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Amyloid Fibrils as Building Blocks for Natural and Artificial

Functional Materials
Tuomas P. J. Knowles* and Raffaele Mezzenga*

to address this challenge has emerged in

Proteinaceous materials based on the amyloid core structure have recently the form of fibrillar protein self-assembly.
been discovered at the origin of biological functionality in a remarkably This is a basic form of self-assembly, both
diverse set of roles, and attention is increasingly turning towards such in terms of the composition - the majority
of such structures are homomolecular in
structures as the basis of artificial self-assembling materials. These roles nature - as well as in its geometry, these
contrast markedly with the original picture of amyloid fibrils as inherently structures consisting of linear, near-one
pathological structures. Here we outline the salient features of this class of dimensional nanostructures.[4] Despite this
functional materials, both in the context of the functional roles that have been apparent simplicity, such structures possess
revealed for amyloid fibrils in nature, as well as in relation to their potential as varied roles in nature, including as catalytic
scaffolds,[57] as media for epigenetic infor-
artificial materials. We discuss how amyloid materials exemplify the
mation storage,[812] and as functional coat-
emergence of function from protein self-assembly at multiple length scales. ings.[13,14] This type of protein self-assembly
We focus on the connections between mesoscale structure and material has recently also led to the development
function, and demonstrate how the natural examples of functional of highly controllable artificial proteina-
amyloids illuminate the potential applications for future artificial protein ceous materials with a variety of functions,
based materials. ranging from slow-release depots for anti-
cancer therapeutic peptides[15] to compo-
nents in active composites,[16] to sensors[17]
and biomimetic functional materials. [1820]
1. Introduction While the medical aspects of amyloid formation are an
increasingly well-established part of modern molecular medi-
With a few exceptions, functional materials in nature are realised cine and the focus of a mature research effort, functional amy-
through proteinaceous structures.[1] Inspired by this ubiquitous loid materials represent an emerging and highly dynamic area
use of proteins in nature, a number of remarkable self-assembling of research which has brought soft matter physics and mate-
artificial material systems have also been developed.[2,3] Progress rials science into a central position in this field, and expanded
towards capitalising on the unique properties and rich chemistry in new and unexpected ways our views on the nature of pro-
of proteins in this context is, however, not straightforward and the teinaceous materials. This area encompasses natural mate-
exploitation of protein self-assembly to generate tailored materials rials which possess key catalytic roles in organisms ranging
has proven to be challenging due to the difficulty of controlling from bacteria to humans as well as artificial materials that are
protein self-assembly on different length-scales. In particular, the already in commercial use, for instance as drug delivery plat-
design rules that have emerged for protein assembly are more forms, as well as emerging applications in solar energy conver-
complex than those that have been established for the production sion, active materials and sensing. Figure 1 summarizes some
of nanostructures from nucleic acids. A particularly fruitful avenue of the emerging features and roles of functional amyloids as
natural and artificial materials.

Prof. T. P. J. Knowles
Department of Chemistry 2. Amyloid Fibrils at all Length Scales
University of Cambridge
Lensfield Road Amyloid materials are characterised by fibrillar morphology on
CB2 1EW Cambridge, United Kingdom
E-mail: tpjk2@cam.ac.uk the nanoscale and generic quaternary protein structure on the
Prof. R. Mezzenga molecular scale consisting of beta-strands aligned perpendicu-
Department of Health Sciences and Technology larly to the fibril axis and bound together in a dense hydrogen
ETH Zurich, Switzerland bonding network.[2123] The structure and molecular assembly
E-mail: raffaele.mezzenga@hest.ethz.ch of amyloid fibrils at the atomistic and molecular level have been
Prof. R. Mezzenga uncovered through a multitude of experimental and simulation-
Department of Materials Science
ETH Zurich, Switzerland
based studies. Even though challenges remain in developing
methods to solve routinely the structures of amyloid fibrils, the
DOI: 10.1002/adma.201505961 atomic and molecular level architecture of amyloid materials are

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Tuomas Knowles studied
Biology at the University
of Geneva and Physics at
ETH Zurich from where
he graduated in 2004. He
obtained a Ph.D. in Biological
Physics from the University
of Cambridge in 2008 and
joined the faculty at the
Department of Chemistry at
Cambridge in 2010 where
he is currently professor of
Physical Chemistry and Biophysics. His work is focused on
the development and application of experimental and theo-
retical methods based on the physical sciences to study
biomolecular behaviour and interactions in the context of
both biological function and malfunction.

Raffaele Mezzenga received

Figure 1. Roadmap to the diversity in functions in amyloids-based
materials.
becoming increasingly well understood and have been the focus
of a number of reviews.[4,24] By contrast, however, their mes-
oscale traits remain less well understood, in large part due to a
large number of polymorphic forms found in these systems and
which allow the basic amyloid cross-beta structure to be realised
through a multitude of possible stacking geometries combining
beta sheets in hierarchical structures.[2123] Interestingly, how-
ever, despite the remarkable variety of polymorphs observed
for amyloid fibrils, some universal features are also starting to
emerge at the mesoscopic level.[25] Figure 2 summarizes the dif-
ferent length scales involved in the structure of amyloid fibrils,
and which define many of their physical properties.
A first basic view of the physical nature of amyloid fibrils
can be approached by comparing them with other characteristic
types of one-dimensional organic macromolecular and colloidal
systems. At length scales above the well-established atomistic
fingerprint of amyloid fibrils, these aggregates exhibit meso-
scopic properties comparable to those of natural polyelectro-
lytes, yet with persistence lengths several orders of magnitude
beyond the Debye length. They thus exhibit features associated
with conventional charged polymers, with, however, a persis-
tence length remarkably independent of the environmental
conditions to which they are exposed;[26] this observation can
be rationalised in the light of the fact that the origin of the
persistence length of amyloid fibrils is primarily the hydrogen-
bonding network at their core[27] which is less affected by solu-
tion conditions than electrostatic repulsion responsible for the
persistence length of conventional charged polymers. Further-
more, in contrast to other polymeric systems, they exhibit a
combined chirality and polarity along their fibrillar main axis,
a characteristic which allows for manipulations through elec-
tric fields of very weak intensities,[28] or by adsorption at inter-
faces as symmetry breaking tool.[29,30] Thus, a distinctive feature
of amyloids at the mesoscale is their intrinsic rigidity, which
his Ph.D. from EPFL Lausanne
in 2001 in the field of Polymer
Physics, focusing on the
thermodynamics of thermoset
reactive blends. He then spent
20012002 as a postdoc-
toral scientist at University
of California, Santa Barbara.
In 2003 he joined the Nestl
Research Center, in Lausanne
as a research scientist, and was
hired in 2005 as an Associate
Professor in Physics at the University of Fribourg. Since 2009
he is Full Professor at ETH Zurich. His research focuses on
the fundamental understanding of self-assembly processes
in liquid crystalline polymers, supramolecular polymers,
lyotropic liquid crystals, food and biological colloidal systems.
together with their chiral, polar and charged nature, provides
these systems with a rich physical behavior in one, two and
three dimensions.
3. Material Properties of Amyloid Fibrils Underlie
their Diverse Roles in Nature
The dense hydrogen bonding network that is characteristic of
the core of amyloid fibrils and that leads to strong intermo-
lecular backbone-backbone interactions between polypeptide
chains within the fibrils, results in these materials pos-
sessing a very high Youngs modulus.[27,31,32] Indeed, amyloid
fibrils have elastic moduli which are amongst the highest
recorded for protein based materials and in particular can
exceed those measured for functional intracellular filaments
such as actin and tubulin, and are comparable to extracel-
lular materials such as collagen, keratin and silk. Proteina-
ceous materials in this latter class which exhibit very high
moduli are typically not required to depolymerise rapidly as

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Figure 2. The different length scales at which the fingerprint of amyloid fibrils structure emerges. Adapted with permission.[21] Copyright 2013, National
Academy of Sciences.
part of their native function[31] - unlike actin and tubulin, for
which dynamic control of polymerisation and depolymerisa-
tion on second timescales is essential for their role in cel-
lular motility. Highly reversible assembly is thus not readily
compatible with the highest elastic moduli in protein-based
materials, which can only be achieved through strong inter-
molecular interactions such as the cross-beta structure under-
lying amyloid materials. In agreement with this idea, the
natural biological roles of amyloid fibrils exploit their robust
material properties and accurate self-assembly in the form of
functional coatings and catalytic materials, but do not rely on
rapid depolymerisation. Moreover, the very high aspect ratio
that is characteristic of amyloid fibrils, with diameters on
the nanometre scale but lengths exceeding a micron, leads
to the possibility of such structures fragmenting and there-
fore proliferating through the further growth of fragments
generated by fission of a larger original structure.[11] Such
fragments can be transmitted from mother cell to daughter
cell during cell division, and thus transfer epigenetic infor-
mation independently of the conventional nucleic acid-based
templates. This type of mechanism is exploited by a range
of single cell organisms to implement epigenetic switches.
Finally, functional amyloid is also found in human tissue,
and documented roles include catalytic scaffolds and storage
of peptide hormones.
4. Natural Uses of Amyloid Materials
The amyloid state of proteins was initially discovered in the
context of disease states where it is associated with a diverse
range of protein misfolding disorders, which include the sys-
temic amyloidosis as well as a number of increasingly prevalent
neurodegenerative disorders such as Alzheimers and Parkin-
sons diseases.[4,33,34] These diseases affect different organs[35]
and are very heterogeneous on a tissue to organism level, yet
share common molecular level features through the uncon-
trolled transition of normally soluble cellular proteins into
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insoluble amyloid aggregates. As such, the amyloid state of pro-
teins was originally viewed as a hallmark of disease.[3638] Thus
the discovery that amyloid structures can also have beneficial
physiological roles and occur naturally as functional materials
was initially surprising. It is now, however, increasingly viewed
as a consequence of the fact that many proteins possess an
intrinsic propensity towards amyloid formation and molecular
evolution has harnessed this inherent feature of polypeptide
molecules to generate unconventional functional folds and
capitalise on the robust material properties that are exhibited
by amyloid fibrils.[39] These discoveries in the past 10 years of
non-conventional functional protein structures have trans-
formed the understanding of the evolutionary requirements for
functional protein materials and the design features that favor
functional protein assembly and disfavor aberrant association.
The molecular level mechanisms that differentiate toxic amy-
loid structures found in the context of disease and beneficial
functional amyloid species are not fully understood; however,
increasing evidence points towards small soluble oligomeric
species as being the primary culprits in cellular toxicity asso-
ciated with aberrant protein aggregation.[4042] It is thus likely
that in cases where nature uses the amyloid fold to generate
functional materials, mechanisms are in place to ensure that
the self-assembly reaction goes to completion and does not give
rise to the accumulation of potentially highly toxic intermedi-
ates such as those found in disease states.
4.1. Functional Amyloid as a Structural Material
The rigidity and strength of amyloid fibrils combined with
their high cohesive energy - which originally suggested that
the amyloid aggregation phenomenon could be a largely irre-
versible process - and the resulting high level of resistance
to degradation through chemical or biological processes are
favorable characteristics for structural materials, and as such
a number of organisms exploit amyloid materials in this role.
In particular the biofilms of many bacteria, including the
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Figure 3. Functional amyloid in bacterial biofilms. (A) an electron micro-
graph of an E. coli cell producing curli amyloid fibrils that aid in the for-
mation of biofilms. Credit: Matthew Chapman, Univ. of Michigan. Atomic
force microscopy height (B) and error (C) image of a strain of Pseu-
domonas overproducing fap amyloid. Reproduced with permission.[46]
Copyright 2015, The Authors.
common Escherichia coli contain as a significant component
amyloid fibrils. In the case of E. coli, the principal fibrous com-
ponent, curli, is assembled from a single protein, CsgA[13,43]
(See Figure 3); however, unlike in the case of pathological
amyloids, the assembly process is tightly controlled, and many
co-factors have been identified that control and direct this
process, including a protein CsgB which favors nucleation of
the filament assembly at the cell membrane and CsgG which
forms a membrane pore and directs transport of CsgA to the
extracellular space. These control mechanisms together sup-
press the formation of amyloid fibrils in the intracellular envi-
ronment where it could be harmful and would likely interfere
with normal cellular function, and ensure effective export of
the aggregating species to the extracellular space. Amyloid
REVIEW
fibrils are also found in the biofilms secreted by other bacteria,
including Salmonella and Bacillus subtilis.[44,45] The presence
of amyloid fibrils in biofilms is likely to promote the mechan-
ical rigidity and strength of the film. Recent measurement of
the mechanical properties of biofilms indicate in the case of
Pseudomonas species that the presence of FapC (see Table 1)
amyloid fibrils increases biofilm stiffness (see Table 2) by more
than an order of magnitude.[46] Similar observations were made
for the case of biofilms of Staphylococcus aureus, where the pres-
ence of amyloid fibrils formed from short peptides was found
to increase the stability of the resulting biofilms.[47]
A recent discovery has identified such species also in the
biofilms of Pseudomonas,[48] where the principal aggregating
component is the protein FapC; there is also evidence for the
existence of a nucleator protein, FapB, with a putative role sim-
ilar to that of CsgB in E. coli. Thus, the control of the nucleation
step of the self-assembly process through a specific protein may
be a generic mechanism for bacteria to exploit functional amy-
loid materials while avoiding the danger of uncontrolled poly-
merisation. Such structural roles of amyloid materials are not
restricted to single-cell organisms. Indeed, insects such as silk-
moths exploit the stability and mechanical properties of the amy-
loid fold in eggshells. Silkmoth chorion proteins constitute the
majority of the mass of silkmoth eggshells and have been shown
to exist largely in an amyloid form in the shell where they ensure
the rigidity and mechanical and chemical separation and protec-
tion of the egg material with respect to the environment.[49]

Table 1. Examples of functional amyloids in natural and artificial roles.

Category Protein Function/Application References

Natural Structural CsgA Curli fibrils in the biofilms of E.coli [13]
FapC Amyloid fibrils in the biofilms of [48]
Pseudomonas
Silkmoth chorion proteins Chemical and mechanical isolation [49]
and protection in eggshells
Information transfer/prion Sup35 Control of translation termination [10]
Ure2p Control of nitrogen catabolism [50]
Catalytic scaffold Pmel17 Catalysis of melanin synthesis in [7]
mammalian melanosomes
Storage Human peptide hormones Controlled release [58]
Artificial Food and/or functional -lactoglobulin Biosensors, photovoltaics, hybrids, [16,17,5966]
nanocomposites, transfection, catalysis
Lysozyme Artificial bones, hybrids, Cell scaffolds [19,6770]
Insulin Optoelectronics [7178]
Recombinant proteins Sup35 Biosensors, hybrids [7981]
with a biological role in
neuropathologies
-synuclein Hybrids, Sensors [8284]
Genetically/Chemically Mfp/C.Coli curli; Underwater Adhesives [85]
Modified
Mouse laminin- RGD; Cell Scaffolds [86]
Amyloidogenic Fragments TTR1, Cell scaffolds, [18]
A(1-42) Light harvesting, [20,87]
Cell scaffolds & cell differentiation

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Table 2. Basic concepts in soft materials science.
Youngs modulus The ratio between stress and strain in an elastic
material. Also known as elastic modulus. Has units
of pressure (Pa).
Strain Relative deformation of a material. A unit-less
quantity.
Stress Force per unit area applied to a material.
Pickering emulsion An emulsion, i.e., a macroscopic mixture between
two liquids that are immiscible on the molecular
scale, stabilised by solid particles that adsorb onto
the interface between the two immiscible phases.
Nematic order Nematics are materials where the component
molecules maintain a long-range directional order
without long range spatial order. This is a common
phase for liquid crystals.
Cohesive energy The energy required to break the bonds stabilising a
material. Also known as binding energy.
Elastomer An elastomer is a material possessing both elastic
and viscous characteristics.
4.2. Materials for Epigenetic Information Transfer
An unexpected connection between the mechanical properties of
amyloid fibrils and their ability to generate functional materials
has emerged through the discovery of the yeast prion phenom-
enon.[50,51] Prions are proteinaceous particles which self-assemble
and are infectious, i.e., are able to proliferate and be transmitted
from one organism to another.[52,53] This type of behavior has
been identified in the context of mammalian prions, which are
invariably deleterious and lead to incurable neurodegenerative
disease, but also in the context of fungi, in particular yeast, where
they can fulfil functional roles. Many if not all yeast prions consist
of amyloid fibrils and their transmission is largely mediated by
their ability to fragment and thus be transmitted to daughter cells
upon cell division.[11] The presence of amyloid aggregates leads
to a specific phenotype which is thus passed on to the progeny
resulting in hereditary but non-Mendelian epigenetic information
transfer. Examples are found in many species of yeast. The exist-
ence of such non-Mendelian information transfer was discovered
already in the 1960s[54] and linked with the amyloid phenomenon
in the 1990.[50] Commonly, proteins that act as prions in yeast are
associated with the control of protein expression, for instance as
transcription factors or repressors. In their soluble non-amyloid
form, they are available to exert specific functions to modulate
protein expression, but this function is switched off by the pres-
ence of amyloid fibrils which recruit the soluble proteins into
aggregates - thus implementing an epigenetic switch. The loss of
aggregates reverses the switch and restores the activity of the sol-
uble form. Two well-established examples in the common yeast
Saccharomyces cerevisiae are Sup35 and Ure2p. The protein
Sup35p is a translation termination factor that mediates the ces-
sation of protein synthesis when a stop codon is reached.[8,50,51,55]
In the aggregated form, termination is inactive, and translation
proceeds through stop codons, generating proteome diversity. By
contrast, Ure2p binds to transcription factors regulating nitrogen
catabolism. The formation of prion fibrils from Ure2p decreases
its availability in free form, and thus impairs its function to block
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DAL5 transcription, which in turn enables yeast to take up poor
nitrogen sources through pathways that are controlled by DAL5.
In the presence of a good nitrogen source, such as ammonia,
yeast blocks the transcription of genes needed for utilization of
poor nitrogen sources; a switch between the inactive prion state
and the active soluble state thus allows the pathways underlying
nitrogen utilisation to be modified. There is significant evidence
that the presence of proteins with the ability to convert to such
prions forms results in selective advantage to yeast, and for
example Sup35 is conserved during evolution.[56] A possible expla-
nation for the importance of such epigenetic information transfer
is the possibly faster ability to adapt to environmental conditions
through switches that do not require the formation or breakage of
covalent bonds as is the case for nucleic acids, but rather function
through non-covalent supramolecular self-assembly.
4.3. Functional Amyloid in Humans
The initial discoveries of functional amyloid in nature were in
single-cell organisms, but it is increasingly apparent that the
amyloid fold is exploited more widely in nature, including mam-
mals and indeed in humans. An example of functional amyloid
discovered within human tissue is that of the polymerisation
of the protein pmel17 associated in mammals with melanin
synthesis into fibrils that form a catalytic scaffold favoring the
polymerisation of reactive quinone precursors into melanin (see
Figure 4).[7] Moreover, the amyloid scaffold is likely to contain
toxic melanin precursors that are formed during the synthesis
and prevent their diffusion outside of melanosomes, the spe-
cialised organelles within which melanin synthesis takes place.
The key features of inertness of mature amyloid fibrils as well
as the large surface area, essential for effective catalysis, which
is achieved through the generation of protein nanostructures
with a high surface to volume ratio, are likely to underlie the
functional nature of pmel17 fibrils. Other examples have more
recently come to light where the stability of the amyloid form or
proteins and peptides allows them to act as depots for peptide
hormones and to control their slow release.[57] Indeed a number
of peptide and protein hormones in secretory granules in pitui-
tary glands of the endocrine system are stored in their amyloid
forms. Such applications exploit the dynamic nature of amyloid
formation: the aggregates are formed readily under the condi-
tions found in the secretory granules at high concentrations, but
dissolve rapidly upon dilution once the aggregates are secreted.
5. Functional Amyloid in Technological Applications
Many of the same characteristics that underpin the natural use of
amyloid materials also make them attractive as the basis of arti-
ficial functional materials. In the following section, we discuss
how protein and peptide nanofibrils are already finding applica-
tions as cell culture scaffolds and as vehicles for long lasting drug
delivery. Furthermore, emerging applications are highlighted in
the areas of solar energy conversion, photoluminescent materials,
light emitting diodes, organic quantum dots, active composite
materials, biosensors and hybrid materials capable of mimicking
biological functions or serving new environmental roles.
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Figure 4. Trafficking, amyloid formation and catalytic activity of Pmel17. Pmel17 is trafficked to melenosome organelles via the Golgi after its synthesis
in the endoplasmic reticulum (ER). Within the melanosomes, cleavage of Pmel17 releases an amyloidogenic fragment Ma, while the transmembrane
fragment that is left behind is degraded. Ma forms amyloid fibrils rapidly and these structures catalyse the bio synthesis of melanin from highly reac-
tive indolequinone precursors. Panel b shows the progressive maturation of melanosomes imaged by electron microscopy. Amyloid fibrils are formed
at stage II, and melanin synthesis begins at stage III and is deposited on the amyloid fibrils; after further melanin production (stage IV), the melanin
fills the inter-fibril space. Reproduced with permission.[5] Copyright 2007, Elsevier.

5.1. Adhesive Amyloids
Amyloid fibrils possess a unique combination of size, aspect
ratio, chemical composition, and mechanical robustness,
which open manifold possible applications in nanotechnology
and artificial functional materials. For example, by examining
the structure and properties of a natural adhesive extracted
by terrestrial algae, Jarvis and colleagues[88] could identify
the presence of amyloid fibrils as the main structural con-
stituent of the adhesive under investigation. They based their

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Figure 5. Sequential steps leading to the formation of amyloid underwater adhesives: a) an amyloidogenic sequence from E. coli curli and an unfolded
sequence from mussel feet, are combined by genetic engineering (b) into a new sequence showing amyloid fibrillization features (c), which further
self-assemble into hierarchically assembled sticky fibrils. Reproduced with permission.[85] Copyright 2014, Nature Publishing Group.

conclusions on analysis of mechanical strength, Congo red
staining as well as histological studies. By fusing mussel foot
proteins with an amyloidogenic sequence of the natural adhe-
sive curli amyloid fibrils found in E. Coli, Lu and colleagues
went further and were able to develop strong underwater
adhesives with adhesive energy of the order of 20 mJ/m2
(see Figure 5).[85]
An additional example on how engineering the amyloido-
genic sequences of CsgA-forming curli found on E. coli can
lead to biofilms with enhanced substrate adhesion is offered by
Joshi and colleagues,[89] who expressed proteins consisting of
an amyloidogenic domain and a functional peptide domain to
design biofilms where adhesion properties can be programmed
to adhere onto specific substrates.
In addition to genetically engineered amyloid fibrils demon-
strating superior adhesion purposes, adhesiveness of amyloid
fibrils tends to be a general fingerprint of these systems. It is
likely that it is primarily the unique combination of the chem-
ical nature of amyloid fibrils composed of several amino acids
with different amphiphilicity-, with their nanoscopic dimen-
sions, which provide them a very strong affinity for a variety
of heterogeneous interfaces, such as the air-water, oil-water
and lipid membranes-water interfaces[9094] -for the liquid-
liquid interfaces-, but also for liquid-solid interfaces, the latter
feature being exploited in the formation of layered compos-
ites,[16,19,59] as discussed below. Indeed, by taking into account
the commonly observed high aspect ratio of amyloid fibrils, the
association energy of amyloid fibrils to liquid interfaces was cal-
culated to be of the order of 60000 KBT,[90] leading essentially to
an irreversible adsorption and binding to the interfaces, with a
stabilization mechanism very similar to Pickering stabilization.
5.2. Amyloid Nanocomposites
Adhesion to solid interfaces of amyloid fibrils can be efficiently
exploited for the production of layered nanocomposites, in
which the amyloid fibrils play the role of connecting bridges
between 2D organic or inorganic stacks. By using this prin-
ciple, Mezzenga and colleagues designed fully organic as well
as hybrid layered materials with unprecedented properties.[16]
By combining graphene and amyloid fibrils from the milk pro-
tein -lactoglobulin, biodegradable nanocomposites could be
generated in which the amyloid fibrils provided the enzyme-
sensing substrate, while the graphene layers contributed with
electron conductivity and mechanical strength (see Figure 6).[16]
These nanocomposite materials were generated by a simple
vacuum-assisted filtration method against a porous mem-
brane. Upon exposure to a solution containing porcine gas-
tric enzyme, the amyloid fibrils were digested, leading to a
change in the resulting nanocomposite conductivity. At short
digestion times, this process was controlled to yield a new way
in which enzyme activity can be measured, and was indeed
able to discriminate correctly folded enzymes from unfolded
enzymes. At longer digestion times, this concept was used to
fully degrade the nanocomposites. Among other remarkable
properties of these materials, the nanocomposites were also
shown to possess shape memory features, which could be used

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Figure 6. Various steps in the formation of graphene-amyloid biodegradable nanocomposites.
a) Conversion of -lactoglobulin into amyloid fibrils. b) Electrostatic aggregation of amyloid
fibrils and graphene oxide. c) Reduction of graphene oxide into graphene with amyloid fibrils
adsorbed onto graphene surfaces. d) Vacuum filtration leads to the layered organization of
amyloid fibrils and graphene sheets into hybrid nanocomposites. Reproduced with permis-
sion.[16] Copyright 2012, Nature Publishing Group.
in water-sensing, for example. By replacing the graphene with
single crystal nanoplatelets, but using the same processing
route, the same group prepared hybrid gold-amyloid nanocom-
posites, this time with plasmonic properties and strong fluores-
cence features.[59] Depending on the gold content, the materials
could be changed from insulating to metal conductive, while
mimicking the physical appearance of homogeneous films of
elemental gold over a large composition window. As discussed
later in this review, a promising extension of this approach is
the use of biomimetic layered materials, opening up the pos-
sibility of constructing artificial tissues and bones.[19]
Fully organic, free-standing films of amyloid fibrils and a
plasticizer, such as PEG or glycerol, were designed by Knowles
et al.[95] The film was shown to possess nematic features, and
thus, anisotropic distribution of polar moments within the
film. This feature of the film, combined with the possibility to
functionalize the fibrils with decorating molecules was further
used to align fluorophores into anisotropic patterns, which
could provide interesting perspectives in optical applications.
Gerrard and colleagues,[96] further showed that a small frac-
tion of amyloid fibrils (0.6 wt%) is sufficient to change the
visual, structural and mechanical properties of polymeric films.
An increase in Young Modulus (see Table 2) was observed,
although this improvement was accompanied by a drop in the
glass transition temperature, possibly due to an increase in
the microscopic free volume in the polymer matrix. Yet, by an
improved control of amyloid-polymer interfaces by chemical
modification of the amyloids, it is reasonable to expect that
blending amyloid fibrils with polymeric films may lead to an
improvement of the overall thermomechanical properties of
such materials.
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A complementary approach in amyloid-
based nanocomposites is to replace the
polymeric matrix by another -sheet rich
polymer: silk. Because amyloid fibrils and
silk fibrils are proteinaceous aggregates
in which the constituent -strands are
aligned either orthogonal or parallel to the
main fibril axis, respectively, complemen-
tary physical properties may be expected,
namely a higher stiffness for the amyloids,
and a higher toughness for the silk. Ling
et al.[97] demonstrated convincingly that
a synergistic effect can be achieved by
combining silk fibroin and amyloid fibrils
within the same film, due to the orthog-
onal orientation of the -strands in the two
types of fibrils, yielding a new material in
which both the Young modulus and the
fracture toughness can be modulated by
the amyloid/silk ratio.[97] The same authors
exploited the higher enzymatic resistance
of silk fibroin to enzymatically etch the
amyloid fibrils from mature hybrid films,
resulting in nanoporous films which can
ideally suit nanofiltration purposes. The
example given by these authors illustrates
convincingly the role in intermolecular
interaction and orientation of -strands
and -sheets in establishing the final macroscopic properties
of the material.
5.3. Amyloid Scaffolds for Biomaterials Applications
and Cell Growth
Perhaps one of the most rapidly emerging applications for amy-
loid fibrils is their use as scaffolds to promote and control cell
growth, proliferation and differentiation.
Kasai and colleagues combined the amyloidogenic frag-
ment 20972108 from the 1 chain of mouse laminin with the
arginine-glycine-aspartic acid tripeptide (RGD) or with glycine-
arginine-glycine-aspartic acid-serine (GRGDS), to promote cell
adhesion on amyloid gel scaffolds.[86] Functionalization by the
RGD or GRGDS peptides was inspired by the sequences of cell-
adhesion active sites in the extracellular fibrils of fibronectin.
The authors found that the human fibroblast cells adhere and
spread on the modified amyloids via integrin mediated cell
attachment, promoted by modifying fibronectin fragments, and
inferring the possible use of these systems in tissue engineering.
Gras and colleagues[18] further expanded in this direction,
by using a fragment derived from the amyloidogenic protein
transthyretin (TTR1) to design model amyloid fibrils which
were further doped by co-fibrillization with a modified TTR1
sequence carrying the same RGD pendant tripeptide, i.e.,
TTR1-RGD. The authors confirmed that adhesion with cells
was improved over the neat TTR1 and mediated by the bioac-
tive integrin-binding RGD.
Reynolds and colleagues have used polymer plasma
deposition on amyloid mats formed from lysozyme fibrils
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patterned by nanolithography to enhance cell adhesion and
spreading.[68] This work further highlighted the importance
of the 2D amyloid fibril topology and topography on which
cells are grown. In a following study, the same authors[69] engi-
neered the 2D amyloid network by growing the amyloid fibrils
to different lateral extents following various incubation times
before depositing the fibrils on a substrate.[69] This approach
resulted in film topologies with great variety, which in turn led
to differential growth and attachment of cultured fibroblast
and epithelial cells. In particular, the high surface coverages
obtained through the use of larger amyloid fibrils, resulted in
a favorable increase in spreading and focal adhesion. The pro-
cessing of amyloid fibrils for tissue engineering purposes can
be further expanded if their surfaces are decorated by biocom-
patible temperature responsive polymers, yielding injectable
gels. For example, Li et al.[98] used PNIPAM-decorated amy-
loid fibrils as a system undergoing a solgel transition below
body temperature (32C), so that the system could flow and be
easily injected at room temperature, while turning into a gel at
body temperature.
Perhaps, however, the scope of amyloid fibrils in tissue engi-
neering and cellular scaffolds can be truly expanded only by
integrating other constitutive building blocks of living matter.
Meier & Welland[67] used wet spinning to produce macroscopic
fibers constituted of gellan gum and amyloid fibrils with very
high yield and good orientation of the amyloids along the
main axis of the fibrils. By performing a biomineralization
on these macrofibrils, the authors were able to obtain hybrid
gellan gum-amyloid-brushite nanofibrils, where the brushite
(a calcium phosphate precursor of hydroxyapatite) platelets,
were meant to provide the analogue counterpart of the mineral
phase in natural bone. These composite fibrils resulted in a
highly improved Young Modulus (9 fold).
However, to ideally serve as bone-mimetic material, an amy-
loid-based hybrid composite should consist of: i) hydroxyapa-
tite lamellae layered with biocompatible amyloid fibrils and ii)
support the cellular growth of human osteoblasts. Li et al.[19]
adopted a similar approach as the one used in graphene-amy-
loid or gold-amyloid composites, [ 16,59 ]
to produce biomimetic
nanocomposites of hydroxyapatite platelets and lysozyme
amyloid fibrils in which the amyloid fibrils alternated up to a
maximum weight fraction of 2030% with
layered hydroxyapatite lamellae (7080%),
in direct analogy with real bone, but with
the remarkable difference, however, that
on these systems the collagen was replaced
by amyloid fibrils. To demonstrate the pos-
sibility to serve in real bone-mimetic appli-
cations, the authors demonstrated that the
resulting biomimetic bone could match the
Youngs modulus and density of real cancel-
lous bone, which is a key factor in avoiding
shielding problems in implants, and more-
over to serve as support for the growth of pri-
mary trabecular bone-derived preosteoblasts
Figure 7. Rationale for bifunctional Sup35-ProteinG-MPH nanofibrils for biosensing applica-
from human bone marrow. Importantly, the
tions. Sup35 constitutes the skeleton of the fibrils; Sup35-ProteinG enables molecular recogni-
cells were shown to grow and spread via tion features, and Sup35-MPH allows, in presence of a substrate, catalytic enzymatic reactions
numerous filopodia and lamellipodia sug- quantifiable by spectrophotometry. Reproduced with permission.[79] Copyright 2009, American
gesting an integrin-mediated connection of Chemical Society.
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the cell cytoskeleton with the substrate. Thus, amyloids possess
both the mechanical and chemical features to sustain cellular
growth in a very diverse set of bio-mimetic environments.
5.4. Amyloid-Based Biosensors
There is increasing evidence that amyloid fibrils can serve in
the design of efficient biosensors.[99101] Functionalization of
amyloid fibrils with suitable biomacromolecules has been
demonstrated convincingly already more than one decade ago.
For example, Baldwin et al.[102] showed that proteins such as
cytochrome can be displayed on amyloid fibrils by a supra-
molecular self-assembly fusion process. This leads to a func-
tionality of the modified fibrils, which original fibrils do not
possess. The same principle can be pushed much forward to
generate biosensors based on amyloid fibrils suitably engi-
neered to fulfil this task.
Men et al.[79] used the yeast prion protein Sup35 to guide 1D
self-assembly of two functional proteins along the same fibril:
a protein G, which has the function of providing molecular
recognition features with the immunoglobulin G (IgG), and
an enzyme, methyl parathion hydrolase (MPH), generating a
measurable signal from catalytic reactions. The integration on
the same fibril of both protein G and MPH was achieved by
incubating seeds of preformed fibrils from Sup35 (sonicated)
with Sup35-Protein-G and Sup35-MPH conjugates. By duly
controlling the composition, authors were able to produce
bi-functional Sup35 fibrils with a large amount of enzyme deco-
rating their surfaces and a few molecules of protein G. In the
presence of the enzyme substrate, a modified ELISA could be
demonstrated using this construct, in which the sensitivity of
detection of protein G antibodies was enhanced by two orders
of magnitude compared to the control using conventional
ELISA (see Figure 7). These results illustrate the opportunities
that molecular engineering with amyloid fibrils may provide in
sensing applications.
In a later work,[80] the same authors proposed a modified
strategy to functionalize amyloid fibrils and make them active
for the same biosensing reaction discussed above. The G pro-
tein was fused in the same way in the amyloids, but the other
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building block was a biotin-acceptor peptide, linked to Sup35:
by incubating these two-components, mixed amyloid fibrils
with both G protein and a biotin acceptor were produced.
The authors then relied on biotinstreptavidin interactions to
mediate coupling between the modified amyloid structures and
the final functional unit, a streptavidin-modified horseradish
peroxidase. This approach led to Sup35 based amyloids with
enzymatic activity, capable to detect via spectrophotometry the
attachment of F1 antigen of Yersinia pestis.
Sasso and colleagues[17] also relied on biotinstreptavidin
interactions to achieve functionalization of amyloids and dem-
onstrated how this approach can serve in the context of glu-
cose sensing. The biotinylation of amyloid fibrils based on
whey protein was first performed via succinimide-based biotin
conjugates reacting with the free primary amine groups on
the surface of mature amyloid fibrils. Decoration of the modi-
fied amyloids with nanoparticles, quantum dots and enzymes
was then achieved by incubating the fibrils with streptavidin-
modified colloidal objects. By using glucose oxidase (GOx)
enzymes coupled with streptavidin, the authors could generate
GOx-coated amyloid fibrils, which were then adsorbed on gold
substrates and attached via free thiol groups available on the
fibrils surfaces. Glucose sensing was measured by cyclic voltam-
mograms monitoring the enzymatic red-ox reaction (See Figure 8).
Yang et al.[82] used -synuclein fibrils modified by
10,12-pentacosadiynoic acid (PCDA) to achieve a colorimetric
response to UV, ethanol, pH and heat. Non-colored dispersions
of -synuclein-PCDA complexes turned first blue, upon expo-
sure to UV light, then pink, after ethanol, pH and/or heat treat-
ment. This is primarily due to the unique chromic properties of
the monomeric PCDA, converted to poly PCDA after UV expo-
sure, since PCDA possesses different absorption features prior
and after polymerization. This strategy could be used to further
develop multi-responsive sensors capable of detecting stimuli
in a sequential manner.
Beside the routes briefly discussed above, expanding the
scope and possibilities of sensing with amyloids as functional
components can be achieved by combining them with other
materials. Recently, demonstrations of this general approach
have emerged with graphene being a prominent example of
the basis for such hybrid materials. Indeed, enzymatic sensing
using amyloid-graphene composites has been discussed above
(see reference[16] and Figure 6) and another promising approach
along similar lines has been proposed by Wang et al.[103] who
used a silver-binding peptide prone to fibrillization to decorate
graphene flakes. An electrochemical H2O2 sensor based on this
Figure 8. Schematic of the amyloid-based glucose biosensor. Modified
glucose oxidase is connected to amyloid fibrils based on biotinstrepta-
vidin coupling. The fibrils are then adsorbed and immobilized on gold
substrates via thiol groups and glucose sensing is achieved by voltam-
metry during the enzymatic red-ox reaction. Reproduced with permis-
sion.[17] Copyright 2014, Royal Society of Chemistry.
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hybrid was then demonstrated by immobilizing the aqueous
dispersion of the graphene-amyloid complexes onto a glassy
carbon electrode and by performing cyclovoltammetric analysis.
A specific aspect of using of amyloid fibrils in bio-sensing
applications is enabled through their capacity to bind and immo-
bilize enzymes in an efficient way. In a first study, Gerrard
and colleagues[104] showed that glucose oxidase (GOx) could be
immobilized on amyloid fibrils of bovine insulin using glutar-
aldehyde as crosslinker and that the resulting conjugates pre-
served both the activity and the antimicrobial properties of GOx.
In a later work, Gerrard and colleagues[105] demonstrated that
also organophosphate hydrolase can be immobilized covalently
on nanoscaffolds made of insulin amyloid fibrils and that the
resulting conjugates exhibited improved thermal stability. Paik
and colleagues[83] have further shown how model enzymes such
as horseradish peroxidase (HRP) can be immobilized within
hydrogels made of alpha-synuclein amyloid fibrils. Remarkably,
the activity of the HRP bound to the hydrogels was increased
four-fold compared to the free enzyme, opening new future pros-
pects for enzymatic reactions from enzyme-amyloid conjugates.
5.5. Amyloids for Transfection and Controlled Drug Release
Recent work suggests that amyloid fibrils can also serve as
unique carriers for viruses, and nanoparticles transfection, as
well as controlled drug release.
Recently Munch et al.[106] noted that several peptide frag-
ments from the HIV-1 glycoprotein gp120 increase HIV-1
infection rates in cells in a significant manner and without
causing any detectable cytotoxicity. In particular, they identified
a small amphiphilic amino acid peptide of 12 residues from
the gp120 sequence, also capable of self-assembling into fibrils,
called enhancing factor C, EF-C, with remarkable transfection
properties. The peptide was shown to associate strongly with
virions, enabling separation of the virions from the suspen-
sion by simple centrifugation. Compared to other transduction
enhancers, EF-C was found to boost infection rates in a much
more efficient way. This boosted retroviral gene transfer was
interpreted in terms of an electrostatic nanobridge between
virions and cells. These EF-C nanofibrils show promise for clin-
ical applications, due to the simplicity of preparation and sys-
temic administration. These findings are further supported by
recent work by Dai et al. in which positively charged amyloids
nanosheets were efficiently used as retrovirus transfection.[107]
Shortly after this work, another amyloid system based on
the non-toxic -lactoglobulin, was shown to enhance transport
of metal nanoparticles in living cells.[61] The authors used two
types of cells, a cell line and dendritic cells, to demonstrate
that the same gold nanoparticles penetrate with a three-fold
stronger efficiency when conjugated to amyloid fibrils com-
pared to bare nanoparticles. These findings were exploited to
design cytotoxicity a priori for silver nanoparticles: by conju-
gating silver nanoparticles to amyloid fibrils, their transfection
was increased compared to the same particles conjugated to
native proteins, thus enhancing the cytotoxic response on both
cell types. This work shows that the efficiency in transfection
mediated by non-toxic amyloid fibrils can be used to control
cellular response to exogenous components.
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A promising concept put forward by Riek and colleagues
is the use of amyloid fibrils for the release of active peptides
from their termini.[58] The authors used gonadotropin-releasing
hormone analogues forming amyloid fibrils and exploited the
supramolecular nature of the fibrils to achieve sustained release.
To show release from the termini of the assembled fibrils, the
authors followed the population of monomeric peptide under
highly dilute conditions, carried out via dialysis experiments.
The authors were also able to control the release by binding
other components, such as glycosaminoglycans, which stabi-
lize the amyloid structures. These results demonstrate that the
release rate is not only dependent on the equilibrium constant
characterising the aggregates of the peptide considered, but
can further be modulated by the presence of other components
occurring in vivo or in vitro. Further work has provided addi-
tional support for the viability of drug delivery approaches via
amyloid based systems: for example Silva and colleagues[108]
used L-diphenylalanine to produce amyloid hollow nanotubes
which can be loaded with model drugs (Rhodamine B), which
could successively be released in a sustained manner.
5.6. Amyloid-mediated Synthesis of Hybrid
Organic/Inorganic Materials
One of the first utilizations of artificial amyloid-like fibrils for
functional purposes has relied on their high aspect ratio and
functional surfaces to produce hybrid nanowires. In particular,
Reches and Gazit[109] used the diphenylalanine structural motif
to generate hollow tubes of few hundreds of nm (<300 nm)
in diameter. The tubes were hollow, and once silver salts
were dissolved in the dispersion solution, following reduction
by sodium citrate, the tubes became filled by metallic silver,
resulting in core-shell silver-peptide nanowires. Silver nano-
wires were then recovered by degrading enzymatically the pep-
tide shell via proteinase K digestion. The resulting nanowires
had a diameter of the order of tens of nm. Since this seminal
work of Reches and Gazit, many contributions followed in
which amyloid fibrils were used templates to reduce metal salts
into metal nanowires, nanoparticles and nanocrystals. Malisau-
skas et al.[110] followed the same strategy proposed by Reches
and Gazit, but in their work, diphenylalanine was replaced
by hen egg lysozyme. By doing so, the template used was the
natural cavity of the amyloid fibrils in between -sheets, which
resulted in ultrathin silver nanowires of a diameter of 1 nm.
Beside the ultrasmall dimensions of the nanowires produced
by this method, the work of Malisauskas et al. also offered an
experimental demonstration of the hollow structure within
lysozyme amyloid fibrils, otherwise postu-
lated only indirectly.[111] A similar concept
was exploited also by Dzwolak,[112] who suc-
ceeded in coordinating iodine within the cav-
ities of insulin amyloid fibrils. The insulin-I2
complexes resulted in intense violet color,
and the iodine could be released from the
insulin amyloid fibrils by perturbing their
structure via exposure to dimethyl sulfoxide.
Figure 9. Amyloid and gold-amyloid hybrid aerogels from -lactoglobulin amyloid fibrils pro-
Bolisetty et al.[62] have shown that duced by supercritical CO . AC) Photographs of an amyloid aerogel (A), a gold nanoparticle
-lactoglobulin amyloid fibrils can be used to amyloid aerogel (B) and a gold crystal amyloid aerogel (C). Reproduced with permission.[113]
reduce gold salt into gold single crystalline Copyright 2016, Wiley-VCH.
6556 wileyonlinelibrary.com 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.MaterialsViews.com
nanoplates with remarkable properties. The amyloids are likely
to assist the orientation of the nanoflakes in suspension due
to competing excluded volumes of the two anisotropic objects.
Furthermore, the gold nanoplates were shown to possess plas-
monic and fluorescence properties which could be used for
efficient sensing. Indeed, Li et al.[59] later employed the same
-lactoglobulin-gold nanoflakes hybrids for humidity and pH
sensing by measuring conductivity and plasmonic features
on the solid-state composites made thereof. Very recently,
Li et al.[64] also showed that by tuning the relative composi-
tion of amyloid -lactoglobulin/gold salt precursors, could
promote growth rates over nucleation rates, leading to single
gold crystal microplates of an unprecedented lateral area.
i.e., of the order of 104 m2. This concept was used to produce
elastomeric polymer-gold microplate composites in which the
conductivity of the material could be translated into pressure
sensors of very high sensitivity, e.g., capable of sensing fingers
contacts or air blow.
Alternatively, Bolisetty et al.[66] have shown again that
-lactoglobulin and metal nanoparticles can be combined to
produce hybrid membranes capable to operate as active layers
for continuous flow catalysis of aqueous solutions passing
through the membranes and allowing performing a complete
catalytic conversion of a molecular precursor solute into its
product within a single passage of the feeding solution through
the membrane.
A particularly promising synergy between -lactoglobulin
amyloid fibrils and gold salt precursors is the one proposed by
Nystrm et al. for the production of gold aerogels with unprec-
edented properties.[113] Nystrm et al. started from gels of
-lactoglobulin, and grew in situ either gold nanoparticles or
gold nano/microcrystals, leading to a perfectly homogeneous
distribution of the inorganic colloids within the gels. They
then exchanged the water phase for ethanol, to produce alco-
gels first and then removed all the liquid phase by using super-
critical CO2 drying. The resulting materials exhibited unprec-
edented physical, structural and visual properties. For instance,
by changing the type of gold (nanoparticles vs crystals), the
color and the visual appearance of the aerogel could be finely
tuned (see Figure 9). Remarkably, using final hydrogels with
98% porosity, and 75% by weight of gold crystals mass (i.e.,
25% by weight of amyloid fibrils), the authors were able to
produce gold of a purity larger or equal to 18 carats, yet, more
than a thousand times lighter than any equivalent gold alloy.
The same authors demonstrated convincingly that the resulting
aerogels exhibit a series of other very unconventional physical
properties, such as catalytic features, plasmonic properties and
2
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pressure-sensing features. This example highlights very well
how the simultaneous structural properties of amyloid fibrils,
combined with their unique surface chemistry features, enable
the design of new hybrid materials with unprecedented phys-
ical and chemical properties.
It should be noted that the possibility of producing gold
single crystals is not a unique property of -lactoglobulin: Lara
et al.[70] showed that the same gold crystals can be produced by
lysozyme amyloid fibrils and amyloid nanotubes.
Poviloniene.,[84] further showed that -synuclein amyloid
fibrils can be used to template the formation of hybrid nano-
structures formed by amyloid fibrils bridged by gold nano-
particles. To this end, the authors modified the -synuclein
building blocks by including thiol groups, which then provided
anchoring points for the gold nanoparticles bridges, finally
resulting in original hybrid nano-ladder structures.
Bolisetty et al.[63] reduced iron salts onto -lactoglobulin amy-
loid fibrils leading to magnetically responsive fibrillar systems,
which could be oriented with weak magnetic fields. This is
an interesting alternative way to control alignment of amyloid
fibrils in suspensions at concentrations well below the onset of
the nematic phase.
A final example on how amyloids can serve inorganic mate-
rial templating is offered by Larsen and colleagues.[114] In this
work, silicon wires were produced using peptide nanotubes
based on diphenylalanine. The nanotubes were then used as an
etching mask to fabricate silicon wires in a rapid and low-cost
manner in combination with plasma treatment. These findings
show that amyloids can also serve as sacrificial templates for
inorganic nanostructures.
5.7. Amyloid-templated Optoelectronic Materials
The use of amyloid fibrils in optoelectronic applications has
been motivated by their high aspect ratio and the possibility
to modify their surfaces with functional polymers, metals
and oxides. Inganas and colleagues have been among the
pioneers of fully organic optoelectronic materials based on
amyloid templates. As they have demonstrated in a series of
recent publications, the most beneficial effect in using amy-
loids in this context for templating optoelectronic materials
is the possibility to use them to orient conjugated polymer
chains.
For example, Herland et al. have shown that electroactive-
polythiophene based polyelectrolytes could be associated to
bovine insulin amyloid fibrils to generate fluorescent green
bundles of amyloids whose fluorescence could be electro-
chemically quenched.[75] Later, the same authors[74] showed
that the positively charged bovine insulin amyloid fibrils can
be complexed electrostatically in water with the negatively
charged poly(thiophene acetic acid) (PTAA) into molecular
nanowires. The authors then used molecular combing to
transfer and align the amyloid-PTAA complexes on patterned
PDMS surfaces. Then authors studied the anisotropy of fluo-
rescence emission of aligned amyloid-PTAA complexes, by
using circular polarized light to excite the conjugated polymer
and to collect the emitted light through a rotating polarizer.
The results of this work demonstrated convincingly that the
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REVIEW
PTAA chains were highly aligned, thus following the direc-
tion imparted by the more rigid amyloid fibrils. The work was
successfully extended to demonstrate the alignment of other
types of non-charged polythiophenes, using the same amyloid
fibril scaffolds, and incubating the polymer and the amyloid
in a water-tetrahydrofuran mixture instead than pure water.[76]
These results demonstrated that hydrophobic and polar inter-
actions can be sufficient to produce the association of amyloid
fibrils and conjugated polymers, and to organize the latter into
one-dimensional nanowires. Alternatively, the same amyloid
fibrils can be used as dispersing agents for polythiophenes
which are otherwise insoluble in water.[71] Irrespective of how
the amyloid-polymer complexes are prepared, the most spec-
tacular effect of this alignment lies perhaps in the resulting
optoelectronic properties. For example, again the group of
Inganas showed that when producing an organic light emit-
ting diode (OLED) by using a polyfluorene alone in the active
layer, or the same polyfluorene attached to the insulin amyloid
fibrils, the performance of the OLED was found to be remark-
ably different:[78] when the amyloid fibrils were present, the
quantum efficiency was increased by one order of magnitude,
an effect originating from the improved electron injection and
charge-carrier mobility in the device as a result of the align-
ment of the polyfluorene chains. Perhaps the most impressive
use made of amyloids in light-emitting devices is the possi-
bility to bring together red, blue and yellow emitting dyes on
the same and single device, thus producing white-light emit-
ting diodes (Figure 10).[77] Inganas and co-workers[77] used
an ingenious approach to achieve this objective: they mixed
insulin fibrils which were modified with Ir-based fluorophores
emitting in the red and yellow wavelengths with polyfluorenes
emitting in the blue wavelength. The yellow emitting fluoro-
phore was (bis(2-(9,9-dihexylfluorenyl)-1-pyridine)(acetylaceto-
nate)iridium(III), (Ir(dhfpy)2(acac)), and the red emitting one
was (tris(1-phenylisoquinoline) iridium(III), Ir(piq)3). When
mixed together in the same active layer of a polymeric emit-
ting device, the color of the resulting light depends on the
combination of the three light-active species, and bicolor and
white light can be readily generated. In comparison to devices
made only by the bare fluorophores and polyfluorene, in
absence of fibrils, both the efficiency and the luminance were
significantly improved by the presence of the insulin amyloids,
once again confirming the key role that these fibrils can play
in organizing charge transport on the nanoscale in optoelec-
tronic active layers.
The same principles can be used to generate different types
of fully organic optoelectronic devices simply by changing
the type of conjugated polymer and the design of the device:
for example, again Inganas and co-workers have shown that
by combining bovine insulin amyloid fibrils and poly(3,4-
ethylenedioxythiophene) (PEDOT), it is possible to produce
functional electrolyte-gated transistors operating at low volt-
ages comprised between 0 and 0.5 V.[73] Finally, Barrau et al.[72]
showed that the same amyloid fibrils can be used as template
for both electron donor (polythiophenes) and electron acceptor
(fullerenes) species, thus allowing intimate mixing within the
same active layer of fully organic photovoltaic devices. The
resulting quantum efficiency, transport and fill factors were
all enhanced by the presence of amyloid fibrils, which were
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Figure 10. Combination of amyloid fibrils and three fluorophores leads to white light organic emitting diodes. Insulin amyloid fibrils are first deco-
rated by Ir(dhfpy)2(acac) and Ir(piq)3, leading to yellow-green and red emission, as shown in the fluorescence microscopy images in (A) and (B),
respectively (scale bars - 20 m). Adding a blue emitting polyfluorene (C) leads to possible chromatic emissions, including white light, as shown by
the corresponding chromaticity diagram (D). In panel (D), X and Y are the CIE spectral coordinate weights. Adapted with permission.[77] Copyright
2010, American Chemical Society.

therefore shown to significantly affect also the exciton transport
needed in donoracceptor blends.
In all of the applications discussed above, amyloid fibrils are
used as hosts for conjugated polymers that are already polymer-
ized. Meier et al.[115] have however recently demonstrated that
polymerization of aniline monomers can be carried out directly
after adsorption onto lysozyme amyloid fibrils, yielding highly
conductive lysozyme-polyaniline core-shell nanowires.
The templating process of conductive nanowires does not
necessarily need to follow a wet-chemistry approach. Andersen
et al.[116] used a ion etching procedure using amyloid fibrils as
dry ion etching masks to produce semiconductive nanowires of
PEDOT doped by p-toluenesulfonate with excellent electronic
properties. By using this route, the solvent based chemistry
associated with conventional lithography could be avoided.
Other original optoelectronic applications demonstrated
using fully organic amyloid-templated systems are light har-
vesting antennae and infrared linear dichroism. In the first
case, assembly of two chromophores on the same fibril was
achieved by functionalizing the internal and external walls of
a protein nanotube based on fragments of the A(1-42) pep-
tide with two chromophores: Rhodamine 110 and Alexa 555.[87]
Light harvesting features for the resulting amyloids were
probed by demonstrating Frster energy transfer from Rhoda-
mine 110 to the adjacent Alexa 555. In the second case, Squires
and colleagues utilized molecular combing to transfer and
orient lysozyme amyloid fibrils into solid substrates and to gen-
erate highly anisotropic infrared linear dichroism signals from
the resulting systems.[117] An alternative interesting application
of complex optical signal generated from dye-labelled amyloid
fibrils, is offered by[118] Dzwolak et al. who used insulin amyloid
fibrils bound to thioflavin T to yield mature fibrils with opposite
chiral signals as detected by circular dichroism.
The scope of amyloids in optoelectronic applications can be
further widened by expanding to organic-inorganic hybrids.
Scheibel et al.[81] demonstrated that gold nanoparticles of
1.5nm could be covalently attached at the surface of sup35p
amyloid fibrils, genetically engineered to maintain cysteine
groups available on their surface. The covalent binding of
colloidal gold generated hybrids core-shell nanowires without
any significant impact on the macroscopic shape of the fibrils,
which preserved their original length. In a final step the
hybrids were modified by depositing on top of them either
silver or gold through a salt reduction process. The resulting
nanowires, 100 nm in diameter, were shown to possess very
low Ohmic resistance. Bolisetty et al.[60] used -lactoglobulin
amyloid fibrils as templates to synthesize core-shell nanowires
of Titanium Dioxide (TiO2). The authors used a TiBALDH pre-
cursor in solution with the -lactoglobulin amyloid fibrils to
produce electron acceptor TiO2 nanowires. Because the same
fibrils can be used as dispersants for electron donor polythio-
phenes, a water-processed active layer of -lactoglobulin-TiO2
nanowires and -lactoglobulin-polythiophenes was produced
and sandwiched between Aluminum and Indium-tin oxide
(ITO) electrodes to generate a photovoltaic cell with a power
conversion efficiency approaching 1%, thus demonstrating
the possibility of using amyloids also in hybrid optoelec-
tronics devices.
5.8. Emerging Applications of Amyloids in Artificial Materials
The nature of amyloid fibrils as supramolecular aggregates
leads to a large number of degrees of freedom available in
the molecular engineering of these systems. MacPhee and
Dobson[119] have demonstrated already fifteen years ago that
different proteins can be mixed to generate hybrid fibrils
with properties that none of the constituent peptides pos-
sess. Barone and colleagues have shown that fine-tuning the
composition of amyloid-forming peptides with different con-
tent of glutamine or lysine can greatly affect the final mor-
phology of the resulting fibrils, inducing changes from flat
to cylindrical cross-sections.[120] Paik and colleagues[121] have
used 2-microglobulin to produce robust films of amyloid

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fibrils which were then degraded on-demand by exposure of
dithiothreitol to produce nanoporous materials, similar to
the enzymatic route followed by Ling et al.[97] Udomprasert
et al.[122] have combined the 1D self-assembly features of amy-
loids with the complex architectures that DNA origami can
generate, to nucleate amyloid-oligonucleotides into structures
not accessible to amyloids alone. By inducing formation of
non-toxic amyloids within single droplets in a microfluidic
setup, Shimanovich et al.[123] have also demonstrated that
amyloid hydrogels can be engineered in content, shape and
concentration, which could serve drug delivery applications
in near future. Finally, in sharp contrast with the unpopular
image which amyloid fibrils once had in the context of neuro-
degenerative diseases, two new applications have recently been
proposed for amyloid fibrils, which open new perspectives in
the struggle to environment protection and preservation. The
application proposed recently by Eisenberg et al.[124,125] for
example, on the capture of CO2 by amyloid fibrils seems par-
ticularly relevant in view of limiting CO2 release in the envi-
ronment and the associated climate changes. Bolisetty and
Mezzenga,[65] on the other hand, have recently demonstrated
how amyloid fibrils may be used to ideally remove radioactive
waste and heavy metal ions from environmentally polluted
waters and industrial wastewater streams, with the additional
appealing possibility of recycling toxic, but expensive heavy
metal ion pollutants into valuable metal particles and films,
turning a urgent global environmental problem into a unique
opportunity for science and technology.
6. Conclusions
In this paper, we have focused on the functional roles that
amyloid fibrils can play, either in nature as cellular and extra-
cellular materials or in an artificial context where they enable
new types of nanobiomaterials to be synthesised from natural
building blocks. These varied roles are underpinned by the
key physical features of the component amyloid fibrils. In
particular, these materials have emerged as being amongst
the most rigid proteinaceous structures, a characteristic that
makes them particularly attractive for structural applications.
Moreover, the assembly of amyloid fibrils takes place under
mild conditions in aqueous solution, in a process which yields
a very highly ordered nanostructure, with individual compo-
nent polypeptide molecules positioned with nanometre accu-
racy. Moreover, a wide variety of very different peptides and
proteins have been observed to be able to undergo assembly
into amyloid fibrils, either as part of their natural function
or under laboratory conditions. Thus, amyloid assembly rep-
resents a general form of nanoscale ordering of polypeptide
molecules that has significant potential as a framework for the
creation of future functional materials. Indeed, many prom-
ising demonstrations of functional materials have already
emerged, with applications ranging from cell culture mate-
rials to sensors and storage structures. These recent devel-
opments show that amyloid materials possess roles that are
much more diverse and polyvalent than originally thought
and that far from being a material inherently associated
with pathology, possess many beneficial features which are
Adv. Mater. 2016, 28, 65466561 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim
www.advmat.de
REVIEW
exploited by nature and are finding artificial applications.
Thus, amyloids, strong in this new role, can promote progress
and development in materials science, nanotechnology and
environmental sciences.
Received: December 1, 2015
Revised: March 15, 2016
Published online: May 11, 2016
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