Charge transport and intrinsic fluorescence in amyloid-like fibrils

Abstract

The self-assembly of polypeptides into stable, conductive, and intrinsically fluorescent biomolecular nanowires is reported. We have studied the morphology and electrical conduction of fibrils made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly). These amyloid-like nanofibrils, with a diameter ranging from 20 to 250 nm, result from self-assembly in aqueous solution at neutral pH. Their morphological properties and conductivity have been investigated by atomic force microscopy, scanning tunneling microscopy, and two-terminal transport experiments at the micro- and nanoscales. We demonstrate that the nanofibrils can sustain significant electrical conduction in the solid state at ambient conditions and have remarkable stability. We also show intrinsic blue-green fluorescence of the nanofibrils by confocal microscopy analyses. These results indicate that direct (label-free) excitation can be used to investigate the aggregation state or the polymorphism of amyloid-like fibrils (and possibly of other proteinaceous material) and open up interesting perspectives for the use of peptide-based nanowire structures, with tunable physical and chemical properties, for a wide range of nanobiotechnological and bioelectronic applications.

• peptide nanostructures
• self-assembling
• atomic force microscopy
• scanning tunneling microscopy
• confocal microscopy

Full text

Charge transport and intrinsic fluorescence
in amyloid-like fibrils
Loretta Laureana del Mercato*
†
, Pier Paolo Pompa*
†
, Giuseppe Maruccio*, Antonio Della Torre*, Stefania Sabella*,
Antonio Mario Tamburro
‡
, Roberto Cingolani*, and Ross Rinaldi*
*National Nanotechnology Laboratory, Istituto Nazionale per la Fisica della Materia-Consiglio Nazionale della Ricerche, and Italian Institute of Technology
(IIT) Research Unit, Scuola Superiore ISUFI, University of Salento, Via per Arnesano, 73100 Lecce, Italy; and
‡
Department of Chemistry, University of
Basilicata, Via N. Sauro, 85100 Potenza, Italy
Edited by David Baker, University of Washington, Seattle, WA, and approved September 20, 2007 (received for review March 27, 2007)
The self-assembly of polypeptides into stable, conductive, and
intrinsically fluorescent biomolecular nanowires is reported. We
have studied the morphology and electrical conduction of fibrils
made of an elastin-related polypeptide, poly(ValGlyGlyLeuGly).
These amyloid-like nanofibrils, with a diameter ranging from 20 to
250 nm, result from self-assembly in aqueous solution at neutral
pH. Their morphological properties and conductivity have been
investigated by atomic force microscopy, scanning tunneling mi-
croscopy, and two-terminal transport experiments at the micro-
and nanoscales. We demonstrate that the nanofibrils can sustain
significant electrical conduction in the solid state at ambient
conditions and have remarkable stability. We also show intrinsic
blue-green fluorescence of the nanofibrils by confocal microscopy
analyses. These results indicate that direct (label-free) excitation
can be used to investigate the aggregation state or the polymor-
phism of amyloid-like fibrils (and possibly of other proteinaceous
material) and open up interesting perspectives for the use of
peptide-based nanowire structures, with tunable physical and
chemical properties, for a wide range of nanobiotechnological and
bioelectronic applications.
peptide nanostructures 兩 self-assembling 兩 atomic force microscopy 兩
scanning tunneling microscopy 兩 confocal microscopy
M
olecular self-assembly is ubiquitous in biological systems
and underlies the formation of a w ide variety of complex
biological str uctures (1). One import ant example of self-
assembly is the amyloid fibril (2, 3). In vivo, amyloids are of ten
associated with disease (4, 5). Amyloid fibrils can be formed by
both nor mal and variant proteins of different origins and with no
primary sequence homolog y (6, 7). They are highly ordered
molecular assemblies with similar biophysical and ultrastructural
characteristics, including a typical x-ray diff raction pattern and
a predominant
␤
-sheet c onformation (5, 8–13). The similarit y
among the different amyloid deposits and their ubiquity suggests
that such str uctures might represent a generic for m of the
nonc ovalent packing of polypeptide chains (6, 7, 14). It may be
possible that the aggregation into such well defined, nano-
ordered assemblies represents a st ate of an efficient min imal
energy arrangement of polypeptide chains (15–17). Recent
results have shown that fibrillar str uctures similar to amyloid
fibrils are for med by sequences like poly(XGlyGlyYGly) (X, Y ⫽
Val, Leu, or Ala), which are highly repeated in the hydrophobic
domains of elastin (18, 19).
Apart from their pathological relevance, amyloid fibrils are
one of several self-assembling peptide systems that are attracting
increasing interest for applications ranging from molecular
electron ics (20, 21) to tissue engineering and material science
(22–27). Met allic nanowires have been produced by different
strategies on templates made from protein nanotubes and nano-
fibrils (20, 21), and the possibility to control both the vertical and
horizont al arrangements of the peptide nanotubes has been
demonstrated recently (28). However, electrical conduction in
unmet allized fibrils has not been reported. In this paper, we
investigated charge transport and intrinsic fluorescence of amy-
loid-like fibrils made from the synthetic polypentapeptide, poly-
(ValGlyGlyLeuGly).
Results and Discussion
Poly(ValGlyGlyLeuGly) amyloid-like fibrils [see supporting in-
for mation (SI) Materials and Methods and SI Fig. 8] were
prepared by resuspension in 0.1 mg/ml ultrapure water and
examined by atomic force microsc opy (AFM) at ambient con-
ditions after deposition onto a SiO
2
substrate and solvent
evaporation. We observed protein structures with different sizes,
revealing a high degree of conformational heterogeneity of the
fibrils upon self-assembling in the water medium (Fig. 1a). The
diameter of the fibrils was found to be in the 20- to 250-nm range.
The presence of some monomers or small aggregates also is
visible in the background of the image (Fig. 1a). These protein
aggregates are thought to fuse into fibrillar structures, although
the detailed mechanism underlying the fibrillogenesis process is
not yet completely understood (3). AFM measurements showed
that poly(ValGlyGlyLeuGly) self-assembles to give fibrils with a
characteristic domain texture. Each fibril consists of several
protofilaments arranged in a roughly t wisted pattern, indicating
that they were formed through the lateral alignment of many
polypeptide molecules (Fig. 1b). A high-resolution AFM image
of a single fibril also is reported in Fig. 1 c. In this case, the
biomolecular nanowire is 120 nm in diameter and ⬇22 nm in
height (see the line profile in Fig. 1d). The length of the fibrils
varied from hundreds of nanometers to several microns. Tam-
burro and colleagues (29) recently reported that the filament
length of the analogue poly(ValGlyGlyValGly) peptide can
reach 70
␮
m, suggesting a possible mechan ism of longitudinal
alignment of the peptides besides a lateral one. Repeated AFM
imaging per formed over the same sample at dif ferent times (up
to several months after sample preparation) revealed no sign if-
icant morphological changes, indicating a remarkable st ability of
the fibrils at ambient conditions, which is in qualitative agree-
ment with our previous data on solid-state protein films (30, 31).
AFM analyses also showed that the poly(ValGlyGlyLeuGly)
fibrils are st able to heat treatment (80°C with atmospheric
pressure for 1 h; 121°C with 1.2 atmospheric pressure for 50 min),
Author contributions: L.L.d.M. and P.P.P. contributed equally to this work; L.L.d.M., P.P.P.,
G.M., and R.R. designed research; L.L.d.M., P.P.P., G.M., and A.D.T. performed research;
A.M.T. contributed new reagents/analytic tools; L.L.d.M., P.P.P., G.M., A.M.T., S.S., R.C., and
R.R. analyzed data; and L.L.d.M. and P.P.P. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Abbreviations: AFM, atomic force microscopy; RH, relative humidity; STM, scanning tun-
neling microscopy.
†
To whom correspondence may be addressed. E-mail: loretta.delmercato@unile.it or
piero.pompa@unile.it.
This article contains supporting information online at www.pnas.org/cgi/content/full/
0702843104/DC1.
© 2007 by The National Academy of Sciences of the USA
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as well as to the organic solvents typically used in lithog raphy
processes, such as acetone and isopropanol (data not shown).
Protein fibrils also were investigated by scann ing tunneling
microsc opy (STM) experiments. Unmodified fibrils were de-
posited onto gold substrate to explore their conductive prop-
erties and to gain deeper insight into their str ucture (thanks to
the higher imaging resolution of the STM). For these ex per-
iments, the 0.1 mg/ml poly(ValGlyGlyLeuGly) fibril suspen-
sions in ultrapure water were deposited onto gold substrates by
the same procedure used with silicon dioxide samples. Mature
fibrils c onsisting of several filaments laterally aligned, which
interact side by side, were detected (Fig. 2a). Each fibril was
found to be c omposed of many polypeptide molecules (Fig.
2b), which is in line with previous reports suggesting that
amyloid-like fibrils are likely to originate f rom extensive
self-interactions of elemental cross
␤
-str uctures (19). We also
were able to observe fine structural features of the fibrils, such
as the characteristic helical t wist. Both right-handed (Fig. 2b)
and lef t-handed helical orient ations were found, which is
c onsistent with a recent study (32).
It is worth noting that the above STM imaging experiments
were performed without any metal coating of the proteins,
indicating the ability of unmodified fibrils to sustain electrical
c onduction. This ex perimental ev idence implies the concept of
charge transport processes in these polypeptides molecules.
Actually, we recently observed similar findings in disorder
multilayers of nonredox proteins (31). Because this class of
biomolecular nanowires also was found to be amenable to direct
STM imaging, protein c onductivity may be considered, in prin-
ciple, as a rather general phenomenon regardless of the redox
functionalit y of the biomolecule. Remarkably, Wang et al. (33)
also were able to perform STM experiments on A
␤
42 amyloid
fibrils w ithout the need for metal coating. Nevertheless, the
authors focused their attention on the aggregation processes of
the protein structure and did not discuss the physical mecha-
n isms responsible for the fibril conductivity.
In line with our AFM data, STM images were essentially
unchanged after several months at ambient conditions, indicat-
ing that both the structure and conductivity of the fibrils are
marginally affected by aging.
The conductive properties of the fibrils were further investi-
gated by t wo-terminal transport experiments at ambient c ondi-
tions. The devices for these measurements consisted of inter-
digit ated electrodes with gaps ranging from 2 to 10
␮
m(a
representative SEM image of a device with a 2-
␮
m interelec-
trode separation is reported in Fig. 3a). The fibrils were depos-
ited onto the electrodes by cast deposition of a 10-
␮
l drop of the
0.1 mg/ml fibrils suspension in ultrapure water. After solvent
evaporation, numerous fibrils were seen to span the interelec-
trode gaps as probed by SEM (Fig. 3b) and AFM (Fig. 3 c and
d) analyses. Electrical conductivity was readily detected in these
samples. High-current values were recorded, typically in the
range of several nA (Fig. 4) depending on the number of fibrils
bridging the electrodes and the environmental c onditions (the
qualit y of the protein–electrode contacts also plays an important
role). Control experiments carried out on empty devices (i.e.,
without proteins) revealed low-current signals (always ⬍1 pA)
(Fig. 4 Inset).
A possible qualitative model for the transport mechanism
suggests that charges can travel through the self-assembled
polypeptides because of the presence of efficient charge-transfer
pathways in the fibrillar structures. In particular, a strong role of
water molecules in the transport mechanism may be envisaged.
It is likely that protein hydration shells are largely retained in the
Fig. 1. AFM characterization of poly(ValGlyGlyLeuGly) amyloid-like fibrils. (a
and b) Two representative AFM images of the fibrillar structures onto SiO
2
substrates. (c) High-resolution 3D image of a single-peptide nanowire. (d) Line
profile of the fibril reported in c. All AFM experiments were performed at
ambient conditions in tapping mode.
Fig. 2. Two typical high-resolution STM images of poly(ValGlyGlyLeuGly)
fibrils deposited onto gold substrates. Experiments were performed in air at
room temperature in constant current mode (50 –150 pA tunneling current,
0.1 V bias voltage, 1 Hz scan rate).
Fig. 3. Charge transport experiments on the nanofibrils. (a) SEM image of
the interdigitated electrodes used for transport experiments. (b) Representa-
tive SEM micrograph of the electrodes after fibrils deposition. (c and d)
Two-dimensional (c) and 3D (d) AFM images of the poly(ValGlyGlyLeuGly)
fibrils across the gold electrodes.
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solid state and significantly support the intra- and intermolecular
charge-transport pathways along the peptides nanowires. There-
fore, the formation of continuous molecular chains in the
solid-st ate fibrils may allow for long-range charge transfer
bet ween electrodes.
Fibrils samples were characterized in different conditions of
humidit y [in the 30–70% relative humidity (RH) range]. A
strong dependence of the charge-transport behavior on this
parameter was observed, with high RH values favoring protein
c onductivity (the current intensities recorded at ⬇70% RH were
t ypically one to two orders of magnitude higher than those
detected at ⬇30%). Moreover, essentially no conductivity was
observed in a vacuum, whereas conductivity was promptly
rec overed when the samples were brought back to ambient
c onditions. These results are in qualitative agreement with
previous studies on the charge transport in proteins multilayer,
where a significant influence of the humidity on the conduct ance
was observed (31). Interestingly, preliminary AFM measure-
ments carried out under vacuum conditions (data not shown)
revealed a remarkable shrink age of the fibrils, which was likely
because of the loss of water molecules f rom the fibrillar struc-
tures upon extensive drying. This evidence is consistent with the
observed dependence of the protein conductivity on the relative
humidit y, confirming the primary role of water molecules (e.g.,
hydration shells) in the charge-transport mechanism.
In addition to electrical c onductivity, we disc overed that
poly(ValGlyGlyLeuGly) nanofibrils display an intrinsic blue-
green fluorescence upon near-UV/blue excitation. Fig. 5 shows
t wo representative fluorescence images of the fibrillar nano-
str uctures obt ained by confocal microscopy (excitation wave-
length, 405 nm). These samples were prepared by means of the
same cast-deposition procedure used for scanning probe mea-
surements by using two different concentrations of the polypen-
t apeptide (Fig. 5a, 1 mg/ml; Fig. 5b, 0.1 mg/ml). Spatially
resolved fluorescence experiments revealed the t ypical struc-
tural features of the fibrils, which are similar to those observed
by AFM. Fig. 5a shows a region of high-fibril density (near the
edge of the deposited drop), in which the presence of large
amorphous aggregates is clearly visible, along with a dense
carpet (layer) of close fibrillar structures. Not ably, in these
regions, the polypeptide aggregates seem to act as fibrillogenesis
nuclei, from which most of the fibrillar structures radiate. This
finding was observed in different regions of the same sample, as
well as in other solid-state samples realized with the same fibril
c oncentration by both confocal and AFM measurements. How-
ever, such peculiar features were not found in less concentrated
samples (see Fig. 5b ), suggesting that such large structures arise
f rom extensive (random) aggregation of many fibril molecules
oc curring at high-protein c oncentrations. At any rate, more
det ailed and specific investigations might further clarif y the
possible role of peptide aggregates in the fibrillogenesis mech-
an ism and/or in the fibrils self-assembling in the solid state.
Single fibrils randomly distributed onto the silicon substrate
and characterized by a remarkable degree of morphological
heterogeneit y are clearly visible in samples prepared from more
diluted suspensions (Fig. 5b). In line with AFM experiments (see
Fig. 1a), the presence of some small aggregates also is detectable
(Fig. 5b). The possibility of imaging such small protein str uctures
reveals the g reat potential of the direct (label-free) excitation of
the self-assembled polypeptides, e.g., to investigate the aggre-
gation state or the polymorphism of amyloid-like fibrils (and
possibly of other proteinaceous material) by fluorescence mi-
crosc opy even in the absence of aromatic residues, such as in the
case of the poly(ValGlyGlyLeuGly).
Some single, bright protein filaments also are detectable in
Fig. 5a, such as the long fibril (arrow). A line profile of this
str ucture is reported in Fig. 6a, revealing a diameter of ⬇1
␮
m
(in this figure, the presence of many less bright fibrillar structures
in the submicrometer range also may be observed). Spectral
analysis of the native fluorescence of the self-assembled polypep-
tides discloses a broadband emission (⬇95-nm spectral width)
centered at ⬇465 nm (Fig. 6b). No significant emission shifts
were observed by analyzing the fluorescence spectra from large
aggregates or single fibrils, although a detailed, high-resolution
examination of the line shapes of the emission spectra still needs
to be carried out, owing to the noisy spectra from most single
fibrils or smaller regions w ithin individual str uctures (brighter
samples by specifically designed ex periments might reveal an
interesting dependence of the spectral features on the confor-
mational/aggregation st ate of the biomolecules). Interestingly,
preliminary time-resolved experiments performed by c ollecting
the fluorescence signals over wide areas (i.e., without spatial
resolution) indicated average lifetimes in the range of a few
nanosec onds, with significant variations depending on the ex-
Fig. 4. Typical current–voltage (I–V) characteristics of the nanofibrils. (Inset) Control experiments carried out on empty devices (i.e., without fibrils), revealing
low-current signals (always ⬍1 pA).
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cited region (the excitation spot size was a few millimeters
squared). The typical lifetime fits were always multiexponential
(usually three or four) and changed markedly according to the
inspected region. This finding reflects the high conformational
heterogeneit y of the sample and suggests a possible correlation
bet ween this photophysical parameter and the polypeptides’
c onformational patterns. If confirmed by spatially resolved,
time-resolved experiments (e.g., by fluorescence lifetime imag-
ing microsc opy techniques), such an intrinsic feature would of fer
an extremely attractive method for the investigation of the
folding properties of proteins and polypeptides. We also have
observed that the intrinsic luminescence of the fibrils can be
photobleached, and that the same fluorescence images can be
obt ained by two-photon excit ation, although with low efficiency
(dat a not shown).
The physical mechanism underlying the intrinsic fluorescence
emission is unknown, but it may be argued that the charge
transport and photoluminescence properties exhibited by un-
modified fibrils could be strictly related. It is possible that direct
excit ation of the fibrils may induce electron ic transitions in
peptides (e.g., in amide groups) because of a partial delocaliza-
tion of peptide electrons elicited by the presence of hydrogen
bonds (amyloid fibers are characterized by the cross-
␤
str ucture,
in which extensive hydrogen bonding occurs, largely mediated by
trapped water molecules) (Fig. 7) (9–12). Such electronic delo-
calization also may oc cur in the solid state thanks to the
sign ificant retention of water molecules at ambient conditions
(such as hydration shells and other water molecules present in
the inner structure of the nanofibrils). Peptide excitations can
partially relax by fluorescence processes, whereas the electronic
delocalization may account for the observed conductivit y of the
fibrils. This hypothesis is consistent with the experimental
evidence of poor conductivity in vacuum conditions. When water
molecules are lost by the fibrillar structures, hydrogen bonds
c ollapse and, in turn, electronic delocalization is strongly re-
duced. Thus, solid-st ate fibrils might support a net work of
delocalized electrons that can tunnel through the peptide back-
bone and hydrogen bond net works, creating charge-transfer
pathways between different parts of a polypeptide or fibril
molecule. Importantly, fluorescence measurements performed
under vacuum conditions strongly support such assumptions
about the fluorescence/transport mechanisms (SI Fig. 9). The
fluorescence emission, in fact, was found to be strongly depen-
dent on the retention of water molecules; that is, the fluores-
cence signal underwent a significant decrease in intensit y under
vacuum conditions (⬎70% of the fluorescence signal was lost,
c ompared w ith the same samples maintained at ambient con-
Fig. 5. Confocal microscopy images of the poly(ValGlyGlyLeuGly) amyloid-
like fibrils. Fluorescence experiments were performed on the same samples
used for AFM characterizations by using two different concentrations of the
fibril suspension. (a) Concentration at 1.0 mg/ml. (b) Concentration at 0.1
mg/ml. The excitation wavelength was 405 nm.
Fig. 6. Fluorescence analysis of the nanofibrils. (a) Fluorescence line profile
of the single fibril indicated by the arrow in Fig. 5a.(b) Intrinsic fluorescence
spectrum of the self-assembled polypeptides (excitation wavelength, 405 nm;
emission bandwidth, 10 nm).
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ditions) (SI Fig. 9). When fibril samples were brought back to
ambient conditions, the fluorescence signal was completely
rec overed. This experimental evidence strongly suggests that the
water molecules bound to the proteins play a key role in the
fluorescence emission of the fibrils. It is import ant to point out
that such behavior is analogous to what we have already
observed and discussed for protein conductivity.
We have performed the same characterization experiments
(AFM, STM, transport, and fluorescence) on the analogue
poly(ValGlyGlyValGly) synthetic polypeptide. Similar results
were obtained in terms of morphological, conduction, and
luminescence properties, thus confirming the general validity of
the presented results. Furthermore, x-ray photoelectron spec-
trosc opy studies on poly(ValGlyGlyValGly) hydrogel (32) dem-
onstrated that the carbonyl groups of the polymers are involved
in an extensive set of hydrogen bonds with water. If the same
model is applied to poly(ValGlyGlyLeuGly), the previous con-
siderations on charge transport and photoluminescence proper-
ties are quite understandable in terms of structure at the atomic
level.
In conclusion, we have shown the conductive properties of the
fibrillar nanostructures self-assembled from the synthetic poly-
(ValGlyGlyLeuGly) peptides by STM and two-terminal trans-
port experiments. We observed a remarkable stability of the
fibrils at ambient conditions in terms of both morphological and
c onduction properties, which obviously represents an important
aspect from the v iewpoint of dev ice implementation. We also
demonstrated native fluorescence of the nanofribrils, which
allows for direct imaging of their folding and aggregation
properties.
The peculiar features of these peptide nanostructures, along
with the possibilit y to specifically modify and man ipulate their
aminoacidic sequence by genetic engineering techniques, may
provide several advantages for their use in future applications in
biomolecular electronics and nanobiotechnology. Experiments
aimed at controlling the assembly of single-peptide fibrils onto
desired locations with precise orient ations (by direct interaction
with SAM patterns, covalent bonds to specific residues, or
microfluidics techniques) and investigating their elastic proper-
ties (by AFM spectroscopy) are currently underway in our
laboratories.
Materials and Methods
Sample Preparation. The polypent apeptides poly( ValGly
GlyLeuGly) and poly(ValGlyGlyValGly) were chemically syn-
thesized ac cording to the procedures previously developed by
Tamburro and colleagues (29, 34).
Fibrils were generated by suspending the dry powder peptide at
a concentration of 1.0 mg/ml in Milli-Q water (Millipore Corpo-
ration, Billerica, MA) and incubating the solution in a vial tube for
3–5 weeks at 27°C to obtain mature amyloid-like fibrils (29).
AFM Measurements. Fibril samples were prepared by cast depo-
sition of a 20-
␮
l drop of 0.1 mg/ml diluted fibril suspension onto
an SiO
2
substrate. After solvent evaporation, the protein struc-
tures were characterized by AFM (in tapping mode). A ll AFM
ex periments were performed in air at ambient conditions (20–
25°C, atmospheric pressure, 50–60% humidity). AFM images
were t aken by using a CP-II scanning probe microscope (Digital
Instr uments, Santa Barbara, CA) equipped with 5- or 100-
␮
m
scanners (0.5/1 Hz scan rate). Standard silicon probes (MPP-
11100; Veeco Probes, Camarillo, CA) with a nominal spring
c onstant of 40 N/m and a resonance frequency of 300 kHz were
used.
STM Measurements. STM images were acquired in air at room
temperature by using a multimode scanning probe microsc ope
(Digit al Instruments) equipped with an E-scanning head (max-
imum scan size, 10
␮
m). For these experiments, the 0.1 mg/ml
fibril suspension in ultrapure water was deposited onto gold
substrates by means of the same procedure used for silicon
dioxide. Before fibril deposition, STM analyses (data not shown)
c onfirmed the presence of atomically flat 111 Au terraces of
⬇0.5-nm height. STM images were taken in constant current
mode with a typical tunneling current of 50–150 pA and a bias
volt age of 0.1 V (0.5–1 Hz scan rate). The samples were scanned
with mechanically prepared platinum/iridium STM tips (PT-
ECM; Veeco Probes). During STM imaging, several control
ex periments were carried out to ensure that the real structures
of the fibrils were observed, instead of artifacts from the
substrate or tip features.
Transport Measurements. The conductive properties of the fibrils
were investigated by two-terminal transport experiments per-
for med onto interdigitated electrodes. The fibrils were deposited
onto the electrodes structures by cast deposition of a 10-
␮
l drop
of the 0.1 mg/ml fibrils suspension in ultrapure water (Millipore).
The sample was then maintained overnight at ambient condi-
tions. Such a procedure resulted in ef ficient fibrils deposition
across the electrodes. The presence of polypent apeptide fibrils
bet ween electrodes was carefully assessed by SEM (150 E-beam
lithography system; Raith, Ronkonkoma, NY) and AFM
analyses.
The devices for our transport experiments consisted of inter-
digit ated electrodes fabricated on thermally oxidized silicon
wafers using standard photolithographic techn iques. The struc-
ture c onsisted of interdigitated gold lines of 2-
␮
m width and
40-nm height and a line-space period of 2–10
␮
m, covering an
active area of 400 ⫻ 500
␮
m
2
. Control ex periments carried out
on empty devices (i.e., without fibrils) revealed low-current
signals (always ⬍1 pA) (Fig. 4 Inset). The sample current was
measured by means of a probe station (Karl Suss, Waterbury
Center, VT) combined with a parameter analyzer (Agilent
Technologies, Palo Alto, CA) and a cryogen ic system (MMR
Technologies, Mountain View, CA).
Confocal Microscopy Experiments. Spatially resolved fluorescence
images of the fibrils were taken by a confocal microscope
(TCS-SP5; Leica, Wetzlar, Germany). These experiments were
performed on the same samples used for AFM characterizations
by using t wo different concentrations of the fibril solution (0.1
and 1.0 mg/ml). The excitation wavelength was 405 nm. Samples
were observed through a 63⫻, 1.40 NA oil-immersion objective.
Fig. 7. Model for the extensive interaction of poly(ValGlyGlyLeuGly) with
water.
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For spectral analysis, the excitation wavelength was 405 nm, and
the emission bandwidth was 10 nm.
We thank F. Calabi for fruitful discussions, M. R. Armenante and G.
Lanza (University of Basilicat a, Potenza, Italy) for a c omputer-
generated model of hydrated poly(VGGLG), and E. D’Amone and P.
Cazzato for technical assist ance. This work was supported by the Italian
Ministry of Research (Fondo per l’Incentivazione della Ricerca di Base
Project RBLA03ER38㛭001) and the European Union (Elastage Con-
tract 018960).
1. Whitesides GM, Mathias JP, Seto CT (1991) Science 254:1312–1319.
2. Dobson CM (1999) Trends Biochem Sci 24:329–332.
3. Rochet JC, Lansbury PT, Jr (2000) Curr Opin Struct Biol 10:60–68.
4. Pepys MB (2001) Philos Trans R Soc London B 356:203–210.
5. Dobson CM (2001) Biochem Soc Symp 68:1–26.
6. Guijarro JI, Sunde M, Jones JA, Campbell ID, Dobson CM (1998) Proc Natl
Acad Sci USA 95:4224–4228.
7. Gross M, Wilk ins DK, Pitkeathly MC, Chung EW, Higham C, Clark A, Dobson
CM (1999) Protein Sci 8:1350–1357.
8. Serpell LC (2000) Biochim Biophys Acta 1502:16–30.
9. Jimenez JL, Guijarro JI, Orlova E, Zurdo J, Dobson CM, Sunde M, Saibil HR
(1999) EMBO J 18:815–821.
10. Perutz MF, Finch JT, Berriman J, Lesk A (2002) Proc Natl Acad Sci USA
99:5591–5595.
11. Makin OS, Atkins E, Sikorski P, Johansson J, Serpell LC (2005) Proc Natl Acad
Sci USA 102:315–320.
12. Kishimoto A, Hasegawa K, Suzuk i H, Taguchi H, Namba K, Yoshida M (2004)
Biochem Biophys Res Commun 315:73–745.
13. Laidman J, Forse GJ, Yeates TO (2006) Acc Chem Res 39:576–583.
14. Gazit E (2002) Angew Chem Int Ed Engl 41:257–259.
15. Dobson CM (2002) Nature 418:729–730.
16. Koga T, Taguchi K, Kobuke Y, Kinoshita T, Higuchi M (2003) Chemistry
9:1146–1156.
17. MacPhee CE, Dobson CM (2000) J Am Chem Soc 122:12707–12713.
18. Kozel BA, Wachi H, Davis EC, Mecham RP (2003) J Biol Chem 278:18491–18498.
19. Tamburro AM, Pepe A, Bochicchio B, Quaglino D, Ronchetti IP (2005) J Biol
Chem 280:2682–2690.
20. Reches M, Gazit E (2003) Science 300:625–627.
21. Scheibel T, Parthasarathy R, Sawicki G, Lin XM, Jaeger H, Lindquist SL (2003)
Proc Natl Acad Sci USA 100:4527–4532.
22. Jayawarna V, Ali M, Jowitt TA, Miller AF, Saiani A, Gough JE, Ulijn R, (2006)
Adv Mater 18:611–614.
23. Zhang S (2003) Nat Biotechnol 21:1171–1178.
24. Ryadnov MG, Woolfson DN (2003) Nat Mater 2:329–332.
25. Kasai S, Ohga Y, Mochizuki M, Nishi N, Kadoya Y, Nomizu M (2004)
Biopolymers 76:27–33.
26. Mesquida P, Ammann DL, MacPhee CE, McKendry RA (2005) Adv Mater
17:893–897.
27. Horii A, Wang X, Gelain F, Zhang S (2007) PLoS ONE 2:e190.
28. Reches M, Gazit E (2006) Nat Nanotech 1:195–200.
29. Flamia R, Zhdan PA, Martino M, Castle JE, Tamburro A M (2004) Biomac-
romolecules 5:1511–1518.
30. Pompa PP, Bramanti A, Maruccio G, del Mercato LL, Cingolani R, Rinaldi R
(2005) Chem Phys Lett 404:59–62.
31. Pompa PP, Della Torre A, del Mercato LL, Chiuri R, Bramanti A, Calabi
F, Mar uc cio G, Cingolan i R, Rinaldi R (2006) J Chem Phys 125:
021103.
32. Flamia R, Salvi AM, D’Alessio L, Castle JE, Tamburro AM (2007) Biomac-
romolecules 8:128–138.
33. Wang Z, Zhou C, Wang C, Wan L, Fang X, Bai C (2003) Ultramicroscopy
97:73–79.
34. Tamburro AM, Guantieri V, Gordini DD (1992) J Biomol Struct Dyn 10:441–
454.
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