Self-assembly of Au Nanoparticle-containing Peptide Nano-rings on Surfaces

Abstract

The peptide nano-rings containing Au nanoparticles inside their cavities were self-assembled on dithiol SAMs patterned as an array by AFM-based nanolithography. The peptide nano-rings were aligned as a line on these SAMs, and Au formed lines with the spacing between these nanoparticles as the peptide nano-rings functioned as spacers. This type of array fabrication will provide improved tunability in their optical properties of resulting nanoparticle-assembled arrays. In addition, optimization of the inter-particle distance of nanoparticles in the array with various spacers may allow one to design new types of photonic crystals with desired optical properties.

Full text

Self-assembly of Au Nanoparticle-containing Peptide Nano-rings
on Surfaces
NURXAT NURAJEa, KAI SUb, JACOPO SAMSONa, AMIT HABOOSHEHa, ROBERT I.
MACCUSPIEa, HIROSHI MATSUIa,*
aDepartment of Chemistry, Hunter College and the Graduate Center, City University of New York,
New York, NY 10021, USA
bDepartment of Chemistry, College of Staten Island, Staten Island, NY 10314, USA
Abstract
The peptide nano-rings containing Au nanoparticles inside their cavities were self-assembled on
dithiol SAMs patterned as an array by AFM-based nanolithography. The peptide nano-rings were
aligned as a line on these SAMs, and Au formed lines with the spacing between these
nanoparticles as the peptide nano-rings functioned as spacers. This type of array fabrication will
provide improved tunability in their optical properties of resulting nanoparticle-assembled arrays.
In addition, optimization of the inter-particle distance of nanoparticles in the array with various
spacers may allow one to design new types of photonic crystals with desired optical properties.
Keywords
Peptide nano-rings; Bionanotechnology; Nanofabrication; Self-assembled monolayers
INTRODUCTION
Recent improved two-dimensional and three-dimensional nanofabrication techniques allow
one to build precisely designed structures in nanoscale for various photonic applications [1–
3]. While the top–down approach has been applied for photonic crystal fabrications, the
bottom– up approach via self-assembly of photonic nano-building blocks is also showing
promising outcomes [4–12]. For the bottom–up approach for the optics fabrications,
synthesis of photonic nanomaterials and their alignments need to be accomplished efficiently
and precisely.
Biomineralization process, where peptides or proteins are utilized to mineralize metals and
semiconductors, has been shown to produce various types of nanocrystals [13–25]. Since the
amino acid sequences are very sensitive to elements for their mineralization, optimized
peptide sequences can produce nanocrystals efficiently [26]. In addition to the effective
crystal growth, the amino acid sequences of mineralizing peptides could also influence the
size, the alignment, and the shape of resulting nanocrystals [27–30]. Further more, in some
*Corresponding author. hmatsui@hunter.cuny.edu.
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cases, peptides could mineralize nanocrystals in solution at room temperature that do not
grow under the ambient condition [31,32]. Because the size, the alignment, the shape, and
the crystalline structure of photonic nanocrystals control optical properties of those
assembled nanocrystals, the tunabilities of these features and the potential of new material
synthesis by using peptides will provide a significant advantage to apply peptides for
photonic material syntheses.
The one of smart approaches to create photonic materials by using mineralizing peptides is
to pattern these peptides on surfaces based on photonic device designs and grow photonic
crystals on the patterned peptides. For example, a hologram was applied to pattern silica-
mineralizing peptides as an array and resulting silica nanostructures exhibited a nearly fifty-
fold increase in diffraction efficiency over a comparable polymer hologram [14]. Recently,
microfluidics and lithography were also used to pattern silica- and silver-mineralizing
peptides for photonic applications [28,33].
In this report, we assembled ring-shaped peptide nanostructures as an array on surfaces.
Previously, we developed the ring-shaped peptide nanostructures by self-assembling a
peptide monomer, bis(
N
-α-amido-glycylglycine)-1,7-heptane dicarboxylate and an organic
Au precursor, trimethylphosphinegold chloride (AuPMe3Cl) in solution [34]. After reduction
of Au ions trapped inside the cavities of nano-rings, the peptide nano-rings could template
Au nanoparticles. In this report, after Au nanoparticles were grown inside the cavities of the
peptide nano-rings, these nano-rings containing Au nanoparticles were aligned on the
chemically functionalized arrays patterned by nano-lithography (Fig. 1). Since these peptide
nano-rings were self-assembled in a closely packed manner along the array of those
dithiolated self-assembled monolayers (SAMs), these Au nanoparticles were positioned in
the equal spacing on each line without touching each other as the peptide nano-rings
functioned as spacers. This type of alignment of nanomaterials with spacers can be very
useful for improved photonic crystal designs.
RESULTS AND DISCUSSION
When the peptide monomers were self-assembled in the presence of the water-insoluble
trimethylphosphinegold chloride (AuPMe3Cl) for 5 days in the dark, the ring-shaped peptide
assemblies were observed in solution. The average outer diameter of nano-ring was 50 nm
and the average inner diameter was 15 nm [34]. Our previous spectroscopic investigation
showed that these peptide nano-rings were self-assembled from the peptide monomers and
the organic Au salts by chelating Au with amide groups of the peptide monomers. After UV
light was irradiated to the nano-ring solution for 20 min Au nanoparticles were grown inside
the cavities of nano-rings, however the organic Au salts, trapped inside the cavities, were
reduced to grow Au nanoparticles in the middle of nano-rings [34,35]. In a TEM image of
the nano-ring after reduction of Au ions, the Au nanoparticle appeared darker at the center
of the nano-ring (Fig. 2a). These particles in the cavities were also confirmed as Au
nanoparticles by electron diffraction before their surface assembly on the dithiol SAMs.
When the peptide nano-rings were previously synthesized, they were observed to be stable
in solution [34] however these nano-rings have not been assembled on surfaces. Therefore, it
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was necessary to examine their stability via the simple surface-assembly. Before fabricating
the structure shown in Fig. 1, we examined whether the Au nanoparticle-containing peptide
nano-rings are rigid enough to be self-assembled on surfaces by spin-coating them on TEM
grids. A height image of AFM in Fig. 2b confirms that the nano-rings were deposited on a
TEM grid. This figure imaged the assembly of the nano-rings on the surface, but the Au
nanoparticles inside the nano-rings could not be resolved in this AFM height image.
However, when this assembly was imaged by the phase mode of AFM as shown in Fig. 2c,
the Au nanoparticles were observed as a brighter contrast inside the nano-rings because
harder surfaces of Au nanoparticles appeared brighter than softer surfaces of the outer
peptide nano-rings. In the magnified phase image of Fig. 2d, these Au nanoparticles were
not exactly located at the center of the nano-rings, which may be due to the deformation of
the nano-ring shape via spin-coating and the multiple orientations of the nano-rings on TEM
grids.
Since the peptide nano-rings were stable enough to be assembled on TEM grids via spin-
coating, we examined the targeted self-assembly of the peptide nano-rings on the
functionalized surfaces. The functionalized array of dithiol SAMs was patterned by three
steps. First, alkyl SAMs were deposited on a Au substrate and the array was patterned by
removing the alkyl SAMs with the AFM cantilever. Then, mercaptohexadecanoic acid was
assembled on the curved array where Au surfaces were exposed. The carboxylic groups on
the top of these SAMs on trenches were substituted by thiol groups, as shown in Scheme 1.
To align the peptide nano-rings containing Au nanoparticles on the dithiol SAM array
patterned on a Au substrate as shown in Fig. 1, these nano-rings were incubated with this
substrate in solution for 8 h. Because the end groups of the SAMs on the trenches were
functionalized by thiol (Scheme 1), this incubation process allowed the nano-rings to self-
assemble onto these trenches with the thiol-Au interaction, as shown in Fig. 3. Figure 3a is
the AFM image of the patterned substrate in the height mode after the nano-rings were
incubated in the substrate-containing solution. The peptide nano-rings were closely packed
to form the array of continuous lines along the trenches in Fig. 3a. As seen in the height
mode AFM image of the spin-coated samples in Fig. 2b, the individual Au nanoparticle
inside the nano-ring could not be identified in Fig. 3a due to their small height difference
between the peptide nano-ring and the Au nanoparticle. However, when the phase mode of
AFM was applied to image this substrate, the deposited spheres on the trenches looked less
continuous in Fig. 3b since the brighter spheres in this phase image are more likely Au
nanoparticles due to their surface hardness as compared to the one for the peptide nano-
rings. While these AFM images in the phase mode in low resolution do not explicitly show
the discrete positioning of Au nanoparticles inside the nano-rings aligned on the trenches as
shown in Fig. 1, the high-resolution image of the single trench containing the peptide nano-
rings in the phase mode assisted visualizing the peptide nano-ring alignment more clearly.
When the single trench was imaged with the height mode image in high-resolution (Fig. 3c),
still only continuous packing of the nano-rings was observable as a line along the trench and
the discrete Au nanoparticles could not resolve. But when the single trench was imaged in
the phase mode in high-resolution, the discrete alignment of Au nanoparticles was observed,
as shown in Fig. 3d. In this high-resolution image, the harder Au nanoparticles appeared to
be brighter and the spacing between these nanoparticles was visible. The most of Au
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nanoparticles in Fig. 3d were self-assembled discretely without touching each other due to
the spacing of the peptide nano-rings (Fig. 1). The darker nano-ring contrast around the Au
nanoparticle, observed in the spin-coated sample in Fig. 2b, was not observed clearly in Fig.
3d, however this phase contrast difference between the spin-coated nano-rings and the self-
assembled nano-rings may be caused by their topological difference. For the spin-coated
sample the nano-rings were forced to pack closely by the external force on very smooth and
flat TEM grids, and the resulting nano-ring monolayer also became relatively flat.
Therefore, the detailed structure of nano-rings and Au nanoparticles was resolved in the
AFM image of the spin-coated nano-ring sample due to the flatness of the coating. However,
for the self-assembled sample, the nano-rings were self-assembled on rough surfaces
consisting of alkyl SAMS and dithiol SAMs in different heights. The trenches were also not
flat because the AFM cantilever could not shave alkyl SAMs smoothly and it further scraped
the Au substrate partially. Shaved SAMs and Au substrate were piled at the edges of
trenches, which also increased the roughness of the substrate. Since all different heights of
SAMs, trenches, and edges contributed unevenness of the surface topology, the AFM
resolution in the phase image on this self-assembled sample was degraded due to its surface
roughness and it prevented to map the locations of the nano-rings and Au nanoparticles in
the phase image. When the neat Au nanoparticles without the peptide nano-rings were
assembled on the same dithiol-functionalized trenches, those Au nanoparticles were aligned
as continuous lines without spacing in their phase AFM image. This observation supports
that the Au nanoparticles observed in Fig. 3d were separated by the peptide nano-rings
otherwise the spacing between Au nanoparticles should not be imaged. It should be noted
that some of these nanoparticles seem to contact each other in Fig. 3d due to the deformation
of the nano-rings during the self-assembly, which was also observed in the spin-coated
sample in Fig. 2.
CONCLUSION
The peptide nano-rings containing Au nanoparticles inside their cavities were aligned on
dithiol SAMs patterned as an array by AFM-based nanolithography. The peptide nano-rings
were self-assembled as lines on these SAMs, and Au nanoparticles inside the nano-rings
also formed lines with the spacing between these nanoparticles as the peptide nano-rings
functioned as spacers. This type of fabrication will provide improved tunability in their
optical properties of resulting nanoparticle-assembled arrays. In addition, optimization of the
inter-particle distance of nanoparticles in the array with various spacers may allow one to
design new types of photonics with desired optical properties. However, for those optical
applications, the stability and the rigidity of the nano-rings will be more desirable to be
improved since some of the nano-rings were deformed during the self-assembly and this
deformation caused the uneven spacing of the nanoparticles on the array. For realistic
photonic applications, the nanoparticle assembly needs to be accomplished without
deformation because they require the perfect alignment of nanoparticles with minimum
defects. The additions of hydrogen-bonding functional groups or polymerizing groups to the
monomer may increase the rigidity of the nano-rings, which may help overcome this
problem.
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EXPERIMENTAL
Materials
Bis(
N
-α-amido-glycylglycine)-1,7-heptane dicarboxylate was synthesized and recrystallized
in our lab by the published manner [36,37]. 1-Ethyl-3-[3-
(dimethylamino)propyl]carbodiimide hydrochloride (EDAC),
N
-hydroxysuccinimide(NHS),
2-mercaptoethylamine, trimethylphosphinegold chloride, 16-mercaptohexa-decanoic acid
and octadecanothiol from Aldrich. Annealed gold substrates from Molecular Imaging. A
series of trenches (100 nm × 1 µm) were made by shaving the alkylthiol SAM with a Si3N4
tip (Veeco Metrology) of the AFM (Nanoscope IIIa and MultiMode microscope, Digital
Instruments). These trenches were made by customized Nanoscript software
(VeecoMetrology). Formvar Film 200 Mesh Cu TEM grids were obtained from Electron
Microscopy Sciences. UV-Lamp (14 mW/cm2, 254 nm).
Preparation of Peptide Nano-rings
After peptide monomer of bis(
N
-α-amido-glycyl-glycine)-1,7-heptane dicarboxylate, 0.028
g, was dissolved in 10 mL of water and the pH of this solution was adjusted to 5.5 with citric
acid, an excess amount of an organic Au precursor, trimethylphosphinegold chloride
(AuPMe3Cl) was added to this solution. After 5 days in the dark, the peptide nano-rings
were observed in an outer diameter of 50 nm and an inner diameter of 15 nm. The nano-ring
solution was washed with deionized water and centrifuged at 14.5 krpm, and then Au ions
trapped inside the peptide nano-rings were reduced by a UV light (14 mW/cm2, 254 nm) for
20 min. The resulting Au nanoparticles were observed to be about 15 nm in an average
diameter from AFM images.
Nanolithography on Au Substrates
In order to pattern the peptide nano-rings containing Au nanoparticles in their cavities as an
array on Au substrates, an array of dithiolated SAMs was patterned by a cantilever of an
atomic force microscope (AFM) with the following sequence. First, 1-octadecanethiol (0.01
mM) was self-assembled on Au substrates in 99% ethanol at room temperature for 12 h.
Then, an array of trenches (100 nm × 1 µm) was created by removing the alkylthiol SAMs
by the tip of AFM via nanolithography technique [38–40]. On these trenches where Au
surfaces were exposed, 16-mercaptohexa-decanoic acid (0.01 mM) was self-assembled for
overnight. After the resulting substrates were washed by deionized water, the end groups of
the SAMs on trenches were functionalized by thiol via substituting carboxylic acid groups
with thiol groups as shown in Scheme 1. In Scheme 1, 400 µL of ethyl-3-[3-
(dimethylamino)propyl]carbodiimide (EDAC, 75 mM) and 400 µL of
N
-hydroxy
succinimide (NHS, 15 mM) were immersed in aqueous solution containing the
functionalized Au substrates for 30 min. Then, as shown in the step 3 in Scheme 1, 800 µL
of 2-mercaptoethylamine (15 mM,) was incubated in the solution for 24 hrs to modify the
ends groups of the SAMs in trenches to thiol groups [32,41].
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Self-assembly of Au Nanoparticle-containing Peptide Nano-rings on Surfaces
When the peptide nano-rings containing Au nanoparticles in their cavities were mixed with
the dithiolated SAM-patterned surfaces in aqueous solution for 8 hrs, the nano-rings were
self-assembled on the trenches with the thiol-Au interaction. After these substrates were
washed with deionized water, the attachment of the peptide nano-rings on the arrayed
trenches was confirmed by AFM. While the height mode of AFM imaging was used to
probe the topology of the nano-ring assembly, the phase mode of AFM imaging was applied
to probe the location of Au nanoparticles since the distinguished hardness of surfaces
between Au nanoparticles and peptide nano-rings allows us to image them respectively in
the trenches.
We also assembled the peptide nano-rings containing Au nanoparticles on TEM grids by
spin-coating to examine whether the nano-rings can be packed on surfaces without
distraction. To obtain smooth surfaces of TEM grids, these grids were fixed on the top of
mica substrates, which were also attached on AFM metal pucks. After the Au nanoparticle-
containing peptide rings were spin-coated on the TEM grids, the resulting coatings were
examined by AFM.
Acknowledgement
This work was supported by the US Department of Energy (DE-FG-02–01ER45935) and the National Science
Foundation CARRER Award (ECS-0103430). Hunter College infrastructure is supported by the National Institutes
of Health, the RCMI program (G12-RR-03037).
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FIGURE 1.
Illustration of Au nanoparticle-containing peptide nano-ring assembly on the patterned Au
substrate.
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FIGURE 2.
(a) TEM image of the peptide nano-ring containing a Au nanoparticle inside the cavity, scale
bar = 50 nm. (b) AFM image of spin-coated peptide nano-rings containing Au nanoparticles
in their cavities on TEM grids in height mode, scale bar = 80 nm (c) AFM image of spin-
coated peptide nano-rings containing Au nanoparticles in their cavities on TEM grids in
phase mode, scale bar = 80 nm (d) the phase AFM phase image in high magnification, scale
bar = 40 nm.
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FIGURE 3.
AFM images of Au nanoparticle-containing peptide nano-rings assembled on the dithiol
SAM-patterned in (a) height mode, scale bar = 300 nm (b) phase mode, scale bar = 300 nm
(c) height mode in high magnification, scale bar = 50 nm, (d) phase mode in high
magnification, scale bar = 50 nm.
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SCHEME 1.
Fabrication of dithiol SAMs on patterned Au substrates.
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