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Towards Selective Functionalization of OWL Nanostructures Using Simple Wet-Etchant
Techniques
Undergraduate Researcher
William A. Gallopp
Skidmore College, Saratoga Springs, NY
Faculty Mentor
Prof. Chad A. Mirkin
Department of Chemistry
Northwestern University
Postdoctoral Mentor
Dr. Matthew J. Rycenga
Department of Chemistry
Northwestern University

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Abstract
A novel approach to selectively functionalize a specific face on a nanorod’s surface with molecules is
proposed in this paper. These Au nanorods are synthesized using on-wire lithography (OWL) and have a
multi-segmented Au-Ni-Au composition (with lengths Au: ~100 nm, ~200 nm, Ni: ~80 nm, and a
diameter of: ~45 nm). The elemental segments of the nanorod can be used to mask specific segment
faces, which can then be selectively functionalized. This method is based upon the protection of the
exposed Au surfaces with a hydrophilic self-assembled monolayer (SAM) such as mercaptohexadecanoic
acid (MHA). The SAM provides a chemical barrier that protects the Au surface from etching solutions
which are used to remove the Ni segment. After removal of the Ni segment, an unfunctionalized Au
surface that was in proximity to the Ni is exposed. This Au surface can then be selectively functionalized
by a variety of molecules. In this paper, a biotinylated SAM is used and then incubated with streptavidin
coated FeO3 nanoparticles (10 nm in diameter) to demonstrate and optimize this unique approach toward
selective functionalization.
Introduction
The unique fundamental properties of nanostructures allow for their potential usefulness in a
variety of technologies, including sensing1
, plasmonics2-3
, biomedical therapy4
and imaging4
. The size,
shape, composition and morphology of these nanostructures are parameters that can be used to tune their
unique properties for a specific application5
. As a result, the interest in the control over the synthesis and
assembly of nanostructures has intensified in the science and engineering communities6,7
. Specifically
nanorods and nanowires have received a great deal of attention due to their unique optical properties and
high aspect ratios. Nanorods can be created through many techniques such as aqueous solution phase8
,
chemical vapor deposition9
, molding, printing and nanolithography10
. However, fine control over these
structures is difficult to achieve with these methods6-10
.

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In 2005, a new method, known as on-wire lithography (OWL), shown schematically in Figure 1,
was developed for controlling the architectural design of nanorods, by incorporating advances in wet-
chemical etching, template directed nanorod synthesis, and electrochemical deposition. OWL has
provided a powerful, simple method for constructing complex nanostructures. In particular, it is an
excellent technique for creating small dielectric gaps between metal segments on a nanorod, and therefore
is a highly successful procedure for fabricating nanorods for sensing application which take advantage of
the unique optical properties of the gap nanostructure11
.
By generating rods with greater complexity (those consisting of multiple elemental compositions
and gaps) scientist and engineers have been able to create new materials for applications such as detection
devices and therapeutic modalities. Consequently, new synthetic challenges have emerged to meet the
requirements of these new applications. Parameters such as diameter, length, and composition of such
structures are major factors that dictate the properties of nanorods. By developing methods to control
these dimensions we can fabricate nanorods with greater flexibility and multiple applications6
.
In addition, another challenge is selective functionalization which is the ability to control the
molecular coverage on a nanoparticels’ surface. For the case of multi-segmented nanorods, segments with
different elemental compositions show selectivity toward different chemical molecules12
. This can be
used advantageously to bind specific molecules to a particular region on the nanorod surface and will etch
in acidic conditions at different rates12,13
. By using etchant techniques to create clean surface suitable for
functionalization, this paper aims to show that a bimetal (i.e. Au-Ni-Au) OWL nanostructure, with ~35nm
in diameter can be selectively functionalized on one face of a Au segment.

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Background
Conventional top down methods for fabricating nanorods include electron beam lithography10
,
dip pen nanolithography14
, focused ion beam lithography10
and nanoimprint lithography15
. Despite their
strong capabilities and attributes, these techniques are limited in regards to material complexity, cost, and
resolution10,11
. These lithography methods also do not provide an excellent method to make nanorods with
multiple segments or dielectric gaps between metal segments. Manufacturing such structures by
conventional techniques proves to be inconsistent, and controlling aspects such as break gap and gap
narrowing are nearly impossible12,16
. OWL has proven it is capable of fabricating nanorods while also
being able to control their diameter, length, composition, and gap size down to ~2 nm.
Since its discovery, OWL has been used for investigating a variety of phenomena that occur at
the nanoscale level. For example, control over nanorod dimensions and gap structure (in terms of size and
surface roughness) can be used to optimize their optical properties, for biodiagnostics and light
manipulation technology6
, encoding17
,and devices to study charge transfer in molecular systems18
. OWL
has also scientist to control the geometric parameters affecting surface-enhanced Raman scattering
(SERS)19
. Such gap structures would greatly benefit from selective functionalization as it would localize
molecules of intrest at the gap region, were near-field enhancements can boost the signal of relevant
molecules for easy identification and detection. When the gap of a dimer is functionalized, it makes it
easier to detect any molecule and can increase the sensitivity and limit of detection. Selective
functionalization could be key to creating powerful sensing and detection devices6,20
.
Approach
Fabricating Nanorods By Electrodeposition
Nanorods were fabricated in a manner similar to that of Banholzer et al6
(Figure 3). For this
project a Au-Ni-Au nanorod will be fabricated. Segments of the nanorod will be created through
electrodepositon, where a metal salt precursors are reduced converting it to their elemental form. A

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alumina template was used to fabricate the nanorods. The template will provide the rod morphology that
is desired (Figure 3). Prior to nanorod fabrication, a thin layer of Ag was evaporated on to the alumina
template. This acts as a backing layer and seals the pores. To achieve the Au-Ni-Au nanorods, different
solutions containing metal salt precursors were electrochemically deposited into a porous alumina
template. Each segment length can be tailored by controlling the charge passed during the
electrodeposition process. The sample is loaded in to a electrochemical cell and connected to a
potentiostat by a three electrode set up similar to that in Figure 3. The potentiostat is connected to a
computer which allows control over the parameters for fabricating the nanorod.
First, a Ag segment was electrodeposited (applied potential: -940 mV, Charge limit: 1000 mC) to
provide a smooth uniform surface for the following segments to grow on. Next, a Ni segment was added
(applied potential: -1100 mV, charge limit: 250 mC), because Au and Ag are very similar and will
exchange electrons resulting in rough nanorods. For this experiment Au-Ni-Au nanorods with one Au
segments of ~100 nm and one ~200 nm segment, Ni segments of ~80 nm and a diameter of ~45 nm were
fabricated (Figure 4). Au segments were deposited using an applied potential of -1000 mV, charge limit
of 7 mC and had the current passed through 20 times. The Ni segment was deposited using applied
potential of -1100 mV and a charge limit of 70 mC6
.
Determining Etchant Parameters for Ni: Unprotected Nanorods
One significant parameter the project aimed to optimize was the etch time needed to remove Ni
with HCl. This is important as it will maintain the Au segment’s morphology while the Ni segment is
being etched. Additionally, the etching occurs after the rods have been coated with MHA (Figure 2B).
The determined parameters will provide the most efficient and most gentle etching conditions. For our
purposes, the Ni segment will be etched in order to expose a clean, uncoated Au surface, which will later
be functionalized using the biotinylated SAM (Figure 2C). Several Experiments were used to determine
the appropriate etchant parameters.

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(Experiment 1)Monitoring Etchant process By SEM To Determine Lowest Etchant Concentration
Several HCl concentrations were used to determine the optimized etchant parameters. A 2.77 M
HCl solution was used to completely etch the Ni from the structures. 10 μL of 2.77 M HCl solution was
added to 10 μL sample of nanorods. SEM samples were prepared over a 3 hour time interval. A sample
was prepared after five minutes and then every 20 minutes until 3 hours elapsed. The process was
repeated using 0.277 M, 27.7 mM 2.77 mM and 0.277 mM HCl solutions. SEM samples were prepared
after five minutes of etching time. These SEM images would reveal whether or not the HCl
concentrations were strong enough to etch the Ni segment in the unprotected rods.
(Experiment 2)Determining Time Frame for Etching Process Using UV-Vis: Unprotected Rod
A Cary 5000 UV-Vis-NIR spectophotometer was used to monitor the etching process of Ni over
a selected time interval. Before monitoring the etching process the absorbance spectrum of unetched,
unprotected (not coated with MHA) nanorods and known etched nanrods were taken. These spectrums
provided a starting point, where the rods are unetched, and an endpoint, where the rods are etched. A 100
μL unetched nanorod sample was placed in a cuvette and loaded into the spectrometer. A UV-Vis
spectrum was collected from 1500 nm to 200 nm. The process was repeated for the known etched
nanorod sample. The maximum peak in the unetched spectrum represents the local surface plasmon
resonance (LSPR), which is the oscillation of valence electrons throughout an entire solid sample
stimulated by light. It is expected that the LSPR will shift as a result oscillation through a shorter rod.
This shift will effectively tell that the nanorods have been successfully etched. The procedure included
taking 5 μL of 0.277 mM HCl solution which was added to a 100 μL nanorod sample. Such a small
amount of acid was used so that the sample would not be significantly diluted. A UV-Vis spectra was
recorded every 5 minutes for ~ 60 minutes to determine the etch time.

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(Experiment 3) Monitoring Etching Process By UV-Vis: MHA Coated Rods
To functionalize he nanorods with MHA, first a sample of nanorods was centrifuged (8000 rpm,
10 min) and the supernatant was removed. The nanorods were resuspended into a 500 μL 1mM MHA
solution and functionalized for ~24 hrs (Figure 2A). Although the nanorods are now coated with MHA
the SAM the MHA coating is not perfect and has deficiencies and holes, which allow the Ni segment to
be etched. A 100 μL MHA coated nanorod sample was placed in a cuvette and loaded into the
spectrometer. A UV-Vis spectrum was collected from 1500 nm to 200 nm. 5 μL of 0.277 mM HCl
solution was added and the nanorod sample and a UV-Vis spectrum was recorded every 5 minutes for
~30 minutes. A small amount of HCl was used to avoid changing the concentration of the sample. This
experiment was repeated to confirm that the 0.277 mM HCl concentration was strong enough to etch the
MHA coated nanorods.
TEM and Energy Dispersion Spectroscopy (EDS) Analysis Of Biotin/ Streptadvin FeO3 Coupling
To determine the success of the selective functionalization technique, a biotin/streptadvin
coupling scheme was used15
. Transmission electron microscopy (TEM) images and elemental mapping
(EDS) were used in an attempt to show that one face of Au rods has been selectively functionalized and
that this occurred for a majority of the sample (> 50%). In a typical procedure a sample of nanorods was
centrifuged and the supernatant removed (8000 rpm, 10 mins).Then a ~100 μL 1mM biotinylated SAM
solution was added to the nanorods for ~24 hrs (Figure 2C). After the 24 hrs the sample was centrifuged
and the supernatant removed (8000 rpm, 10 mins). Then ~100 μL of PBS 7.4 pH solution was added to
the rods followed by ~10 μL of a 1:1000 diluted streptavidin/FeO3 nanoparticles (Figure 2D). The sample
was finally analyzed using TEM and EDS to confirm the selective functionalzation process.

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Results and Discussion
Etchant Parameters
Optimizing the etchant parameters is of high importance as it will allow us to etch the Ni segment
in the shortest time without deforming the nanorod or destroying the MHA SAM on the Au surface. First,
the lowest HCl solution possible of etching the nanorods was determined and confirmed through SEM.
SEM imaging reveled that the 2.77 M HCl solution completely etched the Ni segment after 5 min,
however after 180 min of exposure the structure began to deform and degraded (Figure 5A-B). As a
result, less concentrated HCl solutions were used and observed in a 5 min time frame. SEM’s of the
nanorods using 0.277 M, 27.7 mM 2.77 mM and HCl solutions revealed that all structures were
completely etched (Figure 5C-E). However, the 0.277 mM HCl solution did not etch the nanorods (Figure
5F). It was determined that the 2.77 mM HCl solution was the optimized HCl concentration to etch
unprotected nanorods.
After determining the lowest etchant HCl solution, the time frame was then determined using
UV-Vis spectroscopy. UV-Vis will effectively show the starting point and endpoint of the etching
process. By monitoring the etching process and determining when the end point has been reached, an
optimized etch time can be determined. By comparing the etched and unetched nanorod, UV-Vis
spectrum it can be seen that the LSPR peak at 1287 nm (for the unetched rods) has blue shifted to 870 nm
(for the etched rods), which was expected. (Figure 6A). SEM was used to confirm the rod UV-Vis
spectrums (Figures 6B-C). As a result, the LSPR at 1287 nm was monitored throughout the etching
process. The 2.77 mM HCl solution was used to etch the structures. Figure 6D shows that throughout the
etching process that the peak intensity slowly declined and begins to level out. However, when using the
same etchant solution on the MHA coated nanorods the LSPR at 1287 nm did not steadily decline and
appears to be constant during a 30 min time period (Figure 6D). SEM was used to confirm the UV-Vis
spectrum of the etched and unetched rods. (Figure 6E-F).The optimization process which involved SEM

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and UV-Vis analysis, determined that the best etchant conditions for MHA coated nanorods were a 27.7
mM HCl solution for approximently 1.5 hours. Previous experiments used HCl concentrations of 6 M,
which are two orders of magnitude greater than the optimized conditions. Figure 7 shows images of MHA
coated nanorods that have been etched successfully using the optimized etchant conditions.
EDS Characterization of Biotin/Iron Oxide Streptadvin Coupling
Since the Au surface was coated with the MHA protecting group before the etching process
occured, once the Ni has been etched a clean unprotected Au surface will be exposed. This unprotected
Au surface can then be functionalized with a biotinylated thiolate-SAM. After the nanorod was
functionalized with biotin functional group, streptavidin coated FeO3 particles were added to the rods.
Biotin and streptavidin have a high affinity toward one another and will couple readily when in the
presence of each other21
. From TEM imaging it was determined that coupling between the biotin and
streptavidin was fairly low, < 20% (Figure 8). Although Fe can be detected through EDS the data is
inconclusive due to the high levels of Fe throughout the sample (Figure 8). The next step is to optimize
the coupling between biotin and streptavidin and wash the nanorods to wash away excess Fe particles to
obtain more conclusive
Conclusion
In this paper a proposed systematic approach to selectively funtionalize multi-segmented
nanorods has been proposed. The approach involved: 1) fabricating Au-Ni-Au rods; 2) applying an etch
resistant coating; 3) etching of the Ni segment; 4) functionalization using a biotinylated SAM; and 5)
decorating the functionalized rod with streptavidin coated FeO3 nanoparticles. The etchant parameters
were determined to be a 27.7 mM HCl solution for 1-1.5 hrs, which provided the fastest and most gentle
etching conditions. However, TEM imaging showed that the coupling between the biotinylated SAM Au
surface and streptavidin coated FeO3 nanoparticles was a low < 20%.

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Future work for this project includes optimizing the coupling between biotin and
streptavidin/FeO3 nanoparticles to obtain more conclusive confirmation of selective functionalization.
Once selective functionalization is confirmed a silica backing will be applied to the Au-Ni-Au rods and
then etched to create Au dimer with a selectively functionalized gap. If successful, the nanorods will be
functionalized with other molecules like DNA for detection and assembly.
Acknowledgments
This research was supported primarily by the Nanoscale Science and Engineering Research Experience
for Undergraduates Program under National Science Foundation award number EEC – 0647560. Any
opinions, findings, conclusions, or recommendations expressed in this material are those of the authors
and do not necessarily reflect those of the NSF. The author would also like to thank Dr. Matthew Rycenga
for his mentorship and guidance through the experimental and writing process. He would also like to
thank Dr. Chad Shade and Dr. Gilles Bourret for their support and assistance in fabricating the nanorods.
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Figure 1. The On-Wire Lithograph process. Rods are first systhesized through electrochemical synthesis. The nanowires
are then dispersed onto a glass slide and coated with a layer of silica. The nanorods are then released from the glass by
means of sonication. The final step involves using selective chemical etchant to remove the Ni segments using HCl. The
resulting nanowires will only consist of Au bridged by silica and the desired gap spacing resulting from the etched Ni.

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Figure 2. A strategic approach towards selective functionalization. A) Rods are coated with a protective etch resistant
hydroxyl thiol known as mercaptohexadecanoic acid (MHA). B) The next step involves the etching of the Ni segment.
Although MHA is an etch resistant, MHA does not completely incase the structure and is formed with deficiencies. This
allows for the Ni segment to be etched. C) The etching reveals an uncoated Au surface which is then functionalized with a
biotinylated SAM. D) The biotinylated SAM is then decorated with streptavidin coated FeO3 nanoparticles. Biotin and
streptavidin have a strong affinity for one another and will readily couple together. Detection of this coupling will show
that selective functionalization has been achieved.

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Figure 3. Nanorod fabrication using electrochemical deposition. To obtain nanorods of specific composition, metal salt
precursors were electrochemically deposited onto a porous alumina template. Each segment length can be tailored by
controlling the charge passed during the electrodeposition process. First thin layer of Ag is evaporated on to the alumina
template to seal the pores of the template which is a platform where the metal segments can grow. The template is added
to an electrochemical cell and connected to a potentiostat by a standard three electrode setup. A metal salt precursor is
then added to the cell and a controlled current is applied which deposits the metal segment, the template is then rinsed.
This process can be repeated using different metal salt precursor solutions until the desired rod has been fabricated.

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Figure 4. SEM image of fabricated rods. Au segments: ~100 and ~200 nm in length. Ni segment: ~80 nm in length. Length
of entire nanorod: ~380 nm. Diameter: ~45 nm

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Figure 5. SEM image of HCl etched unprotected rodsA) Rods after etching in 2.77 M HCl solution for 5 minutes. B) Rods
after etching in 2.77 M HCl solution for 180 minutes. C) Rods after etching in 0.277 M HCl solution for 5 minutes. D)
Rods after etching in 27.7 mM HCl solution for 5 minutes. E) Rods after etching in 2.77 mM HCl solution for 5 minutes.
F) Rods after etching in 0.277 mM HCl solution for 5 min.
F)
B)A)
E)
C) D)

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Figure 6. A) UV-Vis spectra of unprotected unetched and etched rods. B.) EM image of unetched rods used for UV-Vis.
C.) SEM image of etched rods used for UV-Vis. D) Peak intensity at 1287nm during the etching process of unprotected
and MHA coated rods using a 2.77 mM HCl solution. E) EM image of unortected rods after etching process. F) Image of
MHA coated rods after etching process with 2.77mM HCl solution.

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Figure 7. SEM Images of etched MHA coated nanorods using 100μL of 27.7mM HCl solution for 1 – 1.5 hrs.

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Figure 8. TEM and EDS analysis of biotinylated functionalized nanorods coupled with streptavidin FeO3 nanoparticles.