs can be chemically synthesized and
applied to assays for further development of integrated sensor systems. Second, engineered target-binding phages can
be mass produced using a bacterial host and used, intact, as
a sensing probe. Serving as a nanometer-scale scaffold, a
phage probe is capable of displaying MREs in high copy
numbers with controlled orientation. Finally, the biological
activity of lytic phages can be utilized for detection of host
bacteria. In this case, high phage replication rates and unique
phage–host interactions enable rapid and specific sensing of
microorganisms.
2.206.4.1. Phage-Based Molecular Recognition
The success of the use of phages for chemical and biological
sensing can be attributed to their broad adaptability and ease
of genetic manipulation as a molecular recognition agent;
capacity to enhance target binding by serving as multivalent
scaffolds for MREs; and natural ability to recognize, replicate
inside of, and sometimes destroy microorganisms. Phages are
thus versatile sensing probes with incredible chemical diversity
and structural complexity. They can be applied for chemical
and biological sensing much in the same way as antibody,105
peptide,106 aptamer,107 or imprinted polymers.108
Both phage display and rationale modification have
emerged as convenient tools for the development of sensing
probes. As discussed in the previous chapter, by insertion
of the randomized gene sequences into phage genome, it is
possible to express billions of peptide-based receptor candidates. These peptide libraries enable rapid evolutionary screening for receptors. There are many reviews related to receptor
development using phage display.19,36,109–112 As an illustration, we briefly introduce one example of rapid receptor discovery performed using trinitrotoluene (TNT) explosive as
a molecular target. Jaworski et al. identified TNT- and DNTbinding peptide motifs using phage display processes.86,113
These two explosive molecules possess very similar chemical
structures with one nitro functional group difference. After four
rounds of TNT screening using phage display, the researchers
identified TNT-binding sequences with the consensus motif of
Trp-His-Trp-X (X: hydroxylated, amine, or positively charged
side chain) at the N terminus of the receptor (Figure 8).86
From competitive screening experiments, 95% of the strong
binding phage exhibited the same tetrapeptide motif: TrpHis-Trp-X. The most abundant sequence was assigned as the
best TNT-binding peptide candidate (Trp-His-Trp-Gln-ArgPro-Leu-Met-Pro-Val-Ser-Ile: TNT-binding peptide (TNT-BP)).
Similarly, consensus DNT-binding motifs were identified after
five rounds of screening (Figure 8(a)). Among the subset
104
Biologically Inspired and Biomolecular Materials and Interfaces
N term
Phage display
TNT target
Select 4th round
Screening results
DNT target
Select 4th round
Screening results
1 2
3
Amino acid
5 6 7 8
4
W H W Q
W H W S
W H W N
W H W K
H P N F
Q R P T
Q R P T
C term % Abundance
9 10 11 12 from 4th round
R P L M P V S
P R T A L Y T
F K P P H D L
P P A P Y V W
S K Y I L H Q
T Q Q G P S M
T Q L G S E Y
I
T
75%
5%
L
W
6%
12%
R
24%
24%
6%
L
A
CH3
TNT
(a)
NO2
0.00010
Level of binding, ratio of phage
(output/input)
High strain
Nonpolar
Positively charged
Negatively charged
H-bonding, hydroxyl
Methionine
Nonpolar aromatic
H-bonding, carboxamide
NO2
O2N
0.00008
TNT substrate
DNT substrate
CH3
O2N
0.00006
0.00004
DNT
0.00002
0.00000
(b)
Nonspecific (PS)
DNT-BP
Receptor on phage
TNT-BP
NO2
Figure 8 Phage display screening against TNT and DNT explosives: (a) Converged amino acid sequences from fourth round of phage display
screening with noted percentage abundance obtained from sequencing results. (b) Selectivity screening of DNT receptor and TNT receptor against DNT
substrates and TNT substrates with level of binding quantified from phage titration. Nonspecific binding levels are identified by polystyrene (PS) binding
phage against TNT and DNT substrate. All data presented as mean standard deviation. Reproduced from Jaworski, J. W.; Raorane, D.; Huh, J. H.;
Majumdar, A.; Lee, S. W. Langmuir 2008, 24, 4938–4943, with permission from American Chemical Society. © 2008 American Chemical Society.
of TNT-binding sequences, several exhibited varying affinity
for DNT. In order to enhance the selectivity between TNT
and DNT targets, negative screening was performed for the
DNT molecules using the isolated TNT-binding peptides. The
TNT-binding peptides with the lowest affinity for DNT exhibited remarkable selectivity for the TNT target (Figure 8(b)).
This result demonstrates that M13-linked receptor sequences
identified from phage display can selectively bind a predetermined target. Through investigation using fluorescent
binding assays and peptides with alanine substitutions in
the N-terminal region, it was shown that the conserved tryptophan and histidine residues are critical for effective TNT
binding. The tryptophan replacement resulted in a 58%
decrease in affinity, while the histidine replacement reduced
binding by 48%. By comparing the identified TNT-binding
sequence, scrambled sequence, histidine substitute, and tryptophan substitute for different substrates, the investigators
provided evidence of substrate selectivity through multivalent binding. Cerruti et al. later demonstrated that these TNT
receptor peptides have a remarkable selectivity for target
TNT molecules following immobilization on quartz crystal
microbalance (QCM) liquid sensing platforms.113 The rapid
receptor discovery technique for the TNT and DNT explosive
introduced here is one of many examples of the application of
the phage display for molecular sensing. With its evolutionary
capabilities, phages can be generalized as tools for the discovery of novel molecular receptors for environment-, terrorism-,
and health-related target toxicants in a rapid and convenient
manner.
2.206.4.2. Phage for a Sensing Platform
Phages have been used in combination with many transducing
modalities not only for the discovery of receptors for various
target analytes but also as functional sensor components.
In order to generate an observable output signal as a result of
a specific molecular recognition event, phage particles have
been coupled with nano- or micromechanical, electrochemical, and optical sensing platforms, which have recently been
reviewed.19 Using many of the self-assembly approaches discussed in the previous section, it can be convenient to fabricate
phage-based sensor systems with highly dense and customizable molecular recognition elements. Both whole-phage and
phage-derived probes can be implemented in several sensing
formats, for example, as alternatives to antibodies in solutionbased and surface-based assays,109,110 or as a biochemical
signal amplifier in bacterial culture-based assays.100,101,114
A variety of useful sensing systems and devices that utilize
phage have emerged since the development of phage display
for molecular biopanning.86,113 These can be based on the
measurement of mechanical, electrical, or optical signals in
response to the presence of molecular or cellular targets
(Table 1).
2.206.4.3. Micro- and Nanomechanical Sensing
Phages have been used to coat materials for various micro- and
nanomechanical sensing platforms. Since their original development for microweighing applications,118 quartz crystal
microbalances (QCMs), nano-cantilevers, and magnetoelastic
Phages as Tools for Functional Nanomaterials Development
Table 1
105
Classes of phage-based sensing systems
Mechanism
Platform
Probe
Target
References
Nano/
micromechanical
Quartz crystal
microbalance
Magnetoelastic
resonator
Amperometric
sensor
Impedance sensor
pVIII-engineered Fd phage
89,91,98,101,114
pVIII-engineered Fd phage
S. typhimurium, b-galactosidase, prostatespecific membrane antigen (PSMA)
S. typhimurium, B. anthracis
Phage l; M13 phagemid/ALP
E. coli K12 (MG1655); E. coli TG-1
99,100
T4 phage, l phage, pVIIIengineered M13 phage
pIII-engineered M13 phage
E. coli, Salmonella spp., anti-M13 IgG, PSMA
91,93,102,115
Human phosphatase of regenerating liver-3
(hPRL-3) marker carcinoma cell (MDAMB231)
87,103
B. anthracis, staphylococcal enterotoxin B
2,4,6-trinitrotoluene, S. typhimurium, hepatitis
B surface antigen
b-Galactosidase, L-monocytogenes
84,88,90,94,116
89,98
Streptavidin
92
Electrochemical
Optical
Light-addressable
potentiometric
sensor
Fluorescence, ELISA
Surface plasmon
resonance
Opto-fluidic ring
resonator
pVIII-engineered Fd phage,
pIII-engineered M13 phage
pVIII-engineered M13 phage,
pVIII-engineered M13
phage
Bifunctional (pVIII and
pIII-engineered) Fd phage
(RGD and HPQ)
resonators have been used widely for real-time, label-free studies of biomolecular interactions and cellular adhesion.119,120
Detection is typically based on the accumulation of mass at the
sensor surface and a corresponding shift in the vibrational
resonance of the transducer.
Commercially available piezoelectric transducers such
as QCMs are commonly functionalized with target-binding
receptors such as engineered phage and implemented for
sensing.92,102,115 On the application of an alternating electric
field, coated QCMs undergo mechanical vibrations at a frequency that is dependent on the amount of accumulated
target. In one comparative study of label-free QCM and standard ELISA-based molecular sensing,115 Nanduri et al. demonstrated the efficacy of physically adsorbed pVIII-engineered
Fd phage as a target-binding agent against a model protein,
b-galactosidase. They found that, while immobilized phage
performed similarly to monoclonal antibodies in a standard
ELISA format, QCM-based measurements yielded a fundamentally different dose–response curve (12-fold lower Kd value
and a 3-fold lower Hill coefficient), perhaps indicating a
relative improvement in target accessibility and thus sensing
capability.
Another means of inducing vibrational resonance in a
mechanical sensing platform is through the use of magnetoelastic (ME) materials. When subjected to megahertz-range
oscillating magnetic fields, submillimeter ME ‘ribbons’ oscillate, generating a corresponding alternating current in a remote
pickup coil. This technique allows for wireless measurement
of frequency shifts and thus remote sensing.96–98 In a
recent demonstration of multiplexed detection of pathogenic
organisms,97 Huang et al. determined the concentrations of
pathogenic Salmonella typhimurium and Bacillus anthracis simultaneously using phage-functionalized ME ribbons with two
different resonance frequencies (Figure 9). Separately functionalized ME platforms were highly specific with no detectable
cross talk between frequency channels.
95–97
Phase-sensitive
impedance measurement
Au
Buffer solution
(b)
p-Ab
n-Ab
M13
Pt
(c)
(d)
(a)
Figure 9 Phages as sensor coating materials. Schematic diagram of
virus-based impedance measurement setup. (b) A dense virus layer was
covalently bonded to the gold surface that produces a dense phage layer
that completely electrically insulates the gold surface from contact
with the buffer solution. (c) Exposure of this virus electrode to a
‘negative’ antibody (n-Ab, blue) causes no change to either the imaginary
component of impedance, ZIm or to ZRe. (d) Exposure to a ‘positive’
antibody (p-Ab, red) that is selectively recognized and bound by the
phage causes a significant increase in the high-frequency ZRe.
Reproduced from Yang, L.-M. C.; Tam, P. Y.; Murray, B. J.; et al. Anal.
Chem. 2006, 78, 3265–3270, with permission from American Chemical
Society. © 2006 American Chemical Society.
2.206.4.4. Electrochemical Sensing
Molecular recognition of specific targets by phage peptide
receptors can be detected using electrochemical sensors. Electrochemical sensing systems are useful because they allow for
direct interrogation of the sample on a compact, integrated
platform. Detection is typically based on the simple readout
of changes in currents, impedances, and potentials associated
with the presence of target species. Amperometric sensing
systems measure current flow in a solution undergoing an
oxidation–reduction reaction. In conjunction with such
106
Biologically Inspired and Biomolecular Materials and Interfaces
systems, phages can be utilized for highly specific detection of
their bacterial host (target) on generation of an electrochemically active reporter. In one example, phage l was used
as a lysing agent for a specific strain of E. coli. Release of
b-D-galactosidase enzyme and subsequent conversion of an
added substrate (p-aminophenyl-b-D-galactopyranoside) to an
electroactive product allowed for detection of cells at concentrations as low as 0.01 colony-forming units per milliliter.101
Detection without lysis of the target microorganism has also
been demonstrated using a phagemid system to express alkaline phosphatase as a reporter.99
Impedance spectroscopy is one of the most well-developed
electrochemical techniques used in combination with engineered phage for sensing applications.92,94,103,116,121 Impedance measurements are easy to conduct over a range of
frequencies and are highly sensitive to perturbations (e.g.,
target binding) at the surface of the sensing electrode. In one
example of such a sensor,92 Yang et al. covalently attached a
dense layer of pVIII-engineered M13 phage to a gold electrode, which was then immersed in a test solution. Measuring time-dependent changes in electrical impedance of
the circuit at the kHz frequency range resulted in a target
(PSMA) detection limit of 120 nM and signal to noise
ratios greater than 10. More recently, the authors used the
same technique to thoroughly characterize binding of antiM13 antibodies to the phage-coated electrode and determined 4–140 kHz as an optimal driving frequency for the
sensing circuit.116 Additional examples of impedance sensing involve detection of E. coli on screen-printed carbon
electrode arrays functionalized with T4 bacteriophage,103
and real-time observation of salmonella infection and lysis
by bacteriophage.94
Phages have also been used as a bioresponsive coating for
light-addressable potentiometric sensors (LAPS).88,104 Composed of a semiconductor–insulator base (e.g., Si–Si3N4),
which can be activated by directed light pulses, these devices
allow for interrogation of surface potentials generated as a
result of ion gradients, redox reactions, or pH changes.122
Such devices have been coated with engineered phage for
label-free detection of human phosphatase marker and cancer
cells.88 More recently, LAPS were covalently modified with four
different phages selected against metastatic SW620 cells.104
When tested with other highly metastatic lines (e.g.,
MDAMB231) added to a plasma sample, the system was able
to detect as few as 100 cancer cells with almost no background
from healthy leukocytes.
2.206.4.5. Optical Sensing
Employing phenomena such as colorimetric amplification,
fluorescence, and photonic resonance, optical sensing modalities are quite versatile. Detection can be based either on signals
associated with optical labels or on changes in the optical
properties of a sensor–sample interface.19 In either case, optical
systems are extremely sensitive, with high signal-to-noise ratios
and, sometimes, single-molecule resolution.
ELISA-like assays are commonly carried out on microtiter
plates using engineered phage or phage-derived affinity
peptides.85,89,91,95,117 This allows for rapid assay development
and the use of standard absorbance or fluorescence-based plate
readers. For example, Sorokulova et al. performed intensive
landscape phage screening against S. typhimurium using
both phage-capture and target bacteria-capture sandwich
ELISAs.117 Secondary labeling of captured target with alkaline
phosphatase-conjugated antibodies allowed for colorimetric
measurements of binding affinities. Fluorescently labeled
phage can also serve as the reporter in these assays.85,123
In addition, fluorescent labels allow for microscopy, FACS
analysis,117 and Förster resonance energy transfer assays,
whereby distance-dependent, nonradiative energy transfer
from a donor fluorophore to an acceptor allows for the detection of highly specific biomolecular interactions (e.g., between
phage-derived antibody fragments and morphine124). Fluorescent quantum dot labels have been used in conjunction with
engineered phage in order to carry out detection of host
bacteria.114 Here, phages were engineered to display a peptide tag, which could be biotinylated by the host’s biotin
ligase protein. After being released into solution, biotinylated phage progenies were then labeled by streptavidinconjugated quantum dots. Viral infection thus served as a
means of signal amplification, as quantum dot fluorescence
was directly correlated with phage infectivity. Because of
rapid replication, a 100-fold amplification was observed
within 1 h, allowing for a detection limit of 10 bacterial
cells per milliliter of sample.
Label-free optical sensing platforms that employ surface
plasmon resonance (SPR), waveguides, interferometers, and
ring resonators allow for real-time detection of target-binding
events.125 These systems measure refractive index changes
at a surface functionalized with MREs. For example, pVIIIengineered phage can be used for highly sensitive SPR
detection of b-galactosidase, a model protein,90 or for kinetic
binding analysis of Listeria monocytogenes, a pathogenic bacterium.99 In these studies, this technique was easily adaptable, as
engineered phages specific to the target of choice were simply
adsorbed onto a commercial SPR chip. Whispering gallery
mode resonators represent another class of versatile optical
biosensors that have been implemented on microspheres,
microtoroids, planar ring waveguides, and cylindrical optofluidic devices.126 Because of recirculation of resonant light
modes, these systems are extremely sensitive. In one example,93 Zhu et al. developed a phage-based optofluidic microring resonator by depositing streptavidin-binding phage on the
interior of a pulled microcapillary tube. The resonator was
coupled to a light source and photodetector via a tapered
optical fiber. Shifts in the resonant frequency peak of circulating whispering gallery modes corresponded linearly to both
phage deposition and target binding, and allowed for a streptavidin detection limit of 100 pM.
Each of the sensing modalities described above exists
because of a unique combination of sensitivity, specificity,
speed, cost, and adaptability. No single system is ‘best’ overall – optimality depends on the particular sensing application of interest. However, at least for the purpose of sensor
development, an engineered phage may be advantageous
over other probes because of its broad adaptability. The
ability to develop MREs specific to almost any molecular or
cellular target of interest is extremely powerful, as it allows
investigators to focus on the detection systems rather than
the probe, alone.
Phages as Tools for Functional Nanomaterials Development
2.206.5.
Phage for Biomedical Application
Over the past century, phages have been used in biomedical
applications including antibiotics, drug delivery, gene therapy,
and tissue engineering. As mentioned, the word ‘bacteriophage’ (directly translated from Greek as ‘bacteria eater’)
implies that we can use it as an antibacterial drug. The first
description of bacteriophages in scientific literature originated
from a British bacteriologist, Ernest Hankin, in 1896, when he
observed that a substance present in the waters of the Ganges
and Jumna rivers in India significantly reduced the titer of
Vibrio cholerae bacteria when grown in culture.127,128 Twenty
years later, two independent scientists, Fredrick Twort in 1915
and Felix d’Herelle in 1917, conducted more conclusive studies
to characterize such bactericidal effects. It was at that time that
d’Herelle reported that they were caused by a bacteria-infecting
virus and coined the term ‘bacteriophage.’ Soon after this
discovery, d’Herelle and his contemporaries began using bacteriophage preparations as a medical therapy against the bacteria that caused diseases such as dysentery and cholera.128,129
Despite early clinical success and even commercialization by
several well-known companies including Eli Lilly128,129 phage
therapy failed to take off. Several factors led to its early demise,
including lack of in-depth knowledge of phage–bacteria interactions, failure to efficiently purify the phage from lysed bacterial solution, and rapid clearance of the phage from the body’s
circulation.128,129 Another factor that led most Western researchers to abandon the advancement of phage therapy was the
coming of an antibiotic era. While most phage-bacteria interactions are species specific, antibiotics can have a blanketing bacteriocidal effect on bacterial populations. Thus, at the time, they
were more effective and attractive for eliminating bacteria.
2.206.5.1. Phage Therapy
During the past 60 years, phage therapy investigations
continued in centers across Eastern Europe and the former
Soviet Union. Today, with the rise of antibiotic-resistant
bacteria130 and further developments in molecular and microbiology, the idea of phage therapy is becoming more popular
among the scientific127,128,131 as well as business communities.
Previous problems associated with phage therapy are now
being addressed, and scientists are expanding on molecular
biology knowledge of phage–host interactions and infective
mechanisms. For example, it has been shown that M13 bacteriophage are only able to infect Gram-negative bacterial
cells that display the F pilus protein on the outer membrane,
such as E. coli. Specific interactions between the TolQRA bacterial receptor and the pIII and pVIII proteins facilitate virus
penetration and DNA transfer.132–134 This specificity allows
for the use of E. coli for effective replication and amplification
of the virus but ensures that M13 will not infect or replicate
within a human patient’s indigenous bacterial population or
eukaryotic cells, which lack the necessary tropism. Another
major problem that previously led to failure of administered
phage therapy was contamination of phage mixtures with
either bacterial particles or endotoxins, which can incite potentially harmful immunogenic reactions.135 At present, advanced
purification methods including ultracentrifugation, precipitation, and ion chromatography allow for solutions of
107
phage to be prepared that are free of bacteria and related
particles.128,129,136
Phages can be cleared from the body through two pathways. Composed of proteins foreign to the body, a virus may
evoke an immune system response that would inactivate and
destroy it. Alternatively, phage particles may be rapidly cleared
by the reticuloendothelial system (RES), which includes filtering organs such as the liver, spleen, kidneys, and cells therein
that are responsible for phagocytosis of bacteriophages and
other foreign particles.137 The RES may be the more prominent
mechanism for phage clearance. For example, when mice or
rats were injected with phages, neutralizing antibodies could
not be detected in the animal’s blood,137,138 suggesting that
phages were removed from the circulation before an immune
response could be initiated. To allow the virus to perform its
designated function and remain in the circulation longer,
directed evolution approaches have proven effective for
bypassing neutralizing antibodies138 as well as reducing RES
clearance.140 In these cases, the virus coat protein was either
allowed to mutate during the selection process140 or chosen
from a recombinant virus library.139 The fittest candidate after
several rounds of stringent selection had only a few amino acid
differences in its coat protein. However, it was either 96 times
more resistant to antibody opsonization139 or was able to
avoid RES entrapment 16 000 times better 24 h after
injection.140
Several recent articles have reviewed the potential of
medicinal phage therapy along with the obstacles that
still need to be overcome before widespread use.127–129,131
One challenge is the intensive regulatory approval process
that any phage-based application will have to face.131 A step
in this direction is the FDA’s recent approval of the use of
a phage mixture to combat Listeria bacterial growth on meat
and cold cuts. The first of its kind, this approval was granted
in August 2006 to Intralytix, Inc. based in Maryland141
and may pave the way for future human-based phage
applications.
2.206.5.2. Phage for Drug and Gene Delivery
In addition to therapies centered on bacterial lysis, medical
applications that harness the ability to alter phage surface
proteins through either genetic engineering or chemical conjugation are also being developed. Several groups are investigating engineered filamentous bacteriophage for in vivo screening
via phage display within organs53 or cancerous tissue,142,143 for
targeted drug delivery,138,144 or as an imaging agent.145 At this
time, behavioral and histological evaluations of animals posttherapy, as well as previous reports of humans treated for
infection with phage solutions, have not indicated the existence of any harmful side effects of phage therapy.128,138,143,145
For gene delivery applications, therapeutic genetic material
can be incorporated into phage DNA and carried into cells
following receptor uptake.146 Phage can be locally targeted to
cell receptors (i.e., via RGD or other ligands) by incorporation
of specific targeting and/or internalization peptides. To make
phage even more effective as DNA delivery vehicles, they can
be further decorated with peptides that facilitate endosomal
escape or nuclear localization motifs that target the nuclear
envelope.147 The most widely used bacteriophages for gene
108
Biologically Inspired and Biomolecular Materials and Interfaces
delivery are M13 filamentous phages148–150 and lambda
phages.151 In general, these can be engineered to incorporate
targeting ligands without undergoing significant structural
change. To enhance gene delivery efficiency, phage with multifunctional peptides can be produced using a phagemid system,
which facilitates manipulation of expressed proteins on the
viral vectors.149 Phage display technology has allowed for
identification of novel homing peptides that target unknown
cell surface proteins. The targeting peptides can be incorporated
into bacteriophage coat proteins through the genetic engineering techniques described previously.146 These include peptides
(RGD, glioma-binding peptide),149,152 HER2 receptor targeting
antibody,150 growth factors (EGF, FGF2),148,153,154 and the penton base of adenovirus.151
Eukaryotic viruses such as adeno-associated virus (AAV)
have fantastic transgene delivery capabilities, but they require
elimination of native tropism for mammalian cells. In contrast,
M13 bacteriophages have no tropism for mammalian cells;
however, their gene delivery efficiency is poor. Thus, there has
been an effort to combine the advantageous aspects of AAV and
M13 bacteriophages into a single system.146 Hajitou et al. constructed hybrid phage with two genes from phage and nucleus
integrating gene from AAV, called inverted terminal repeats.
Additionally, these phages were engineered integrin-binding
peptides on minor coat proteins. Therefore, the RGD peptideinduced internalization of the phage through integrin-mediated
endocytosis process and the inverted terminal repeats (ITR) led
to improved transgene delivery in the cytoplasm. The resulting
AAV/phage system provided superior tumor transduction over
phage alone. Nucleic acid cargo can also be incorporated into
scaffolding materials for delivery to cells. It has been shown that
DNA materials that are tethered to a matrix, rather than simply
encapsulated, are more effectively transferred to the cell.155
2.206.5.3. Phage for Tissue-Engineering Materials
The unique biochemical and structural features of genetically
engineered phage can be also used in the context of tissue
engineering in order to control cellular growth (Figure 10).
Merzlyak et al., for example, have explored the use of genetically modified M13 phage as a novel building block for tissue
engineering materials.21 This was accomplished by engineering
the phage to display specific cell signaling motifs and then
assembling the viral particles into a macroscopic scaffolding
material.21 Many peptide expression systems have previously
been demonstrated on the various capsid proteins of the phage
through creation of peptide libraries.36,44,156 However, as
biological particles for peptide display, phages possess the
inherent limitation of having to be successfully expressed and
assembled within the E. coli bacteria host, which restricts the
type and number of peptides that can be displayed.45,157–159 In
order to expand the utility of M13 for phage display, Merzlyak
et al. developed a novel cloning approach for display of an
integrin-binding RGD motif on every copy of the pVIII major
coat protein. The researchers constructed the phage using a
partial library, in which an engineered octamer insert for
pVIII included a constrained RGD group that was surrounded
by flanking degenerate residues.21 This allowed for expression
of inserts that retained the desired function of the RGD motif
and yet were biologically compatible with E. coli during
the intricate phage replication process. After construction
of engineered phage that stably displayed either RGD- or
IKVAV-peptide groups on every copy of the pVIII protein,
Merzlyak et al. constructed aligned two- and three-dimensional
scaffolding materials containing phages and tested their applicability for tissue engineering. First, the investigators verified
the biocompatibility of the materials by growing NIH-3T3
fibroblast and neural progenitor cells on phage films and in
phage containing media. Both cell types showed normal morphology and proliferation when in direct contact with phage
materials. Neural progenitor cells either retained their progenitor state or differentiated toward the neural cell phenotype
depending on media conditions. It was then demonstrated that
three-dimensional phage materials could support proliferation
and differentiation of neural progenitor cells. Both RGD- and
IKVAV-phage matrices facilitated colony formation of neural
progenitor cells, which sustained a viability of over 85% during
the 7-day observation period. In comparison to RGE and
wild-type phage controls, RGD and IKVAV phage resulted in
enhanced binding and spreading of neural progenitor cells
with high specificity. Finally, by simple extrusion or spinning
of phage solution, the researchers constructed aligned threedimensional phage fiber matrices with embedded neural progenitor cells. The resulting phage fibers encouraged neural cell
differentiation and directed cell growth parallel to the long axis
of the fibers.21 Mechanical shearing of phage solution on a
glass substrate resulted in two-dimensional directionally oriented films. These oriented films were shown to direct the
alignment and morphology of fibroblasts, osteoblasts, and
neural cells.20 The Grinstaff group has worked in panning
phage-displayed combinatorial peptide libraries against
biomaterials160 and implantable biomedical devices161,162 to
identify binding peptides usable in surface modification.
They have synthesized heterobifunctional peptides which
include a cell-adhesion promoting sequence (RGD) along
with an identified binding sequence for the target surfaces.
The interfacial biomaterial coating for polymers (polyglycolic
acid, polystyrene)160,163 and the metal surface (titanium)162
using as-prepared peptides enabled the material to guide
endothelialization on the coated surface. In the same groups,
the HKH tripeptide motif was identified as the titanium-binding
contributor using a phage display screening process.161
A synthetic peptide containing three repeats of HKH was
conjugated with PEG. The pegylated analogue of the peptide
was shown to adsorb to the titanium surface, preventing nonspecific protein adsorption and bacterial colonization which
can cause orthopedic implant failure.
2.206.6.
Summary and Future Perspectives
Viruses are unique in their intrinsic ability to self-replicate
within a cellular host and self-assemble into highly ordered
two- and three-dimensional nanostructures. By combining
these self-replicating and self-assembling functions, virusbased materials can be used to construct nanomaterials and
devices with novel structure and function. Moreover, in order
to improve the performance of the desired function, it is
possible either to evolve these materials toward a directed
endpoint or to incorporate rational design modifications
Phages as Tools for Functional Nanomaterials Development
109
Display signaling motifs
Phage engineering
Construct target libraries
AXXXIKVAVDP and
AXXXRGDXXDP
plX
AEDSIKVAVDP and
ADSGRGDTEDP
pVIII
pVIII
M13 phage
genome
plII
Amplify and
purify the
phage
Tissue engineering
Evaluate cell response
to phage materials
Proliferation
Differentiation
Form aligned
LC scaffolds
with NPCs
Apoptosis
Grow cells within phage
liquid crystal scaffolds
Proliferation
Phage
NPC
Neural cell
Differentiation
(a)
6 days
50 mm
(b)
(c)
50 mm
(d)
Figure 10 Phage-based tissue engineering materials. (a) Schematic diagram of developing M13 phage tissue engineering process to depict phage
engineering, cell response characterization, and aligned fiber matrix fabrication. Directional guidance of neural cells using the liquid crystalline phage
matrices. (b) Photograph of the phage microfiber (1 cm in length) spun in agarose, shown with a centimeter scale ruler. (c) POM image shows nematic
liquid crystalline structure of the phage microfiber, scale bar ¼ 50 mm. (d) Polarized optical microscopy images of the differentiated neural cells within
aligned RGD-phage matrices after 6 days. Reproduced from Merzlyak, A.; Indrakanti, S.; Lee, S. W. Nano Lett. 2009, 9, 846–852, with permission from
American Chemical Society. © 2009 American Chemical Society.
through genetic engineering. Over the past couple of decades,
we have witnessed the emergence of a new class of virus-based
nanomaterials and nanotechnologies. As discussed in this
chapter, viruses have been developed as templates for fabrication of exquisite nanostructures including electronic or semiconductor materials, energy storage materials, sensors, and
biomedical materials. They can be deposited as functional coatings for existing devices used for applications such as biological
and chemical sensing. The resulting novel materials and devices
benefit from the unique biological, physiochemical, and structural features of viruses such as self-replication, self-assembly,
directed-evolution, and target recognition.14,21,55,76,86
Through eons of evolution, nature has arguably mastered
the process of biosynthesis. There are several reasons for this:
First, biological systems can replicate with remarkable precision at molecular, cellular, and macroscopic scales. Second,
these systems can self-organize in a hierarchical fashion, generating structures and exhibiting behaviors that extend beyond
the capabilities of a single subunit. Third, through diversification (mutation) and selection processes, these systems progressively evolve and adapt in order to maintain fitness in the
presence of environmental pressures. This information is
recorded genetically and passed to future generations. Inspired
by these exquisite biological systems and their apparent ability
110
Biologically Inspired and Biomolecular Materials and Interfaces
to self ‘design,’ Dr. Eric Drexler proposed several characteristics
of future materials and machinery in his book Engines of
Creation in 1983.164 He coined the term ‘Nanobot (Nanoscale
Robot)’ for systems that can self-assemble, self-evolve, and selfreplicate – serving as fabricators of the next generation of
improved nanobots. To our knowledge, synthetic systems
that exhibit these behaviors do not currently exist, and it is
difficult to imagine them in the near future. Virus-based materials and devices, on the other hand, may be the best representations of the nanobot concept proposed by Dr. Drexler.
Through the application of techniques described in this chapter, along with methodologies that have not yet been developed, we might witness a real nanoscale ‘robot’ that is capable
of performing intricate programmable tasks. We have termed
this emerging science and engineering field ‘virotronics’ because
it seeks to exploit the unique properties of viruses for human
benefit.59 Virotronics represents a novel virus-based design technology that can be used to create new materials with precise
molecular-level structural and functional control. As we
described throughout this chapter, virotronics incorporates the
unique biological advantages of viruses, such as specific molecular recognition, evolution, and self-replication with engineering principles including information mining, data storage and
processing, as well as structural self-assembly, self-templating,
and organization of various materials into functional devices at
multiple length scales. In the near future, we strongly believe
that products derived from virotronics will be used regularly
for data mining, storage, and computation, for generation of
clean and green energy, for sensing of chemical toxicants and
biological pathogens, for regeneration of damaged tissues, and
for other unforeseen applications which will have an impact
on human health and quality of life.
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