Article

pubs.acs.org/Langmuir

Fibrillar Structures Formed by Covalently Bound, Short, Stranded

Peptides on Self-Assembled Monolayers
Jason W. Dugger and Lauren J. Webb*
Department of Chemistry, Center for Nano- and Molecular Science and Technology, and Institute for Cell and Molecular Biology,
The University of Texas at Austin, 1 University Station, A5300, Austin, Texas 78712, United States
S Supporting Information
*

ABSTRACT: The ability to maintain or reproduce biomolecular

structures on inorganic substrates has the potential to impact diverse
elds such as sensing and molecular electronics, as well as the study of
biological self-assembly and structurefunction relationships. Because
the structure and self-assembly of biomolecules are exquisitely sensitive
to their local chemical and electrostatic environment, the goal of
reproducing or mimicking biological function in an abiological
environment, including at a surface, is challenging. However, simple
and well-characterized chemical modications of prepared surfaces can
be used to tune surface chemistry, structure, electrostatics, and
reactivity of inorganic materials to facilitate biofunctionalization and
function. Here, we describe the covalent attachment of 13-residue stranded peptides containing alkyne groups to a at gold surface
functionalized with an azide-terminated self-assembled monolayer
through a Huisgen cycloaddition, or click, reaction. The chemical composition and structural morphology of these surfaces
were characterized using X-ray photoelectron spectroscopy, grazing incidence angle reectionabsorption infrared spectroscopy,
surface circular dichroism, and atomic force microscopy. The surface-bound -strands self-assemble into antiparallel -sheets to
form brillar structures 24.9 1.6 nm in diameter and 2.83 0.74 nm in height on the reactive surface. The results herein
provide a platform for studying and controlling the self-assembly process of biomolecules into larger supermolecular structures
while allowing tunable control through chemical functionalization of the surface. Interest in the mechanisms of formation of
brillar structures has most commonly been associated with neurodegenerative diseases, such as Alzheimers and Parkinsons, but
brils may actually represent the thermodynamic low-energy conformation of a much larger class of peptides and proteins. The
protocol developed here is an important step toward uncovering not only the factors that dictate self-assembly but also the
mechanisms by which this brillar class of superstructures forms.

nding surface chemistries that integrate the biomolecule with

the surface in such a way that the chemical and electrostatic
properties of the surface act in concert with the biomolecule to
generate a novel biological/abiological material. To this end,
our group has explored the use of self-assembled monolayers
(SAM) on gold surfaces to facilitate the covalent attachment of
structured -helical peptides orientated parallel to the
substrate.11 This method exploits the known structure and
regularity of the SAM to induce a desired secondary structure
in the biomolecules while preserving desirable properties
characteristic of the metal surface such as conductivity.12,13
Here, we report an adaptation of this methodology to
covalently bound, short (13-residue), -stranded peptides to
the gold surface, which in turn nucleate the self-assembly of
large supermolecular brillar structures in a controlled way.

INTRODUCTION
The chemical functionalization of inorganic surfaces and
substrates with folded and functional biological molecules has
the potential for a large variety of applications, including
sensors, electrochemistry, and molecular electronics, as well as
providing a template for studying and controlling the selfassembly of biomolecules into supermolecular structures. The
immobilization of biomolecules on surfaces enables the
formation and examination of biomolecular structures that
would not be possible in solution, such as single-molecule
studies,1 the formation of asymmetric and two-dimensional
materials,24 and the generation of novel materials that
incorporate both biological and abiological functions.57 A
chief obstacle to the incorporation of biomolecules onto
inorganic substrates is the preservation of the structure of the
molecule, which is inherently related to its function and
stability. One strategy that has been employed successfully is to
cover the underlying inorganic substrate with a thick polymer,
such as polyethylene glycol or polylysine, reducing the surface
to a passive support.810 Our laboratory has been interested in
2015 American Chemical Society

Received: December 23, 2014

Revised: February 20, 2015
Published: March 4, 2015
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well as self-assembly into antiparallel -sheets. Additionally,

structural morphology and heterogeneity observed using
atomic force microscopy (AFM) show that these -sheets
organize into larger brillar structures 24.9 1.6 nm in
diameter and 2.83 0.74 nm in height. We hypothesize that
single peptide strands covalently bound to the SAM act as
nucleation sites for the formation of -sheets, which then
proceed to organize into brils in as little as 15 min. Samples
with longer reaction times (18 h) contain similarly sized
structures with a higher degree of surface coverage, creating a
meshlike network of brils. These studies establish the
capability to nucleate noncovalently assembled superstructures
through the controlled immobilization of short peptides to
inorganic substrates. This strategy inherently enables tuning of
chemical reactivity, environmental conditions, and interface
heterogeneity and generalization to other biomolecules.

Biological and synthetic brils and brillar structures have

shown promise in applications such as sensors, ordered
nanomaterials, mimics of extracellular matrices, and scaolds
for cell cultures.1418 Biological brils are also of immense
current medical interest because they are classically associated
with neurodegenerative diseases such as Alzheimers, Parkinsons, and type II diabetes.1921 Because of this, there has been
extensive investigation into the circumstances under which
naturally occurring biological peptides spontaneously selfassemble into aggregates, brils, and plaques in certain tissues
throughout the body, and it is widely believed that one or more
aspects of this self-assembly process may be causing the disease
state associated with that bril. However, it has recently
become clear that the formation of these brillar structures is
not a unique property of a particular biological peptide, but
instead seems to be a common feature of amide-based
polypeptides in general.22 Indeed, recent computational and
experimental work on the thermodynamic stability of brils and
kinetic barriers to the assembly of these structures has
suggested that brillar conformations may be the low-energy
structure of polypeptides in general, regardless of sequence or
length.2326 These observations mean that the basic understanding of biological bril formation, stability, and function is a
research area that lies at a crossroads between materials science,
biophysical chemistry, and medicine, inuencing elds as
diverse as human health and molecular electronics.
Various factors that regulate bril formation and growth,
including kinetics, inhibition, promotion, sequence variation,
and environmental conditions, have been investigated,2731 and
it has been observed repeatedly that there are a large variety of
conformations that a single protein sequence can adopt.19 It has
been shown that brils formed from amyloidogenic peptides of
dierent sequences can adopt structures comprised of parallel
or antiparallel -sheets, and the resulting brils can be
polymorphic.21,3234 For instance, brils have been seen to
adopt a triangular structure with a large central cavity,35 a thin
ribbon-like structure with diameters ranging from 3.5 to tens of
nanometers,36 and even nanotube-like structures over 100 nm
in diameter resulting from the helical twisting of ribbon-like
brils.30,37 Despite the extensive characterization of a variety of
these brillar structures, there is signicant ambiguity
concerning how the complex interactions dictated by peptide
sequence, hydrophobicity, hydrophilicity, and environment
regulate the nucleation and self-assembly of small biomolecules
into more complex supermolecular structures. Understanding
these factors is a necessary prerequisite to generate controlled
materials containing desired properties that can be exploited for
interesting or novel purposes. Taking a surface-mediated
approach to immobilizing these biomolecules allows for
unprecedented control over a number of variables that can be
used to possibly tune bril growth, size, structure, surface
density, and chemical reactivity.
In the work described here, we demonstrate the self-assembly
of superstructures using a bottom-up method to bril formation
through the covalent attachment of short (4.4 nm, 13-residue
long) -stranded peptides to a SAM on a gold surface. This is
accomplished with a triazole linkage formed from a Huisgen
cycloaddition click reaction between one or two alkyne
groups on the peptide and adjacent terminal azide groups on
the SAM. Grazing incidence angle reectionabsorption
infrared spectroscopy (GRAS-IR), X-ray photoelectron spectroscopy (XPS), and surface circular dichroic (CD) spectroscopy conrm the presence of the peptide on reacted surfaces as

MATERIALS AND METHODS

Surface Preparation. Peptide-terminated SAM surfaces were

prepared on silicon (111) wafers for GRAS-IR and XPS measurements, on quartz slides for surface CD experiments, and on gold-onmica substrates (150 nm of gold on mica, SPI Supplies) for AFM
measurements. The dierent substrates were used to exploit their
unique properties in each experiment, with silicon wafers used for their
macroscopic atness in the spectroscopic reection experiments,
quartz for its UV transparency in CD studies, and mica for its
microscopic atness in AFM. Silicon wafers (NOVA Electronic
Materials) were 500 m thick, polished on one side, sealed in N2(g),
and stored in a dry N2(g) atmosphere glovebox until use. A 10 nm
layer of chromium, followed by 100 nm of gold (99.95% pure), was
vapor-deposited on the silicon wafers using a thermal evaporator-II
(Denton) instrument at a pressure of 105 Torr. The samples were
covered in clean-room-rated silicon wafer tape (ICROS TAPE) and
cut into approximately 1 cm2 pieces with a programmable Disco 321
wafer-dicing saw. After removing the tape, the samples were rinsed in
methanol, sonicated in acetone for 10 min, rinsed in high purity water
(HPW) with an impedance of >18 M cm (Barnstead NANOpure
Diamond Life Science UV/UF) and then rinsed again in methanol,
dried under a stream of N2(g), and stored in Paralm-covered
containers until use. This cleaning was done to remove any residue
that may have been left behind by the wafer tape used during the
cutting process. Quartz substrates precut into 2.5 0.95 cm slides
were rinsed in ethanol and dried under N2(g) before the deposition of
3 nm of chromium, followed by 11 nm of gold, and subsequently
stored in Paralm-covered containers until use. The quartz samples
had the thinnest layer of gold of all the substrates in order to increase
transmission in the CD experiments. Previous studies have shown that
SAMs are still formed on gold layers as thin as 7 nm,38 and SAMs are
commonly used to functionalize gold surfaces on a variety of
supporting substrates, including those employed in these studies.
The choice of substrate and thickness of the gold layer is not expected
to aect SAM formation or subsequent reaction with the peptides.
All chemicals were purchased from Sigma-Aldrich unless otherwise
noted. Gold substrates deposited on silicon were cleaned using a
piranha solution (1:3 30% hydrogen peroxide/concentrated sulfuric
acid) (Caution! Explosive in the presence of organic contaminants.) for 1
min; rinsed in a sequence of HPW, concentrated hydrochloric acid,
HPW, and nally ethanol; and dried under a stream of N2(g). These
surfaces were then annealed for 5 min using a H2(g) ame. Gold-onmica substrates were kept in manufacturers packaging (glass vials lled
with argon gas, capped, and sealed with Paralm) until use, where they
were then annealed for 5 min with a H2(g) ame. Quartz substrates
were only rinsed in ethanol and dried under N2(g) because the thinner
layer of gold could not withstand the harsher cleaning methods
described above. All subsequent steps were identical for the silicon-,
quartz-, and mica-supported substrates. To prepare the SAM, the
samples were submerged in a solution of 1 mM 11-azido-1undecanethiol diluted in anhydrous ethanol for 24 h in the dark to
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qualitatively unchanged (254 scans). For the 6-reacted surfaces, a

total of 410 scans were accumulated, and beyond background
subtraction, no other data manipulation was done to the spectra.
X-ray Photoelectron Spectroscopy. To further characterize the
chemical composition of the surfaces, X-ray photoelectron spectra
were collected with a Kratos Azis Ultra XPS equipped with a
monochromatic Al K source illuminating the sample at 1486.5 eV.
Samples were grounded using copper tape and introduced into the
chamber at a pressure below 2 109 Torr. High-resolution spectra of
the carbon 1s region were collected at a resolution of 0.1 eV with a 20
eV pass lter. Ejected photoelectrons were collected with a
hemispherical electron energy analyzer positioned normal to the
sample surface. Due to sample charging from photoelectron loss, the
adventitious carbon peaks were detected at a higher than normal
binding energy. To correct for this, the spectra were shifted to
reposition the adventitious carbon peak to 284.5 eV. Spectral shifts
and decompositions were done using a linear baseline followed by
peak tting to Gaussian curves using the CasaXPS software package.
Atomic Force Microscopy (AFM). An Asylum Research MFP-3D
AFM was used to image the dry, reacted, gold-on-mica substrates in
ambient air. AFM cantilevers (Mikromasch) with typical probe radii of
8 nm, 65 kHz resonance frequencies, and 0.5 N/m force constants
were used in tapping mode to minimize shear forces and tipsample
interactions. Height and width measurements were obtained by
averaging three line proles oriented normal to the long axis of each
bril. A total of 25 measurements for each reaction time point were
taken from images 1 1 m in size. All image processing and line
proles were done using the Gwyddion SPM software package.

fabricate a gold surface covered in 100% azide-terminated SAM. The

11 methylene groups of the thiol participate in van der Waals
interactions with the hydrophobic portions of surrounding thiols,
inducing an ordered SAM that leaves the azide groups exposed at the
SAM/aqueous interface. Unbound thiols were removed by sequentially rinsing with HPW, dimethylformamide (DMF), HPW, and
ethanol and dried under a stream of N2(g).
Azide-terminated SAM surfaces were exposed to the peptide
KLKXKLLLKXKLK (hereafter 6; WuXi AppTec). It has been
shown that sequences of alternating leucine and lysine residues adopt
-stranded conformations in aqueous solutions and at air/water
interfaces.39 The 6 peptide was designed to approximate this
periodicity with the exception of the three sequential hydrophobic
residues to minimize -sheet formation in solution. Two unnatural
propargylglycine residues (X; Wuxi AppTec) contained reactive alkyne
groups for a Huisgen cycloaddition (click) reaction, where the alkyne
groups in the peptide react with the azide groups at the surface to form
triazole linkages using Cu(I) as a catalyst. Sodium ascorbate was used
to reduce Cu(II) to Cu(I), and TBTA {tris[(1-benzyl-1H-1,2,3-triazol4-yl)methyl]amine} was present to stabilize the Cu(I) in the aqueous
solution environment.40 Solutions with a total volume of 5 mL were
prepared by adding 2:1 tert-butanol/HPW solvent, followed by TBTA
(0.5 mol), 6 peptide (0.5 mol), sodium ascorbate (0.6 mol), the
azide-terminated surface, and nally CuSO4 (0.1 mol). Before
addition to the reaction solution, the peptide was vortexed and then
stirred for 20 min in the 2:1 tert-butanol/HPW solvent at
concentrations below 1.3 mg/mL to ensure complete solubility.
After addition of all reagents, vials were immediately placed in an oven
at 70 C for reaction times ranging from 15 min to 18 h. Upon
removal, samples were allowed to cool in the reaction solution for 10
min, and then any physisorbed reactants were removed by sequentially
rinsing with 2:1 t-butanol/HPW, HPW, phosphate-buered saline
(PBS), HPW, and ethanol and then dried under a stream of N2(g).
Dried samples were stored in the dark until measured. All control
samples were azide-terminated surfaces that where immersed in the
reaction solution lacking CuSO4 and its reducing agent, sodium
ascorbate, to eliminate the presence of any Cu(I) catalyst.
Grazing Incidence Angle ReectionAbsorption Infrared
Spectroscopy. Surface vibrational spectroscopy was collected with a
Bruker Vertex 70 FTIR spectrometer equipped with a A518/Q
horizontal reection unit (Bruker) for illuminating the sample at a
grazing angle of 80 with respect to the surface normal. The sample
chamber was purged with N2(g) for 1 h after samples were introduced
to the instrument to reduce background noise from H2O and CO2. A
total of 400 scans of p-polarized light were collected for each sample
before and after reaction with the 6 peptide. A mercury cadmium
telluride (MCT) detector collected signals from the 400-4000 cm1
region, while an indium antimonide (InSb) detector collected signals
from 1870 to 4000 cm1 to take advantage of its higher sensitivity to
the azide, methyl, and methylene stretches in that region. A bare gold
substrate (cleaned and annealed as described above with each set of
samples) was used as a common reference in order to obtain absolute
dierences in absorbance between samples. Following background
subtraction, the spectra were attened using a rubber band correction
baseline function in the instruments OPUS software.
Circular Dichroic Spectroscopy. The conformation of the 6
peptide in solution and on the surface was characterized using a
JASCO J-815 CD spectrometer.12,41,42 Samples were illuminated
between 190 and 250 nm at a scan rate of 50 nm/min, 1 nm
resolution, and a 4 s response time. For solution studies, a 130 M
solution of peptide dissolved in HPW in a 1 mm quartz cuvette was
used to collect three scans, using a HPW solution for background
subtraction. Surface CD spectra were obtained by stacking three 6reacted quartz slides in a 1 cm cuvette lled with HPW, oriented
normal to and facing the UV source. Unreacted azide-terminated
quartz slides set up in the same manner were used for background
subtraction. Due to poor UV transmission through the gold and
relatively low surface concentration of peptide compared to solution, a
large number of scans were acquired to establish the spectra. Scans of
the background slides were taken until the spectra appeared to be

RESULTS
Chemical Composition of Peptide-Reacted Surfaces.
GRAS-IR spectra were acquired for azide-terminated surfaces
before and after reaction with the 6 peptide and for control
samples. Representative spectra of the CH stretching region
for the surfaces discussed here are shown in Figure 1. Azideterminated surfaces (Figure 1, green) were dominated by
features at 2851 and 2924 cm1, corresponding to symmetric
and asymmetric methylene stretches, respectively.11,43,44
Peptide-terminated surfaces reacted for 30 min (Figure 1,
blue) showed an asymmetric methyl stretch at 2958 cm1, as
well as an increase in intensity of the methylene stretches due
to the presence of additional CH groups contributed by the
surface-bound peptides. The peptide-terminated surface reacted
for 18 h (Figure 1, red) also showed an increase in methylene
peak intensities along with the development of symmetric and
asymmetric methyl peaks at 2870 and 2958 cm1. The reacted
surfaces were signicantly dierent from control surfaces
prepared by immersing the azide-terminated SAM surface in
the Huisgen cycloaddition reaction solution without any Cu(I)
catalyst (Figure 1, black). These surfaces displayed methylene
stretches at 2857 and 2928 cm1, indicating preservation of the
SAM, but except for a small methyl peak at 2962 cm1,
corresponding to a small amount of physisorbed peptide, they
did not show any modication from immersion in the control
reaction solution.
While the methyl region of the IR spectrum provides
evidence of the bound peptide on the surface, the amide region
(14001800 cm1) provides additional information regarding
the secondary structure of the peptides bound to the
surface.11,4548 Figure 2 shows representative infrared spectra
of the amide region containing peptide backbone stretches of
the carbonyl group (amide I, centered around 1650 cm1) and
stretching of the CN bond coupled with NH bending
(amide II, near 1545 cm1). A spectrum of the azide-terminated
surface before reaction (Figure 2, green) lacked any peaks in
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sheets in an antiparallel conguration and arises from transition

dipole coupling of carbonyl groups along the peptide
backbone.45,46,4850 The less-intense peaks at 1666 and 1675
cm1 were attributed to random or -turn conformations.45,51,52 Similar to the 18 h reaction, the 30 min reacted
sample (Figure 2, blue) also showed splitting of the amide I
peak at 1637 and 1694 cm1 along with random and -turn
absorbances at 1662 and 1679 cm1. The signal-to-noise ratio
of this sample is lower than the 18 h reaction due to low
peptide surface coverage and represents the shortest reaction
time point that allows detection by IR spectroscopy.
Overall, the increased methyl and methylene peaks, as well as
the presence of amide I and II stretches in the reacted sample
spectra, provide evidence of both the presence and the
structure of surface-bound peptides. The splitting of the
amide I peak indicates that the peptides are self-assembled
into a -sheet conformation in an antiparallel alignment. The
less intense stretches in the amide I region attributed to
random or -turn structures could arise either from peptides
adopting conformations that stabilize the -sheets or because
the SAM surfaces were 100% functionalized in reactive azide
groups, and it was expected that some peptides could bind to
the surface in orientations that do not facilitate the formation of
organized -sheets. Spectra of the control samples showed only
very small increases in methyl/methylene signals and negligible
absorbance in the amide region, indicating that peptides only
bind to the surface in the presence of the Cu(I) catalyst in the
reaction solution. Collectively, the FTIR data demonstrate the
presence of surface-bound peptide in a largely antiparallel sheet conformation, which requires the Huisgen Cu(I) catalyst
to form the triazole linkage that tethers the peptide to the
underlying SAM. In addition to the methyl, methylene, and
amide regions, the azide peak (2103 cm1) of the SAM
functionalized surfaces was also monitored to conrm SAM
formation, but peak degradation was not used for quantitative
analysis due to decomposition pathways unrelated to the
Huisgen reaction.
In addition to vibrational spectroscopy, XPS was also used to
conrm the presence of the surface-bound peptide. Figure 3
shows representative spectra of the C 1s region from an 18 h
6-reacted sample and an unreacted azide-terminated SAM,
including component ts. The C 1s signal from the unreacted
azide surface (Figure 3, bottom) showed peaks corresponding
to CC (284.5 eV) and CN (285.8 eV) bonds, representing
the alkyl chains of the SAM as well as the CN bonds of the
terminal azide groups. The 6-reacted surface (Figure 3, top)
contained peaks arising from photoelectron emission from C
C, C(N,O), and CO bonds near 284.5, 285.8, and 287.7
eV, respectively.53 The development of the carbonyl peak as
well as the increase in detected C(N,O) photoelectrons on
the 6-reacted samples relative to the unreacted surface are
consistent with the presence of amide groups on the surface.
These results indicate the successful attachment of peptides to
the gold surface and are consistent with previous XPS analysis
of surface-associated peptides.4,41
Structural Characterization of Surface-Bound Peptides. CD spectra were collected to identify any dierences in
secondary structure that the peptide adopts in solution and on
the surface. Figure 4 shows the measured ellipticity of the 6
peptide in HPW solvent (red), with a negative band <200 nm
and a small positive absorption near 219 nm. These signals are
consistent with peptides that adopt a random coil conguration
and indicate that in water the peptide is essentially

Figure 1. GRAS-IR spectra of the CH stretching region of an

unreacted azide-terminated SAM (green), an 18 h control reaction
lacking the click Cu(I) catalyst (black), and an azide-terminated SAM
reacted with the 6 peptide for 30 min (blue) and 18 h (red). The
SAM spectrum shows two peaks corresponding to the symmetric and
asymmetric methylene stretches at 2851 and 2924 cm1, respectively.
The 30 min reacted sample shows an increase in methylene stretch
intensity as well as the development of an asymmetric methyl peak at
2958 cm1. The 18 h reacted sample has both an increase in
methylene intensities as well as symmetric and asymmetric methyl
peaks at 2870 and 2958 cm1. Spectra are shifted vertically for aid in
visualization.

Figure 2. Spectra of the amide region of GRAS-IR scans from an

azide-terminated SAM (green), an 18 h control reaction lacking the
click catalyst (black), and azide-terminated SAMs reacted for 30 min
(blue) and 18 h (red). The amide I band is split into two peaks near
1630 and 1695 cm1 (dashed lines) for the 18 h reacted sample,
indicative of an antiparallel -sheet conformation. The 30 min reaction
(absorbance shown at 5 due to low surface concentration) also
shows amide I peak splitting at 1637 and 1694 cm1. Spectra have
been shifted vertically to aid in visualization.

this region, and the control sample (Figure 2, black) showed

negligible amide I and II signals. The 18 h reacted sample
(Figure 2, red) displayed a single large amide II peak at 1541
cm1 and two peaks in the amide I region at 1630 and 1695
cm1. This splitting of the amide I peak is characteristic of 3444

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of the alkyne-containing residues, immobilization may

eectively increase the local concentration of peptides at the
surface, which facilitates the subsequent addition of other
peptides to form brillar structures.
AFM was performed to characterize the surface morphology,
heterogeneity, and supramolecular structure of the peptidefunctionalized samples. Figure 5 shows representative AFM
images (top, height; middle, phase; bottom, height prole)
from an azide-terminated SAM reacted with the 6 peptide for
15 min (Figure 5A), 30 min (Figure 5B), and 18 h (Figure 5C)
and an 18 h control sample that lacked any Cu(I) catalyst
(Figure 5D). Height and phase images of a bare gold sample
and an azide-terminated surface are shown in Figure S1 in the
Supporting Information. The surface topography of the 15 min
reacted samples (Figure 5A, top) displayed only sparse
coverage of brillar structures, while the phase image (Figure
5A, middle) showed a clear contrast between the gold grains of
the underlying surface and the relatively soft brils, which are
composed of peptides. The broad, very bright features in the
phase image are troughs between the gold grains, which
correspond to the black, low-lying regions of the topographic
image. While the bril surface coverage of these 15 min reacted
samples was too low to investigate using ensemble spectroscopic methods like GRAS-IR or CD, the AFM images clearly
showed that bril formation occurs on the surface in as little as
15 min. The 30 min reacted sample also showed similarly sized
brillar structures but with a higher degree of surface coverage
in the topographic image (Figure 5B, top), along with similar
contrast in the phase image (Figure 5B, middle). The 18 h
reacted sample showed the highest degree of surface coverage
evident in both the height and phase images (Figure 5C, top
and middle).
Notably, the 18 h reacted samples diered from the 15 and
30 min reacted samples because of the presence of spherical
aggregates, 70 nm in width, with brillar structures emanating
from them. This could indicate an alternative pathway to bril
formation other than nucleation by a single surface-bound
peptide. To conrm this hypothesis, 20 L aliquots of the
reaction solution were removed in 15 min intervals, drop-cast
onto bare gold surfaces, immediately dried under a stream of
N2 gas, and imaged using AFM to determine if brils were
forming in solution. We observed that spherical aggregates were
present in the reaction solution at all time points, but at 45 min
small brils began to form (Figure S2, Supporting Information). While it is possible that drying the drop-cast solutions
with N2 gas induced some degree of brillation on the surface,
this result strongly supports the hypothesis that any brils
present on the surface at reaction times <45 min were
nucleated from surface-bound peptides. Therefore, in the 18 h
reacted samples, it is possible that the brillar appendages on
the aggregates formed in solution and subsequently bound to
the azide-terminated SAM through either covalent or noncovalent mechanisms. Figure 5D shows a representative image
of the control samples [prepared in reaction solutions
containing no Cu(I) catalyst] reacted for 18 h. These surfaces
showed only small spherical aggregates of polypeptide material.
Occasionally, globular aggregates of peptide with brillar
branches were seen on control surfaces, but their presence
was sparse, and the brils were only found emanating from
those aggregates, not independently formed from the surface as
in the 15 and 30 min reaction samples. Overall, the topographic
images indicate that bril formation occurs only on surfaces
exposed to reaction solutions containing the Huisgen catalyst,

Figure 3. XP spectra of the C 1s region of a 6-reacted azideterminated SAM (top) and an unreacted azide-terminated SAM
(bottom). The spectra were collected at a resolution of 0.1 eV with a
20 eV pass energy. The raw spectra (black dots) are shown along with
component and total Gaussian ts (black). The components with
peaks near 284.5, 285.8, and 287.7 eV correspond to aliphatic CC
(blue), C(N,O) (red), and carbonyl CO (green) bonds. Spectra
were oset for clarity.

Figure 4. CD spectra of the 6 peptide in high-purity water (red) and

a 6-reacted azide-terminated SAM (black). The left vertical axis
corresponds to the ellipticity on the surface, while the right vertical axis
displays ellipticity in solution. The negative peak at 218 nm (dashed
line) indicates a -stranded conformation of the peptide on the reacted
surface.

unstructured.39,54,55 When the peptide was bound to the

surface, its CD spectrum changed dramatically. A small positive
absorption was present at <200 nm along with a substantial
negative absorbance at 218 nm (Figure 4, black). These
signatures are characteristic of -sheet conformations.55,56 The
CD results indicate that while the peptide lacks a dened
secondary structure in solution, peptides found on the reacted
surfaces have a strong propensity to adopt a -sheet
conguration. This variation in conformation between solution
and surface-bound structures suggests that the surface plays a
role in inducing the formation of -sheet structures. By
tethering peptides to azide groups on the SAM at one or both
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Figure 5. Azide-terminated SAMs reacted with the 6 peptide for 15 min (A), 30 min (B), and 18 h (C) and an 18 h control sample that lacked the
click catalyst (D). All columns contain height (top), phase (middle), and line proles (bottom). Scale bars are all 400 nm, and green arrows
correspond to the length and direction of the line proles.

Figure 6. Distribution of bril heights (left column) and widths (right column) for 15 min (top), 30 min (middle), and 18 h (bottom) reactions.
Reaction time does not aect the height and width dimensions of brils, but each time point contains a range of bril sizes with averages of 24.9
1.6 nm in diameter and 2.83 0.74 nm in height.

with bril formation occurring in as little as 15 min. These

results are consistent with the hypothesis that brillation occurs
via nucleation at peptide strands covalently immobilized to the
underlying SAM.

Height and width measurements of 25 brils from each

reaction time point were taken using line proles oriented
perpendicular to the long axis of each bril from images 1 1
m in size. Collectively, the brils had an average diameter of
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assemble into brils, or (2) an increase in local concentration of

peptides at the surface that overcomes the critical concentration
normally required for brillation to occur in solution. As the
61X peptide is tethered at only one point, yet still forms brils
on the surface, it is unlikely that its conformation is locked in
an orientation that promotes additional self-assembly of
peptides in the same manner as the 6 peptide. Therefore,
the more likely hypothesis is that the reactive surface acts as an
aggregator of peptides, creating a high density of immobilized
peptides at which the further self-assembly of peptides into
brils can take place. In fact, brillation through increased local
concentration has been observed on lipid bilayers,57 hydrophilic
mica,58 and at air/water interfaces.59

24.9 1.6 nm and an average height of 2.83 0.74 nm with

lengths ranging from tens of nanometers to microns.
Specically, bril heights for the 15 min, 30 min, and 18 h
reactions were 2.26 0.68, 3.74 0.78, and 2.48 0.76 nm,
respectively. Fibril widths were 25.5 1.6, 27.2 1.7, and 21.9
1.3 nm for the 15 min, 30 min, and 18 h reactions. On the
basis of the distribution of widths and heights, reaction time
does not seem to have any bearing on these dimensions of the
brils (Figure 6), but bril length and surface density did
increase with extended reaction times.
Immobilization of the Nucleating Peptide. As part of a
preliminary investigation of the nucleation mechanism
associated with the covalently bound brillar structures, we
tested the reaction conditions with another short, -stranded
peptide that contained only one propargylglycine residue
(61X, Wuxi AppTec). The 61X peptide preserved the
same alternating leucine/lysine sequence (KLKXKLLLKLKLK) as the 6 peptide but contained only one reactive
alkyne group that could covalently link the peptide to the azideterminated SAM. It was determined that the 61X peptide was
bound to the surface and adopted an antiparallel -sheet
conformation, as conrmed by an increase of methyl/
methylene peaks (Figure S3, Supporting Information) as well
as amide I peak splitting (Figure S4, Supporting Information)
through the use of GRAS-IR. Also, solution and surface CD
experiments showed that 61X adopted a random coil
conguration in solution but forms -sheets when bound to
the surface (Figure S5, Supporting Information). Additionally,
AFM experiments determined that the 61X peptide also
formed brils of similar size and surface coverage as the 6
peptide (Figure S6, Supporting Information). On the basis of
this information, it was determined that the -sheets formed on
the azide-terminated surfaces may be nucleated by surfacebound peptides that are tethered at either one or both alkyne
groups present in the 6 sequence. This is qualitatively
represented in Figure 7.
The 61X experiments also provide insight into the question
of how the surface accelerates the formation of the brillar
structures on the surface. Two likely hypotheses for this include
(1) the induced conformation of surface-bound peptides, which
would allow subsequent addition of other peptides to self-

DISCUSSION
This research represents an exciting rst step in our pursuit to
create functional abiological structures from biological materials
based on carefully controlled chemical inputs. This work aims
to covalently attach -stranded peptides to a SAM supported
on a gold surface, where the surface-bound peptides then act as
nucleation sites for the formation of noncovalently assembled
brillar superstructures (Figure 7). Covalent immobilization of
the peptide is accomplished through a Cu(I)-catalyzed Huisgen
cycloaddition reaction, where a triazole linkage is formed from
reacting one or both alkyne groups on the peptide with
terminal azide groups on the SAM. GRAS-IR experiments
veried the presence of the surface-bound peptide by
comparing spectra of samples before and after reaction, as
well as controls. The vibrational spectra showed that 6peptide-reacted samples contained a greater number of methyl
and methylene groups than both unreacted and control
[lacking Cu(I) catalyst] samples. Additionally, absorption in
the amide region of the IR spectrum (absent in unreacted and
control samples) showed splitting of the amide I peak into
stretches at 1630 and 1695 cm1, which arise from transition
dipole coupling of carbonyl groups and point to an antiparallel
arrangement of peptides within -sheets found on the surface.
Surface-bound peptides were also conrmed through XPS
studies that collected a higher intensity of C(N,O) and CO
photoelectrons from reacted surfaces than unreacted surfaces.
Secondary structure information obtained through solution and
surface CD experiments showed that the 6 peptide was
randomly coiled in solution but adopted a -stranded structure
when surface-bound. This indicates that the surface is not
merely a passive support but that its chemical reactivity acts as a
medium that accelerates brillation. Morphology and heterogeneity of the surfaces were characterized using AFM and
showed that peptide-reacted substrates develop surfacenucleated brillar structures in as little as 15 min of reaction
time. Extending reaction times to 30 min produced surfaces
with a higher degree of bril coverage, and 18 h reacted samples
were densely covered in a combination of surface-nucleated
brils as well as brillar structures formed in solution that
subsequently interacted with the azide-terminated SAM.
Control samples that were reacted for 18 h with no Cu(I)
catalyst showed no uniform surface coverage of brils, and any
brils that did occur were rare and present only as appendages
of disordered oligomeric aggregates that likely formed in
solution.
On the basis of the lack of any uniform distribution of
brillar structures on control surfaces, we suggest that
chemically bonding the peptide to the surface acts as a
nucleation site that then induces the formation of brillar

Figure 7. Schematic representation of single peptides tethered to the

SAM through triazole linkages at both (top) or one (bottom) alkynecontaining residue. Additional peptides proceed to self-assemble to the
bound peptide through hydrogen bonding, forming the -sheets that
comprise the brillar structures found on the reacted surfaces.
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employ the use of electron microscopy and two-dimensional

diraction experiments to determine how the antiparallel sheets are organizing themselves into brillar forms.
To summarize, this work characterizes the binding of short,
-stranded peptides to an azide-terminated SAM on a gold
surface through a Huisgen cycloaddition reaction. We have
found that the peptide orients itself in an antiparallel
conguration within -sheets, as determined through splitting
of the amide I peak in the vibrational spectrum as well as
surface CD. While the peptide has a random coil conformation
in solution, the surface induces the peptide to adopt a stranded structure through covalent immobilization. Additionally, surface topography shows that the peptide self-assembles
into brillar structures 24.9 1.6 nm in diameter and 2.83
0.74 nm in height. When subjected to longer reaction times (18
h), surfaces with a higher density of brillar structures are
produced. Studies are ongoing in our laboratory to further
characterize the structure of these brils as well as to determine
the nucleation mechanism by which they form and continue to
assemble on the surface.
These reacted surfaces demonstrate the production of
supermolecular structures through the use of tunable variables,
including peptide sequence, concentration, solution chemistry,
and surface reactivity. This work provides a general platform for
studying and controlling the natural self-assembly processes of
biomolecules at an interface, with potential applicability toward
studying how peptides and proteins aggregate or assemble into
their respective thermodynamic low-energy conformations.
Understanding the mechanisms by which this self-assembly
occurs is a crucial step in the exploitation of these biomolecular
superstructures in elds such as sensors, molecular electronics,
and small molecule immobilization. This research provides a
unique platform to study these factors and further develop the
use of biofunctionalized inorganic surfaces as practical devices.

structures through the addition of subsequent peptide strands

that associate through hydrogen bonding. The resulting brils
are comprised of an antiparallel arrangement of noncovalently
associated peptide sheets. Peptides tethered to the surface
through one or both triazole linkages are able to orient in a
manner that facilitates -sheet formation and catalyzes
brillation in as little as 15 min. Extending reaction times to
30 min leads to a higher degree of surface coverage, dominated
by brillar structures of consistent height and width. At 45 min,
brils begin to form in the reaction solution, which
subsequently bond covalently to the azide-terminated SAM.
This results in surfaces terminated by a combination of surfacenucleated and solution-formed brils. Conventionally, bril
growth in solution is known to be concentration-dependent;
however, others have noted that the presence of a surface
allows for weakly adsorbed peptides to locally concentrate and
promote brillation in environments below the critical
concentration for the formation of brils.60,61 The results of
the 61X experiments indicate that brillation occurs whether
peptides are bound at one or two locations to the surface,
which supports the hypothesis that increased local concentration of peptides, rather than surface-imposed conformations,
give rise to the surface-formed brils. This assembly hypothesis
also provides a kinetic explanation for why bril formation at
the surface occurs more rapidly than in solution, as the
increased surface concentration of peptides overcomes the
critical concentration required for brillation to occur. Once
brils have begun forming, they may elongate through simple
monomer addition of non-surface-bound peptides and they
may also fragment, creating sites for secondary nucleation.33
The exact nucleation mechanism by which the peptides selfassemble is unclear at this point, but is likely inuenced by
peptide sequence and interactions with the solution/SAM
interface, as well as the solution environment. Previous studies
have shown how the conformation of immobilized peptides is
inuenced by interactions between the tethering medium,
unbound portions of the peptide, and interface chemistry, as
well as solution chemistry.6264 The method by which the
particular peptides discussed here nucleate and elongate to
form brils is unknown, but studies are ongoing in our
laboratory to investigate how this complex environment
dictates this surface-mediated self-assembly mechanism.
The formation of these brillar structures on the surface is an
important step in understanding the factors that dictate the selfassembly of small biomolecules. Fibrillar structures arise from
strands of -sheets interacting to form larger brils, where
individual strands are oriented perpendicular to the long axis of
the bril.19,21,33 Often, these brils are reported as having
diameters ranging from 3 to 20 nm, although it has been
demonstrated that twisting of the brils can lead to the
formation of helical ribbon structures that resemble nanotubes,
which can have diameters up to 160 nm.3,30,37,65 It has been
shown that sequence symmetry may favor antiparallel over
parallel sheet assembly of individual -strands, and examination
of the distribution of polar/nonpolar (P/N) binary patterns of
pentapeptide sequences found in the Protein Data Bank found
that NPNPN and PNPNP have the highest relative frequency
among antiparallel -strands.30,66 Therefore, the symmetric,
alternating aliphatic sequence of the 6 peptide is known to
have a high propensity for orienting into antiparallel -sheets
that proceed to self-assemble into structures within the size
range of commonly characterized brils. Further characterization of these brillar structures is ongoing in our lab and will

ASSOCIATED CONTENT

S Supporting Information
*

AFM of bare gold and SAM-covered gold (Figure S1); AFM of

brils formed in solution (Figure S2); and GRAS-IR, CD, and
AFM of the 61X peptide studies (Figures S3S6). This
material is available free of charge via the Internet at http://
pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*E-mail: lwebb@cm.utexas.edu.
Notes

The authors declare no competing nancial interest.

ACKNOWLEDGMENTS
The authors are grateful for funding from the NSF (Grant No.
CHE-1361252), the Army Research Oce (Grant No.
W911NF-10-1-0280), and the Norman Hackerman Advanced
Research Program. L.J.W. holds a Career Award at the Scientic
Interface from the Burroughs Wellcome Fund and is an Alfred
P. Sloan Foundation Research Fellow. We thank the Texas
Institute for Drug and Diagnostic Development (TI3D)
Automation Facility for use of their circular dichroic
spectrometer, the Texas Materials Institute for the support of
the clean room facility, the Center for Nano- and Molecular
Science and Technology (CNM) for the support of the Asylum
Research AFM, and the National Science Foundation for
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funding the Kratos X-ray photoelectron spectrometer (Grant

No. 0618242).

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DOI: 10.1021/la5049369
Langmuir 2015, 31, 34413450