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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, plasmonics

2-3, biomedical therapy

4 and imaging

4. 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 nanolithography

10. 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, encoding

17 ,and

devices to study charge transfer in molecular systems

18. 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.