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PROGRESS REPORT
Ligand Exchange on Gold Nanorods: Going Back
to the Future
A. Swarnapali D. S. Indrasekara, Robert C. Wadams, and Laura Fabris*

surfactants and reducing agents have been

Ligand exchange on gold nanorods (NRs) is still too often dismissed or not employed to produce gold NRs, but have
given the importance it should deserve. The many applications of gold NRs, been shown to not be able to yield the
mainly in plasmonics, biological imaging, and sensing, are made possible by same finely tunable control over the mor-
phology if used without CTAB.[14] In addi-
finely tuning not only the optical properties of the metallic core but also the
tion, it has been shown that the purity of
tethered functional groups. Gold NRs are mainly synthesized by using CTAB CTAB highly influences the ability to syn-
as the morphology-guiding surfactant, and an intimate relationship between thesize gold NRs.[15]
the crystallographic facets of the rod and the CTAB bilayer exists. Because of Recent work by Wadams et al.[16] has
this, it is imperative to fully understand the ligand exchange mechanisms that demonstrated that the NR growth is
mostly susceptible to the action of CTAB
allow replacing CTAB with functional ligands, including the energetic contri-
during the early stages, dominated by
butions. Here, the major applications of gold NRs are briefly overviewed, and epitaxial micellar adsorption of the sur-
what is known about ligand exchange mechanisms is summarized, as well factant and by adatom reorganization.
as why it is important to achieve complete removal of CTAB, including the The same authors have also shown that
techniques that are used to characterize the exchange reaction products. The each stage is characterized by thermody-
concept of interface in gold NRs is briefly examined, and explained why the namically stable crystallographic facets
at the tips and the sides of the rods that
scientific community should focus more on understanding and characterizing
evolve during growth (see Figure 1).[17]
it. Starting from the published literature, the reader is guided through the Because of these discoveries, the above
reasons why it is thought that ligand exchange on gold NRs is perhaps the studies have opened a Pandora’s box for
next grand challenge in the nanoparticle field. the scientists interested in gold NRs.
Because CTAB is so intimately con-
nected to our synthetic ability to tune the
nanoparticle’s morphology, we cannot consider it only a mere
1. Introduction surface stabilizer, and hence its replacement with other cap-
ping agents through ligand exchange should be considered a
Gold nanorods (NRs) are a versatile type of nanoparticle that complex reaction, whose mechanism needs to be thoroughly
since their inception has found a wide variety of applica- understood via carefully devised thermodynamic studies.
tions,[1–8] mainly owing to their well understood plasmonic Ligand exchange is often employed as a tool to functionalize
properties that can be easily tuned by varying the aspect ratio.[9] the NRs for a specific application and it is only rarely the pri-
The seed-mediated bottom-up synthesis of gold NRs, initially mary subject of investigation. Many of the applications of gold
devised by Murphy and El-Sayed and coworkers,[10–12] is strictly NRs, whether in plasmonics or biological imaging, are closely
dependent upon the use of cetyltrimethylammonium bromide dependent upon our ability to tailor their surface functionali-
(CTAB) as the capping agent, which despite the significant pro- zation and tune their properties. However, the lack of under-
gress in the synthetic protocols[13] has remained the main sur- standing of ligand exchange will most likely negatively impact
factant used in the synthesis of this type of nanoparticle. Other our ability to employ NRs in technological applications of high
complexity unless we design surface characterization experi-
ments able to thoroughly identify the chemical species bound
A. S. D. S. Indrasekara, Dr. R. C. Wadams to or adsorbed on the NR surface and to study the NR inter-
Department of Materials Science and Engineering face before and after ligand exchange. There are numerous
Rutgers University
607 Taylor Road, Piscataway, New Jersey 08854, USA
chemical characterization techniques available nowadays and
Prof. L. Fabris
it is imperative that we start employing them to study gold
Department of Materials Science and Engineering NRs if we want them to become a relevant technological tool,
Institute for Advanced Materials even if this means that we have to carry out hard and complex
Devices and Nanotechnology experiments.
Rutgers University Despite the availability of many excellent reviews on gold
607 Taylor Road, Piscataway, New Jersey 08854, USA
E-mail: lfabris@rci.rutgers.edu NRs and their applications,[18–21] to our knowledge none of
them focuses exclusively on the intricacies of ligand exchange.
DOI: 10.1002/ppsc.201400006 Because we believe that this topic should require some attention

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PROGRESS REPORT

A. Swarnapali D. S.
Indrasekara is currently a
Ph.D. student at Rutgers
University. She earned
her M.Sc. in chemistry
from North Carolina State
University in 2011 and her
B.S. in chemistry from the
University of Peradeniya, Sri
Lanka, in 2007. Her research
interests primarily focus on
controlled gold nanoparticle
Figure 1. The shape evolution of gold NRs at the early stages of growth assembly, surface enhanced Raman spectroscopy, chemical
(I through V) is strictly dependent upon the interaction of the growing sensing, and bioimaging.
particle with CTAB’s micelle. This intimate interaction is reflected on the
selective evolution at either tips or sides, eventually leading to the ther-
modynamically stable morphology that is commonly observed. Repro- Robert C. Wadams obtained
duced with permission.[17] Copyright 2013, American Chemical Society. his Ph.D. in materials science
and engineering from Rutgers
University, where he studied
from the scientific community, in this Progress Report, we will bottom-up nanoparticle syn-
attempt to critically summarize some of the work that has been thesis and functionalization,
carried out to advance this field of research. We have structured and plasmonic nanoparticle
this review to initially introduce the reader to the main applica- enhancement of organic
tions, as they are the reason why many researchers focus their photovoltaic solar cell perfor-
efforts onto this type of material, while progressively setting the mance. His expertise includes
stage to the core topic, that is a critical analysis of the methods surfactant and polymer chem-
used to characterize the surface composition of gold NRs and istry, nanoparticle synthesis
the effectiveness of the ligand exchange protocols. We will high- and functionalization, and functional nanoparticle compos-
light the main areas of research that in our opinion need to ites. His interests are in applications of 2D and 3D nano-
become the focus of intensive research, while identifying the structures in energy harvesting and storage, in biomimetic
major pitfalls one can incur while functionalizing gold NRs for materials, as well as in multifunctional metamaterials.
specific applications. Our final considerations will be devoted
to the interface in gold NRs: what it is, why it is important, and
why we should really take it more into account. Laura Fabris received her doc-
torate degree in chemistry at
the University of Padova, Italy,
2006, and was a postdoctoral
2. Overview of Applications researcher at the University
In this section, we will briefly review some of the most impor- of California at Santa Barbara.
tant applications of gold NRs, that are those applications for In 2009, she joined the
which their intrinsic morphological and optical properties are Department of Materials
truly game changers. The reason why we initially focus on Science and Engineering at
them is the fact that in the majority of the cases, gold NRs Rutgers University, where she
are employed as tools to achieve specific functions rather is currently an assistant pro-
than being the main subject of the investigation. By including fessor. Her research interests
this overview, we hope that those scientists who primarily include the synthesis and assembly of plasmonic nano-
focus on applications will find this review a helpful source of particles, the study of ligand exchange, and the develop-
information. ment of SERS-based approaches for sensing and imaging.
Although applications of gold NRs are heavily influenced by
the chemical composition of the functionalized surface, only
seldom their description is accompanied by a thorough char- examining the relevant experiments that have (or should have)
acterization of its properties. In this overview, we will initially been carried out to characterize the properties of the NR surface.
focus on plasmonic devices, to then move to describe some
of the main biological application of NRs, namely sensing,
imaging, and therapeutic approaches. For all these applica- 2.1. Plasmonic Devices
tions, we will highlight the features that convey the importance
of understanding the role of CTAB, the definition of the inter- Owing to their outstanding and easily tunable optical proper-
face, and the importance of ligand exchange, while critically ties, gold NRs are an ideal candidate for the fabrication of

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PROGRESS REPORT
plasmonic devices. When thinking about device fabrication, it although not yet vastly explored in the biomedical field, it could
is however mandatory to consider the fact that the NRs need prove relevant to, for example, explore orientation-dependent
necessarily to be integrated with other materials, most likely of mechanisms of cellular uptake of NRs by binding cell-targeting
different nature and composition, to obtain tools of technolog- moieties such as RGD to either the tips or the sides of the NR.
ical significance. In doing so, the first hurdle to overcome is the The first example we chose to describe how modulating the
immiscibility, and hence the phase segregation, of the NRs with surface properties of the NRs and their interparticle interac-
the matrix material. Directly following are considerations rela- tions can lead to applications in plasmonic devices was reported
tive to the expected function of the device and the conditions at by Nepal et al. In this work, the authors showed that, by using
which it needs to operate. monodispersed bifunctional gold NRs synthesized from the
In this section, we will describe two excellent examples bottom-up, and by inducing and deterministically arresting their
reported in the literature where the strategic functionalization flocculation, it is possible to obtain predetermined side-by-side
of gold NRs has led to self-assembled materials that display assemblies of gold NRs that can be used for high temperature
novel plasmonic properties. These two examples have been plasmonics.[22] The authors have demonstrated that by carefully
chosen because, while supporting our thesis that a careful con- tuning the properties of the interface, it is possible to assemble
trol of the NR surface functionalization can bring about new the NRs and keep them assembled while carrying out further
exciting applications, they also demonstrate that molecular level surface modifications (such as silica coating) yielding highly
characterization is fundamental to reproducibly and repeatably stable materials that can be heated up to 700 °C. Key steps of the
synthesize the materials of interest and hence render them functionalization protocol include the ligand exchange of CTAB
technologically viable. The key aspect that has been exploited at the tips of the rods with a suitable alkylthiol, followed by the
in the experiments described below is the fact that ligand addition of ethanol (a good solvent for CTAB), which partially
exchange on gold NRs proceeds at different rates depending destabilizes the CTAB double layer and induces side-by-side
on whether it occurs at the tips or at the sides of the NR, with assembly of the rods, that can be maintained by further addition
the former being more reactive than the latter (Figure 2). By of CTAB to reestablish the passivating double layer (see Figure 3).
taking advantage of this fact, it is possible to selectively and het- Despite the fact that the observed results elegantly reproduce the
erogeneously functionalize the tips and the side facets of the predicted outcomes, this work, along with many others that deal
rods. This selective functionalization can be taken advantage of with NR assembly,[22,23,25–32] focuses prevalently on the morpho-
to assemble the NRs either tip-to-tip or side-by-side,[22,23] and logical characterization of the assembled nanostructures [e.g.,
has been widely exploited in the device community. Moreover, carried out by transmission electron microscopy (TEM) and
scanning electron microscopy (SEM)], rather
than on the analysis of the chemical composi-
tion of the surface. Although it may be argued
that the correct assembly can be achieved only
upon selective functionalization, the commu-
nity should, in view of the promise of the field
to produce fancier and more complex devices,
expand the characterization toolkit to encom-
pass techniques able to identify surface com-
position, density of the capping ligands, and
how their structure can influence their spatial
distribution.
The second selected example that high-
lights the importance of achieving regiospe-
cific ligand exchange on gold NRs to produce
materials with novel plasmonic properties was
reported by Kotov and coworkers.[33] In this
work, it is possible to appreciate how by pro-
tecting the tips of the NRs with dithiothreitol
(DTT) and manipulating exclusively the sides
Figure 2. The influence of nanorod surface curvature and epitaxy on ligand exchange. Figure 2a) of the NRs by capping them with forward or
illustrates how two neighboring ligands occupy the planar surface on the longitudinal body of
a NR, highlighting the footprint of each ligand and graphically demonstrating how the steric
reverse PCR primers, the authors were able to
interactions limit the potential packing density of high-molecular-weight species. Figure 2b) pair gold NRs side-by-side while giving them a
depicts the potential increase of ligand packing density on the NR surface due to curvature dihedral tilt angle, thus offsetting the tips and
effects, where the steric interaction is represented by the deflection angle (θ), as defined by breaking the centrosymmetric nature of the
Hill et al.[24] and the ligands can be imagined to occupy a conical portion of the space, instead parallel assemblies. By tilting the rods with
of a cylinder. Figure 2b) also highlights the variation in curvature between the NR tip and body, a “forward” or “reverse” angle, the resulting
where the radius of curvature of the body and tip is represented by rbody and rtip, respectively. As
assemblies are enantiomeric in nature, thus
varying epitaxial surfaces can influence micellar and molecular adsorption energy, the potential
for spatial variations in ligand exchange energetics is illustrated in the end-on view of the NR giving rise to chirality effects in each of the
in Figure 2c), which shows the longitudinal {250} facets, alternating {110} and {111} tip facets, assembled dimers. This work, although not
and the presence of a {001} tip facet that is seen when BDAC is used in the NR synthesis. directly determining the composition of the

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PROGRESS REPORT

Figure 3. Selective side-by-side dimerization can be achieved by replacing CTAB at the NR tips with alkylthiols followed by destabilization of the CTAB
micelle. 1) Stable gold NRs are suspended in water. 2) Ligand exchange is carried out to selectively replace CTAB at the tips with thiolated alkyl chains.
This step can be successfully carried out only by carefully controlling the relative concentrations of NRs and thiols, and the kinetics of the reaction. 3)
By adding ethanol (a good solvent for CTAB), the critical micelle concentration (cmc) of CTAB is increased, thus destabilizing the micelle around the
NR. This leads to side-by-side dimerization. 4) The system is again stabilized by addition of a fresh solution of CTAB in water that reestablishes the
bilayer around the NRs. Reproduced with permission.[223]Copyright 2012, Wiley.

surface monolayer and heavily focusing on optical characteriza-
tion, proposes an interesting approach toward the determination
of the monolayer composition by comparing dynamic light scat-
tering (DLS) and TEM data and fitting them to a model that, in
addition to the NR surface, takes into account the Stern layer
and the slipping plane between interacting NRs in a dimer.
In the two reported examples, successful NR functionaliza-
tion was assumed indirectly on the basis of the assembly results
rather than on direct surface characterization. For example,
by comparing the two articles, the selective functionalization
of the NR tips is allowed to occur during time scales that are
drastically different: 5 min for Nepal et al.[22] and 8 h for Kotov
and coworkers.[33] Even if we account for the differences in
the NR surface morphologies and the linkers used by the two
groups, how can we, as we try to adapt these procedures to our
experiments, know what incubation times we should use if we
cannot base our analysis on surface chemical composition data?
Because exciting applications such as these will most likely rep-
resent stepping stones in the future of gold NRs in plasmonic
devices, it is mandatory that, as we devise new applications,
spend some time thinking about thorough surface-specific
characterization as well.
2.2. Biological Applications
2.2.1. Sensing
NRs have also been implemented as optical sensors for sur-
face enhanced Raman scattering (SERS)-based detection.[40] The
high electromagnetic field enhancement observed at the tips of
the NRs, and at the junction of NR assemblies, has been used
to increase the SERS signal enhancement, which in turn boosts
the sensitivity of SERS-based sensing assays. For instance, Peng
et al.[27] have demonstrated the ultrasensitive SERS-detection of
food contaminants such as melamine and plasticizers in orange
juice at femtomolar level by using gold NR assemblies. A recent
study by Kotov and coworkers has also demonstrated ultrasen-
sitive attomolar DNA detection by side-to-side NR assemblies.
Assembly has been achieved by selectively functionalizing the
surface of 4-aminothiophenol (SERS reporter) tagged-NRs with
primers and then performing polymerase chain reaction (PCR)
under controlled conditions.[41] The SERS signal enhancement
due to the hot spot created upon NR assembly in combina-
tion with the CD spectral responses of DNA has enabled DNA
detection at concentrations as low as 3.7 aM. The level of sensi-
tivity offered by this approach shows promise in the use of NR
assemblies for reliable DNA biomarker detection and disease
diagnosis.
Considering the nature of NR-based sensing approaches, it
is obvious that the NR surface should be approachable and flex-
ible enough to enable effective NR-analyte interactions. There-
fore, careful control of the NR surface chemistry by ligand
exchange is essential and crucial for sensitive and effective
sensing applications.

Many NR-based sensing methods have been developed based

on the changes in the longitudinal plasmon resonance fre-
quency (LSPR) of NRs due to the high sensitivity of this reso-
nance band to the refractive index of the surrounding medium
and the distance between NRs.[34–36] Binding of biological mol-
ecules, such as proteins or antigens, to the surface of suitably
functionalized NRs translates into changes in the refractive
index of the medium, which is then reflected on the magni-
tude of the LSPR wavelength shift.[37,38] Fluctuations in the
LSPR due to the analyte-mediated NR assembly, such as DNA
hybridization or antibody–antigen interactions, have been used
in many instances as a read-out method for both quantitative
and qualitative analyte detection.[39] A recent study by Truong et
al.[37] has shown 111 aM detection of prostate-specific antigen
in a carboxylate-functionalized single NR-based bioassay corre-
sponding to a wavelength shift of 2.8 nm.
2.2.2. Imaging
Biological imaging using gold NRs is made possible by virtue
of the tunable longitudinal plasmon band of gold NRs, that can
be moved from roughly 620 nm all the way into the infrared
by increasing the AR of the rods. NRs have been extensively
used to develop contrast agents for imaging due to their excel-
lent optical properties, such as (a) highly efficient plasmon
resonance absorption in the near-infrared (NIR) region, which
is the biologically transparent window of the electromagnetic
spectrum, (b) large linear and nonlinear absorption cross sec-
tions, and (c) reduced photobleaching compared to conven-
tional contrast agents.[42–44] Although each technique exploiting
the interaction of NRs with light to deliver successful imaging
or therapeutic functions is intrinsically limited by the short

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penetration depth of light in biological tissues, the NIR remains
the best region of the electromagnetic spectrum to target, as it is
indeed the one for which the least tissue absorption takes place.
The strong scattering of NRs upon their interaction with light
has been successfully used in dark field microscopy (DFM)-
based cancer cell imaging.[42,45,46] In these experiments, bright
staining of cancer cells exposed to NRs has been observed, as
opposed to no signal from healthy cells.[46] NRs have also been
used to improve the contrast of imaging modalities such as
optoacoustic and photoacoustic tomography.[47,48] In a in vivo
optoacoustic imaging system, Eghterdari and coworkers[47]
have demonstrated that areas injected with functionalized NRs
generate brighter features compared with those without NRs.
Two-photon luminescence (TPL) imaging has employed NRs
due to the minimum damping of its surface plasmon.[49–51]
Cancer cells, where targeted NRs are accumulated, have shown
remarkably bright TPL images in comparison to two-photon
auto-florescence images of unlabeled cells (without NRs).[49] For
instance, Wang et al.[52] have identified in in vivo imaging TPL
signals of single NRs flowing through blood vessels in mouse
earlobes with intensities 3 and 58 times higher than the auto-
fluorescence from the surrounding tissues and the two-photon
fluorescence from single rhodamine molecules, respectively.
NRs tagged with Raman active molecules have been used
in SERS-based cell imaging. In SERS spectroscopy, the weak
signals of Raman active molecules, due to the inherently low
scattering cross sections, are overcome by leveraging the
intrinsic optical properties of plasmonic nanoparticles such as
NRs. In SERS-based imaging, the accumulation of NRs at tar-
geted tissues has been confirmed by monitoring and mapping
the distribution of the SERS signal from NR-bound reporter
molecules.[53,54] Multiplexed SERS imaging has been achieved
by labeling NRs of different aspect ratios with various SERS
reporter molecules. For instance, intra- and post-operative
in vivo tumor imaging and in vivo identification of different
cancer cell lines have been achieved by NR-based SERS mul-
tiplexing.[55,56] Both covalent binding and surface adsorption
of SERS active molecules to NRs have been employed, but the
former is preferred since it leads to reproducible, strong SERS
signals for highly sensitive intracellular NR detection. Thus, for
effective SERS-based imaging, the surface immobilization of
SERS reporters on gold NRs should be ensured through opti-
mization of ligand exchange methods.
It should also be noted that suitable NR surface modifi-
cations are crucial for the effectiveness of the above imaging
modalities toward specific cell targeting, disease diagnosis, and
image-guided therapeutic applications. Thus, it is important
to pay special attention to the characterization of the surface-
functionalized NRs following ligand exchange protocols, to
exactly determine the composition of the targeting/biocompat-
ible monolayer.
2.2.3. Therapy
The excellent optical properties of NRs (in particular, the fact
that the LSPR lies in the NIR region) and the ability to perform
surface modifications via favorable gold-thiol chemistry have
drawn significant attention toward NRs for their biomedical
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PROGRESS REPORT
applications, such as disease diagnosis, targeted drug delivery,
and therapy.[57,58]
Plasmon-assisted photothermal therapy (PTT) is a noninva-
sive treatment where nanoparticles such as NRs act as an effi-
cient photothermal transducer, moderately increasing the local
cellular temperature, and hence inducing the denaturation of
proteins and disorganization of cellular components that lead
to cell death.[42,59–61] The first PTT application of gold NRs
was demonstrated by El-Sayed and coworkers,[62] where the
complete destruction of human oral cancer cells treated with
anti-EGFR-functionalized NRs was reported after NIR laser
irradiation. A recent study by Bhatia and coworkers[56] has also
showed an excellent example of in vivo PTT, where a single
intravenous injection of polyethylene glycol (PEG)-capped
NRs in human xenograft tumors in mice completely eradi-
cated the tumor without regrowth for over 50 d. PTT has also
been used in conjunction to chemotherapy for enhanced thera-
peutic efficacy.[63–67] It has been reported that the cytotoxicity of
the chemotherapeutic agents is enhanced due to the elevated
localized temperature during PTT. For instance, Hauck et al.[63]
have observed enhanced cytotoxicity from the synergy between
NR-meadiated PTT and cisplatin (a chemotherapeutic drug) as
opposed to the combined effect of the independent treatments.
This synergy will facilitate the use of lower dosages of chemo-
therapeutic drugs and thereby alleviate the inherent side effects
of traditional chemotherapeutics.
Cellular uptake-mediated anticancer drug delivery and photo-
triggered gene therapy are two other common therapeutic
applications associated with NRs.[67–70] The NRs are function-
alized with tissue-specific aptamers, peptides, or proteins that
improve targeting and uptake of NRs. In photo-triggered gene
therapy, NRs functionalized with plasmid DNA are introduced
in specific diseased cells of interest followed by laser-stimulated
release of plasmid DNA, which will then code the specific pro-
tein expression required for therapeutic purposes.[67] Takahashi
et al.[67] reported the release of NR-conjugated plasmid DNA
only upon its exposure to high-energy pulsed laser irradiation
as evidenced by gel electrophoretic analysis.
For therapeutic applications, it is very important to ensure
the complete removal of CTAB by robust surface modifications
to produce stable NR suspensions that would facilitate longer
circulation lifetimes and specific targeting of the cancer cells
without leading to CTAB-induced cytotoxicity in healthy tissues.
In addition, the NR surface modification is essential for the
conjugation of specific cell-targeting and cell-penetrating moi-
eties.[61] The presence of an adequate amount of cell-targeting
moieties and their presentation on the NR surface is critical to
facilitate their optimal interaction with cell membrane receptors
for successful cellular internalization, even though the extent of
uptake is heavily dependent upon the study carried out (in vitro
vs in vivo) and on the type of cell targeted.[42]
3. The Micellar Behavior of CTAB and Its Influence
on Au NR Growth
Since the initial publications on the seed-mediated protocol by
Murphy and El-Sayed,[10–12] CTAB, along with other chemical
species that aid in anisotropic crystal growth, has been used as
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PROGRESS REPORT
the main surfactant for NR growth. Reagents, other than CTAB,
exhibiting the most influence on NR growth, include silver
nitrate, benzyldimethylhexadecylammonium chloride (BDAC),
halides, and salicylic acid derivatives.[20,71–74]
Although several articles have been published in the lit-
erature that attempt to synthesize gold NRs avoiding CTAB,[14]
they have all failed to yield the reproducibility and sample
homogeneity that are typically achievable when employing
it. The significance of CTAB in affecting NR formation was
indicated through molecular dynamics simulations by Gro-
chola et al.[75] This work showed that the anisotropic growth
of NRs is facilitated through epitaxial adsorption of CTAB to
the developing gold facets, and that as a consequence of the
heterogeneous adsorption energies of CTAB to gold surfaces,
CTAB’s passivation of (110) and (100) surfaces mediates ani-
sotropic crystal growth through preferential reduction at (111)
facets. Additional evidence by Nikoobakht and El-Sayed,[12] Ye
et al.,[76] and Wadams et al.,[16] as well as the pioneering works
by Murphy and coworkers,[74] and Liz-Marzan and coworkers[77]
suggest that the micellar state (i.e., micelle morphology and
surface chemistry) strongly influences the NR morphology as
well, where one expects the micelle morphology to influence
the ability of CTAB micelles to effectively passivate epitaxial Au
surfaces, which in turn is dictated by ionic strength, counterion
population, and surfactant chemistry.[11]
CTAB’s epitaxial behavior, regarding preferential passiva-
tion and binding energy, is also expected to vary according to
whether the seed-mediated synthesis employs silver nitrate,
as the introduction of Ag(I) to the growth solution has shown
to improve the morphological monodispersity and yield of
nanorods.[12] Crystallographic analysis between rods synthe-
sized by both methods showed that NRs made in the pres-
ence of Ag(I) are single crystals, where the longitudinal growth
occurs in the <001> direction, the tip is bound by {110} and
{111} facets, and the diameter of the rod by eight higher index
{250} facets, according to work by Liz-Marzan and coworkers.[78]
Furthermore, Park et al.[17] indicated that the {250} facets are
developed through adatom migration as the Au NR matures
during synthesis, and that the NR is bound by alternating
{100} and {110} facets at early stages. Au NR synthesis in the
absence of Ag(I) produces a penta-twinned particle, with the
rod growing in the <001> direction. The longitudinal surface is
bound by {100} and {110} facets, and the tip by {111} facets.[20]
Variations in the epitaxial surfaces presented by the NR product
between these two synthetic strategies are expected to present
different binding potential to surfactant adsorbates, as recently
demonstrated by Feng et al.,[79] whose computational studies on
the soft-epitaxial adsorption of molecular species onto gold sur-
faces suggest a strong dependence of the binding energy on the
adsorbate’s molecular structure and the atomic spacing of the
epitaxial surface.
The use of Ag(I) is likely to have a more direct effect on the
adsorption behavior of CTA+. Extended X-ray fine structure
measurements of Au NRs indicate that silver is present in
elemental form on the NR surface.[80,81] Liu and Guyot-Sion-
nest[82] derive that this is likely due to under-potential deposi-
tion of silver on the Au NR surface, and that silver reduction
is more likely to occur on the longitudinal {110} facets, thus
acting to passivate these facets in order to bring about aniso-
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tropic growth, and providing a different surface onto which
CTAB adsorbs. In a separate study by Hubert et al.,[83] XPS
indicated that the CTA+AgBr2− complex is potentially the active
surfactant, which is expected to alter both micellization and epi-
taxial adsorption in comparison to CTAB.
Consolidating surfactant self-assembly and theoretical
micellar morphology studies with experimental results, Missel
et al. developed a thermodynamic model that predicts the
sphere-to-rod micellar transitions, which are of fundamental
importance for the successful synthesis of gold NRs.[84–89]
Their work included the derivation of an explicit equation [see
Equation (1)] for a thermodynamic parameter, K, which meas-
ures the micellar morphology evolution from spherical to rod-
like, and contains both electrostatic and hydrophobic contribu-
tions. The coefficient α nC2 , where α is a constant and nc is the
carbon chain length, is derived from Israelachvili’s calculation
of the critical aggregation number (Nc) for a spherical micelle,
which is defined as follows: Nc = 4πR3/3v = 4πR2/ao ≈ α nC2 ,
where R is the fully extended amphiphile carbon chain length, v
is the volume of the micellar core, and ao is the surface area of
the surfactant head group at the liquid micelle interface.[84,85,89]
According to this analysis, spherical micelles are only favored
when the geometric parameter G = v/aoR ≤ 1/3.[89] Based on
these results, the micellar growth from spherical to elongated
is affected by the molecular structure and chemistry of the sur-
factant, by the ionic strength of the solution, and by the sur-
factant counterion’s ability to screen micellar surface charge
density.
⎡ ΔF ( A − AC ) ⎤
logK = α nc2 ⎢ el + γ HI S ⎥⎦ (1)
⎣ RT RT
Empirical evidence confirming the influence of the counte-
rion on CTA+ micellar adsorption and growth was investigated
by Ha et al, Velegol et al., and Magid et al. who showed that
bromide exhibits a fivefold higher binding affinity to the CTA+
micellar surface compared with chloride, and that in the pres-
ence of both chloride and bromide, the latter is able to displace
the former and assume control over the micellar morphology
evolution.[71,90,91]
The counterion’s contributions toward solution-phase
micellar growth of CTAB also show significant effects on the
morphology and the density of CTAB adsorbed onto epitaxial
surfaces. Velegol et al.[90] compared the adsorption of CTA+ onto
silica surfaces when chloride and bromide are employed as the
counterion. Above the critical micelle concentration of both
CTA+-Br− and CTA+-Cl−, employing bromide yields a surface
concentration of CTA+, which is 60% higher than that observed
with chloride. The surfactant disparity in turn affects the mor-
phology of the adsorbed micellar aggregates: CTA+-Br− aggre-
gates adsorb as worm-like micelles with an observed proclivity
to coalesce into a laterally homogeneous structure, presumably
a bilayer, while the morphology of the adsorbed CTA+-Cl− aggre-
gates appear as circular projections. Because the effect of CTA+
is mostly exerted on the growing Au NRs through micellar
passivation of the evolving surfaces, one can therefore expect
that systems employing a bromide counterion will promote
enhanced epitaxial passivation of {110} and {100} facets, and
therefore promote anisotropic growth. Indeed, this is supported
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in recent work by Wadams and coworkers[16] who showed that
when the Br− to CTA+ ratio is reduced below unity, through
the addition of chloride in the form of CTA+Cl−, Au NR yields
are significantly reduced, with a simultaneous increase in the
amount of isotropic by-products.
In this section, we have highlighted the importance of
CTAB’s micelle in guiding the anisotropic NR growth and in
passivating its surface by tightly binding to the evolving facets.
Thus, the role of CTAB is mechanistically far more important
than that of a surface passivating surfactant and the above
energetic considerations demonstrate that its interaction with
gold is strong and has important implications. As a conse-
quence, the removal of CTAB from the NR’s surface, via ligand
exchange reactions, is not a mere exercise of chemistry, and
should be understood and studied employing a set of comple-
mentary quantitative techniques capable of providing not only
the identity and relative amounts of the molecules present on
the NR surface but also kinetic and thermodynamic parameters
that thoroughly describe the ligand exchange reaction.
4. Toxicity of CTAB
As described above, there is a wide array of biological applica-
tions in which gold NRs are employed and, as a consequence,
the well-known toxicity of CTAB (and consequently of gold
NRs) should be a serious concern not only for the community
interested in these applications but also for the scientists whose
expertise could contribute to the identification of its causes.
Because the addition of thiolated PEG to the NR suspension,
commonly employed to render the NRs biocompatible, is not
sufficient to assume complete replacement of CTAB, it is of
fundamental importance to evaluate in depth the safety of the
NRs and the origin of their toxicity via a universally approved
and standardized set of experiments. Most importantly, it is
imperative to devise carefully selected experimental practices to
thoroughly detoxify the NRs and to set in place characterization
protocols to directly study their surface properties.
CTAB forms a chemisorbed bilayer on the surface of the
NRs with the cationic CTA+ head groups pointing toward the
external environment, thus rendering them dispersible in
water. However, despite their compatibility with aqueous envi-
ronments and hence physiological solutions, CTAB-capped
NRs have shown to induce toxicity in cells, a major challenge
for their successful application in biological environments.[92–96]
Therefore, much research has been dedicated to understand
the source of NR toxicity, but it still remains a controversy
within the scientific community. Does it originate from free
CTAB molecules in suspension or from NR-bound CTAB? Do
residual contaminations from the starting materials promote
cytotoxicity? Does the positive NR surface charge from the
CTA+ group play a role in cytotoxicity?[93,97–99] The cytotoxicity of
CTAB-capped NRs has been evaluated largely by standard cell
viability, cell proliferation, and oxidative stress assays. In addi-
tion, efforts have been made to understand the effect of CTAB-
capped NRs at the genetic level as well as on cellular membrane
integrity. In this section, we will briefly highlight the origins of
NR cytotoxicity and their proposed mechanisms, some of the
common approaches to evaluate the sources of toxicity, along
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PROGRESS REPORT
with the approaches to mitigate NR toxicity by ligand exchange.
Some of the protocols used can be considered valuable in iden-
tifying and characterizing the sources of cytotoxicity, while
others, specifically those proposing the use of polyelectrolyte
overcoating to either mask or hold CTAB in place and prevent
it from leaching, would benefit from more in-depth surface
characterization.
The surface charge of NRs is one of the proposed sources
of cytotoxicity, and this relationship has been studied by
many groups by manipulating the overall surface charge
of the NRs through polyelectrolyte coating and/or ligand
exchange.[56,92,97,100–102] The overall surface charge has been
assessed by zeta-potential measurements, and standard cell
viability and cell proliferation assays have been conducted to
evaluate the NR toxicity. It has been reported that CTAB-capped
NRs significantly reduce cell viability compared with either
NRs coated with polyelectrolytes (both anionic and cationic)
or NRs capped with ligands such as PEG and alkanethiols,
in which cases only a negligible loss of cell viability has been
reported.[97,103–105] These observations have led to the conclu-
sion that there is no correlation between the overall surface
charge and the cytotoxicity of NRs.[97,100] Many studies have
suggested that the layer-by-layer polyelectrolyte coating reduces
the interaction between NR-bound CTAB with its external envi-
ronment, presumably preventing CTAB from desorbing and
thereby leading to the observed reduced toxicity in comparison
to CTAB-capped NRs.[64,92,95] However, this hypothesis has not
yet been supported by experimental evidence, and it needs to be
validated by investigating CTAB’s desorption kinetics.
Another proposed source of NR toxicity is the free CTAB pre-
sent in NR suspensions that can derive from insufficient purifi-
cation protocols and/or can be due to the dynamic surface des-
orption of CTAB from the NRs.[106,107] Therefore, it is necessary
to differentiate the cytotoxicity of CTAB free in the medium from
that of NR-bound CTAB. In order to investigate the origin of the
free CTAB, CTAB-capped NRs were centrifuged, and the toxicity
of the supernatant was compared to that of the NR pellet resus-
pended in water and to the original CTAB-capped NR suspen-
sion.[92] Standard cell viability tests such as MTT and MTS have
been used to evaluate the toxicity of free CTAB versus NR-bound
CTAB. The cytotoxicity of the supernatant was found to be com-
parable to that of the free CTAB alone and of the original CTAB-
capped NR suspension, thereby revealing that free CTAB in the
NR suspension is a dominating factor influencing NR toxicity.
Many of these studies have relied only on the cell viability data
without any quantitative estimations of the NR-bound CTAB
and on the free CTAB in the supernatant. However, Alkilany
et al.[92] have used liquid chromatography-mass spectrometry
(LC-MS) to estimate CTAB’s concentration in the superna-
tant (0.2–0.3 × 10−6 M) and the amount of NR-bound CTAB
(1.2–5.5 × 10−6 M) for a given range of NR aspect ratios. They have
also performed similar experiments with polyelectrolyte-coated
NRs, for which the CTAB concentration in the supernatant was
found to be below the detection limits of the mass spectrometer,
and showed little to no toxicity. Compared with polyelctrolyte-
coated NRs, CTAB-capped NRs have shown significant toxicity
and cell growth inhibition, both attributed to the presence of free
CTAB.[62,103] Based on similar results by different studies, it was
concluded that the cytotoxicity arises from free CTAB molecules
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Figure 4. MTT and LDH assays establishing the viability and membrane
integrity, respectively, of LLC-PK1 cells after incubation over 48 h with the
following: 45 µg mL−1 gold NR stock solution; 100-kDa retentate (after
resuspension in fresh media); 100-kDa filtrate. Percentage values are nor-
malized with respect to a media-only control. Reproduced with permis-
sion.[60] Copyright 2008, American Chemical Society.
in the suspension but not from the surface charge of the NRs
nor from the NRs themselves, and that the polyelectrolyte over-
coating could possibly reduce the exposure of the cells to the
desorbed CTAB (see Figure 4).[60,92,97,100] Similarly, it has been
reported that replacing CTAB with PEG via ligand exchange
dramatically decreases the toxicity.[103,105,108] In contrast, Son-
nichsen’s group[109] has reported a significantly reduced meta-
bolic activity in cells exposed to CTAB-capped NR suspensions
compared to that of those incubated with pure CTAB solutions
at an equivalent concentration. They proposed that NRs act as a
CTAB carrier, releasing surface-bound CTAB upon cellular inter-
nalization, thereby causing more toxicity than the free-floating
CTAB in solution that directly interacts with only the cell exte-
rior having a limited ability to cross the cell membrane. How-
ever, these arguments need to be supported by experimental evi-
dences through careful characterization methods.
There are few reported studies that have investigated the
effect of CTAB-NRs on the integrity and interfacial features of
cell membranes and organelles, another measure of toxicity.
Lactate dehydrogenase (LDH) assays, which detect the release
of LDH from the cytoplasm as a response to cell membrane
damage has often been used to understand the effect of the sur-
face chemistry of NRs on the cell membrane. A significantly
elevated secretion of LDH in cells treated with CTAB-capped
NRs has been observed compared with other surface-modified
NRs and the corresponding NR supernatants.[101] Wang et al.[98]
have proposed that the CTAB bilayer on the NRs can also be
internalized, when bound to gold NRs, through mechanisms
and interactions with the cell membrane that are different from
those taking place with free CTAB and CTAB micelles. It was
also proposed that CTAB-capped NRs can adsorb proteins in
the biological media via opsonization, but release them upon
cell internalization and transport them into lysosomes as a
response to the acidic environment, thereby exposing the CTAB
bilayer on the NRs to the membrane structures.[92,110,111] It has
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been reported in many other studies that the cationic CTAB
itself can directly diffuse through the cell membrane, enter the
mitochondria, and induce apoptosis.[98,112] Thus, thorough puri-
fication protocols and quantitative characterization techniques
should be performed to minimize the introduction of free
CTAB with the NR samples for biological applications.
While cell viability assays have extensively been used to
evaluate cytotoxicity, only very few studies that focused on the
effects of long-term exposure of cells to NRs (in particular
on oxidative stress, inflammation, and specific gene expres-
sions) can be found in the literature.[93,97,100,113] These aspects
should be pursued in depth since the effects of NRs on the
genetic material must be investigated, if clinical applications
of NRs are to be launched in the future. A direct relationship
between NRs and the immune system has been hypothesized
since it has been experimentally shown that the cellular uptake
of NRs involves macrophage cells, which are the major compo-
nent of the immune system that participate in the inflamma-
tory processes. Pissuwan et al.[100] have investigated the impact
of NRs on the inflammatory response in macrophage cells
(Raw264.7) by measuring the secretion of cytokines (an inflam-
matory mediator), interleukin-6, and tumor necrosis factor
alpha. In cells exposed to CTAB-NRs, an elevated degree of cell
death was recorded as the concentration of NRs increased from
5 to 100 μg mL−1, accompanied however by very minimal to no
secretion of cytokines. This result was explained as due to the
presence of significantly low amounts of live cells because of the
high cytotoxicity of CTAB. On the other hand, when cells were
exposed to polyelectrolyte-coated NRs, no cytokine stimulation
or cell death was observed. It was also concluded that ligand
exchange of CTAB on NRs impacts the inflammatory response
of the macrophage cells the least leading to higher cell via-
bility.[113] Grabinski et al.[93] have studied the influence of the NR
surface chemistry on the stress response-related gene expres-
sion, and the toxicity to human keratinocyte cells by real-time
reverse transcriptase polymerase chain reaction (RT-PCR). Sig-
nificantly elevated reactive oxygen species (ROS) were reported
in human keratinocyte cells exposed to CTAB-NRs in compar-
ison to mercaptohexadecanoic acid- and PEG-capped NRs (see
Figure 5). This observation also supports that the free CTAB
present in the NR suspension is the major cause of NR toxicity,
and it can be reduced to a large extent by ligand exchange.
However, it should be noted that it is not an easy and fair
task to draw generalized conclusions on NR cytotoxicity based
solely on the literature. These studies have used a wide range
of NR concentrations, aspect ratios, incubation times, and most
importantly different cell lines and cell models (in vitro and in
vivo), which mainly dictate the level of cytotoxicity. One relevant
question that should be asked is: can in vitro results be trans-
lated to in vivo conditions? For instance, it was reported that the
toxicity of both citrate-capped nanoparticles and CTAB-capped
NRs is significantly lower in 3D cell culture constructs (much
closer to the in vivo model) compared with 2D constructs.[92,114]
Therefore, it is time to set up standardized toxicological param-
eters to systematically analyze the interactions between NRs
and biological materials, which will allow us to draw accurate
conclusions.
In summary, there are many studies that report that the
cytotoxicity of NRs can be significantly reduced by performing
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PROGRESS REPORT
Figure 5. Oxidative stress production after exposure to CTAB-capped gold NRs (GNR-CTAB). a) Negative control; b) positive control (0.03% H2O2);
(c) 5 µg mL−1 GNR-CTAB; d) 50 µg mL−1 GNR-PEG; e) 50 µg mL−1 GNR-MHDA. Nuclei are stained with Hoechst, and the DCFH probe fluoresces
green in the presence of ROS. White arrows indicate green fluorescence produced in positive control. f) Oxidative stress production after exposure
to GNR-CTAB and g) GNR-MHDA and GNR-PEG. Data normalized FITC intensity to Hoechst intensity to account for cell number. Reproduced with
permission.[93] Copyright 2011, American Chemical Society.

ligand exchange and polyelectrolyte-overcoating. However, more
clarification on the main source of NR cytotoxicity with stronger
experimental evidences is still needed. It could be achieved
by careful and thorough quantitative and qualitative charac-
terization of the nanoparticle interface before and after surface
modifications by ligand exchange protocols. This will facilitate
the development of effective methods for detoxifying NRs and
introducing new functionalities for their biological applications.
5. Tuning Nanorod Surface Chemistry and
Ligand Exchange
Among those described above, the biological applications of gold
NRs have arguably received the most attention. Here, while not
discounting the importance of all the other realms of science in
which NRs can be successfully employed, we will focus on the
important task of rendering gold NRs biocompatible and stable
in physiological conditions (where for example CTAB-capped
gold NRs would aggregate and coalesce). The versatility of Au
NRs in biological applications is not only mediated by their tun-
able plasmonic response but also via the exotic chemistries,
which can be grafted onto their surface by leveraging the affinity
of thiols for gold.[115,116] Moreover, recent advances in molecular
synthesis and bioconjugation have provided a surplus of thi-
olated biological and biologically compatible molecules including
RGD peptides, tailored deoxyribonucleic acid (DNA), and PEG,
ultimately providing the opportunity to tether multiple chemis-
tries to the surface of the NRs, thus creating biocompatible mul-
tifunctional gold NRs, which do not exhibit cytotoxicity.[47,117]

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Numerous strategies have been employed to tune the NR
surface chemistry. The most popular methods used for bio-
logical applications employ either the layer-by-layer assembly
of zwitterionic molecules onto the CTAB-coated NR, or the
direct displacement of the CTAB bilayer with amphiphiles or
molecules, which bind to the NR surface via Au–S bonds. The
use of layer-by-layer techniques for NR functionalization was
developed by Gole and Murphy,[118] where alternating layers
of polyelectrolytes were adsorbed onto the positively charged
CTAB bilayer of the NR, and has subsequently been shown by
others.[97,119,120]
A more preferable approach is to remove CTAB from the
NR surface altogether via ligand exchange reactions. By taking
advantage of the affinity of sulfur for gold, one can perform
ligand exchange on the Au NR surface, where a desired incoming
molecule forms a self-assembled monolayer on the NR surface,
ideally displacing the CTAB bilayer completely. The advantage of
ligand exchange strategies over layer-by-layer techniques is the
potential to completely remove CTAB, therefore increasing stabi-
lization and avoiding the risk of polyelectrolyte leaching.
However, in contrast to the efficiency and ease with which
one can carry out layer-by-layer deposition, ligand exchange has
met difficulties in providing procedures for high-yield ligand
exchange products that effectively remove CTAB. NRs often
aggregate irreversibly during ligand exchange from excessive
destabilization of the CTAB bilayer before the NR surface is
effectively passivated with the incoming ligand.[20] With regards
to incomplete removal of CTAB, many protocols indicate that
CTAB remains trapped within the polymer layer on the NR
surface, especially when employing large molecules such as
PEG.[20] However, recent literature has shown progress in
developing facile, reproducible ligand exchange protocols for
selective tuning of Au NR surface chemistry and simultaneous
removal of CTAB. Here, we will review the different strategies
toward ligand exchange on Au NRs, their advantages and disad-
vantages as related to CTAB removal and direct application in
bio-nanotechnology.
The premise behind ligand exchange is to employ a molecule
which will displace CTAB from the NR surface, will favorably
bind to the NR surface, and will subsequently remain tightly
bound to the Au surface under several solution conditions.
A variety of organic species have been utilized for binding to
the gold surface during surfactant-based ligand exchange,
including amphiphiles such as phospholipids and cationic sur-
factants, along with molecules that bear pendant thiol moie-
ties or form dithiocarbamates.[20,121–124] Successful demonstra-
tions of surfactant exchange are illustrated in work by Alkilany
et al.,[96] who employed phosphatidylcholine to reduce residual
CTAB concentrations to ≈10%.[96]
However, the majority of recent literature has focused on
developing strategies for using thiolated molecules, dithiocarba-
mates, and cyclic disulfides as a means for attaching molecules
to gold nanoparticle surfaces.The strength of the semi-covalent
Au-S bond, where studies have indicated the bond energy to be
on the order of 45 kcal mol−1, enhances the binding strength
of the tethered molecule.[125] Moreover, the attractiveness of the
gold thiol chemistry has lead to a rapid increase in the number
of available molecules bearing pendant thiol groups suitable for
ligand exchange.
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Successful approaches for ligand exchange on Au NRs
can primarily be divided into three categories: one-pot, phase
transfer, and solid-phase ligand exchange.[117,126–131] One-pot
ligand exchange is carried out by introducing the thiolated
ligand into the aqueous NR solution, and leaving the ligand
exchange product in the reaction medium. Phase transfer is
carried out in a similar fashion where an organic solution of a
hydrophobic ligand is introduced into the aqueous NR solution.
Upon ligand exchange, the NRs are extracted from the aqueous
phase, and are rendered soluble in organic solvent. Of course,
phase transfer can be carried out in the opposite fashion, but
since the typical seed-mediated, bottom-up synthesis of Au NRs
renders the particles soluble in water, we use the convention
that phase transfer implies an aqueous-to-organic transition
unless otherwise specified. Ligand exchange can also be per-
formed in solid phase through immobilization of the NRs onto
an ion exchange resin during the exchange process.
One-pot ligand exchange is the most straightforward
approach; following the place exchange methods developed
by Hostetler et al.[126] for gold nanospheres, ligands are added
directly to an aqueous NR solution, and the functionalized par-
ticles maintain their solubility during the process. Recently,
Vigderman et al.[127] indicated that one-pot exchange using a
thiolated CTAB analogue, (16-mercaptohexadecyl) trimethylam-
monium bromide (MTAB) could provide an effective means of
NR functionalization and removal of CTAB. By simply adding
MTAB to a concentrated NR solution, and purifying solution-
phase surfactants after a 48 h incubation, CTAB can be com-
pletely removed from the NR surface, as indicated by 1H NMR
spectroscopy. Additionally, MTAB-capped NRs were found to be
nontoxic, and can readily be taken up by mammalian cancer
cells, thus indicating that MTAB-NRs can be leveraged as a plat-
form for subsequent biofunctionalization (vide infra).
Another significant advancement in one-pot ligand exchange
protocols was recently published by Kinnear et al.,[128] where an
ethanolic solution of thiolated PEG (PEG-SH) is employed to
facilitate ligand exchange. The choice of PEG-SH as the ligand
of study is due to the biocompatibility imparted by PEG; In
these experiments, the introduction of PEG-SH is enabled by
the introduction of a cosolvent, which significantly raises the
critical micelle concentration (cmc) of CTAB, hence destabi-
lizing the CTAB bilayer on the NRs, and facilitating CTAB’s dis-
placement by PEG-SH. Furthermore, Kinnear et al. employed
a two-step functionalization procedure where the first step is
performed by adding 10 PEG-SH/nm2 (expressed in terms of
NR surface area) to a NR solution of reduced CTAB concentra-
tion; the resulting NR is expected to have a surface composi-
tion containing both PEG and residual CTAB.[128] Complete
grafting of the NR surface with PEG is performed in the second
functionalization step where an ethanolic solution (90% v/v
EtOH in water) of 10 PEG-SH/nm2 is added to the NR solution
([CTAB] = 1 × 10−3 M), followed by a 24-h incubation, after
which ligand exchange is complete. In this work, the pres-
ence of residual CTAB was indicated by 1H NMR, however, the
cytotoxicity was significantly reduced in comparison to the
one-step, one-pot ligand exchange.
Phase transfer methods have also shown promising results
for ligand exchange on Au NRs. In a typical phase transfer reac-
tion, a hydrophobic ligand dispersed in an organic solvent is
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added to an aqueous NR solution. As before, solvents known
for increasing CTAB’s cmc are employed to facilitate ligand
exchange, and, upon successful grafting of the NR surface, the
NRs undergo immediate precipitation from solution, where
they can be subsequently dispersed in organic solvents. Illus-
trative studies by Composto and coworkers[129] have shown that
Au NRs can be successfully grafted with hydrophobic ligands
such as thiolated polystyrene, where a range of molecular
weight can be used. However, despite the broad application of
this functionalization strategy, Au NRs-bearing ligands, which
allow their dispersion in organic solvents are not directly appli-
cable in biological systems, and would require subsequent
functionalization to render them soluble in aqueous solvents.
Furthermore, ligand exchange by phase transfer raises ques-
tions regarding the effectiveness of these strategies in removing
CTAB; initiation of ligand exchange induces rapid precipitation
of functionalized particles. It is therefore likely that CTAB can
be trapped between the polymer molecules on the NR surface.
Additionally, when enough polymer is tethered to the NR sur-
face to render the particles hydrophobic, the NRs will precipi-
tate, and likely arrest the ligand exchange reaction prematurely.
However, Gentili et al. provided strong evidence that phase
transfer exchange effectively removes CTAB. They introduced
ethanolic solutions of ethyl-11-mercaptoundecanoate to NRs
phase transferred from water into chloroform,[130] evidencing
that very little residual CTAB remained, falling below the detec-
tion limits of 1H NMR and XPS.
In 2008, Wijaya and Hamad-Schifferli[117] investigated the
efficacy of aqueous-organic-aqueous phase transfer in ren-
dering NRs viable for biological applications through what is
more commonly known as round-trip ligand exchange. In their
protocol, CTAB-capped aqueous gold NRs at high concentration
(≈10−8M) are treated with dodecanethiol (DDT) in the presence
of acetone. Upon ligand exchange, the gold NRs are extracted
directly into the organic phase leaving the aqueous, CTAB-con-
taining solution colorless, therefore indicating the full phase
transfer of NRs. Organic-to-aqueous phase transfer of DDT-
capped gold NRs was subsequently performed by refluxing
the gold NRs in toluene with a variety of mercaptocarboxylic
acids (C6, C11, and C16), to yield gold NRs having terminal car-
boxylic acid groups, which allowed gold NR dispersion within
tris-borate-EDTA buffers. The novelty in Wijaya and Hamad-
Schifferli’s[117] round-trip phase transfer protocol is that while
effectively removing the gold NRs from the CTAB-based solu-
tion, the mercaptocarboxylic acid-capped rods are readily sus-
ceptible to further place-exchange with biological molecules
such as thiolated DNA and biologically compatible molecules
such as PEG, even though the complete removal of the first
thiolated ligand has not been demonstrated and cannot be
Table 1. Comparison of ligand exchange protocols.
Exchange protocol Au-ligand interaction
One-pot surfactant exchange Electrostatic
One-pot, Murray-type reaction Semi-covalent
Phase transfer Semi-covalent
Solid phase (ion exchange resin) Semi-covalent
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warranted based on the proposed synthetic protocol. Fourier
transform infrared (FTIR) spectroscopy was performed on the
ligand-exchanged sample to probe the self-assembled mon-
olayer’s composition, and indicated that it was primarily com-
posed of the incoming ligand, but still showed the presence of
residual CTAB.
Another approach to ligand exchange, which was illustrated
in 2008 by Dai et al.,[131] utilized ion exchange resins to immo-
bilize CTAB-capped rods prior to the ligand exchange reaction;
therefore, diffusion-mediated NR aggregation that remains
prevalent in liquid phase reactions is avoided. Specifically,
CTAB-capped NRs are first immobilized onto the anionic ion
exchange resin Amberlite IRA-67, after which the polymeric
resin beads are washed, dried, and subsequently incubated in
a chloroform solution containing 11-mercaptoundecanoic acid
(MUDA). Upon a sufficient degree of ligand exchange, the NRs
desorb from the exchange resin into chloroform. Dai et al.[131]
examined the self-assembled monolayer (SAM) composition
using XPS, which provided a strong evidence that the residual
CTAB concentration was negligible. Furthermore, the carbox-
ylic acid moiety-terminated MUDA provides the opportunity to
further functionalize AuNRs using conjugation chemistry via
the pendant carboxylic acid group.
Table 1 illustrates the primary categories of ligand exchange
protocols that show promise for the manipulation of Au NRs
surfaces. By comparing the various approaches, one-pot
methods are advantageous due to the ease with which they can
be carried out. In one-pot exchange using amphiphiles, demon-
stration of their stability in comparison to thiolated analogues
is warranted. However, demonstrations that a broad range of
chemistries can be employed, while keeping aggregation losses
and residual CTAB low are needed. In contrast, despite a more
complicated procedure, ligand exchange using phase transfer
has shown versatility in the variety of biological molecules that
can be used, namely for the round-trip ligand exchange. Solid-
phase exchange gains advantage in preventing aggregation of
particles during functionalization, but the versatility of this pro-
cedure in fabricating water-soluble ligand exchange products
must be demonstrated.
All of the procedures outlined here show promise for pro-
viding functionalized particles, both in the sense of successful
ligand exchange and removal of CTAB. However, at the current
state of the art, an acceptable level of residual CTAB must be
established. The works presented here outline strategies that
take residual CTAB concentrations below the detection limits of
NMR and XPS. However, would it be acceptable to bring these
developing technologies to the marketplace?
These recent advances in gold NR functionalization have
facilitated the investigation of their applicability in sensing,
Primary benefits Primary disadvantages Cited works
No reports of particle aggregation Monodentate species less strongly [20,120–123]
bound than thiols
Ease of synthesis; strongly bound Limited chemistry has proven viable [126]
Range of viable chemistry Many synthetic steps [118,128,129]
Prevents aggregation Many synthetic steps [131]
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imaging, and drug-delivery technologies. However, bench-
top to marketplace transitions will require knowledge of the
molecular composition of the surface of the NRs, not only to
prevent the introduction of chemical species not removed by
ligand exchange (i.e., CTAB) but also to determine that the
desired surface functionalization has been achieved. Such pre-
cision requires investigations into the molecular-driving forces
of ligand exchange to understand the conditions under which
ligand exchange will occur, as well as what contributions dictate
the steady-state monolayer composition of multi-functionalized
particles.
5.1. Energetic and Morphological Considerations for
Ligand Exchange
The importance of understanding the thermodynamic-driving
forces for ligand exchange was indicated by Hostetler et al.[126]
who showed that ligand exchange rates on monolayer-pro-
tected gold clusters are significantly influenced by the molec-
ular structure of the initial protecting ligand, where the rates
decreased with increasing carbon chain length and steric bulk
of the initial capping layer. Furthermore, they observed that the
reactivity also varied with binding sites, where edge and vertex
sites showed higher exchange rates than terrace sites. These
results highlight the impact of the intermolecular forces, which
contribute to the energetic stabilization of the initial protecting
layer, and the nanoparticle’s surface characteristics in defining
surface reactivity. This is especially true in the case of Au NRs,
for which the surface curvature varies between the longitudinal
and the transverse surfaces, as well as the tips (which can be
either truncated or sphero-cylindrical depending on the syn-
thetic procedure) and likely gives rise to a significant disparity
in ligand exchange conditions, mechanisms, and yields (see
Figure 2 above).[16] Additionally, the inhomogeneity in the den-
sity of the adsorbed CTAB bilayer, due to the Au NR surface
curvature, has been indicated in recent publications, where the
curved tip exhibits a lower density of CTAB than the longitudinal
facets, making the functionalization of the tips energetically
more favorable than that of the sides.[132] Figure 2 demonstrates
the effect of Au NR morphology and crystallography on ligand
exchange. Figure 2a) exemplifies the steric constraints imposed
by a planar surface on ligand density, where steric interactions
increase the molecule footprint (dashed circles) in comparison
to a curved surface, Figure 2b), where θ is the deflection angle
of the ligand.[24] Au NRs exhibit both planar and curved regions;
longitudinal {250} facets have planar regions, which exhibit
curvature at their intersection.[78] Furthermore, the NR tip is
often hemispherical in syntheses strictly employing CTAB and
Ag (I).[16] However, Wadams et al. indicated that adding BDAC
at different times during Au NR growth can alter the tip cur-
vature; moreover, employing BDAC at early stages of Au NR
growth yields NRs with truncated tips, which are presumably
{001} facets.[16] Therefore, the radius of curvature of the tip
(Rtip) and the body’s cross section (Rbody) will often be different
from one another, and can lead to variations in grafting density
and ligand exchange energetics. The longitudinal and tip facets
are also different, which lead to different soft-epitaxial adsorp-
tion energies of surfactants (Figure 2c).[79] The variations in sur-
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face curvature between the rod’s body and tip, along with the
varying epitaxial surfaces of these regions are what separates
the case of ligand exchange on Au NRs from that on isotropic
bodies such as cubes and spheres, where surface curvature and
epitaxy remain constant, respectively.
Evidence of site-specific functionalization of Au NR tips was
initially indicated in 2003 by Caswell et al.,[132] who were able
to specifically functionalize the Au NR tip with biotin disulfide,
and subsequently to induce end-to-end NR assembly through
the introduction of the protein streptavidin, which is well
known to tightly bind up to four biotins per protein. Subse-
quently, Nepal et al.[22] demonstrated the use of Au NR tip func-
tionalization with hexadecanethiol (HDT) to fabricate side-by-
side Au NR dimers, in a study that demonstrated the disparity
in reactivity between longitudinal facets and tip regions of the
Au NR. While the tips were readily functionalized with HDT,
thereby displacing CTAB in these regions, the CTAB bilayer
on the longitudinal facets remained relatively intact, demon-
strating that a drastic difference exists between ligand exchange
reactions carried out on spherical (or more generally isotropic)
nanoparticles and that taking place on the anisotropic counter-
parts such as NRs.
The molecular structure of a ligand, whether a simple
alkylthiol or a complex protein, defines its physicochemical
interactions between itself and its identical counterparts, other
competing ligands on the nanoparticle surface, as well as
their interactions with the solvent. These interactions define
whether ligand exchange is favorable, and if so, determine
the steady-state molecular composition of the monolayer.
Oyerokun et al.[133,134] showed that the competition between
short-chain linear alkylthiols for adsorption to epitaxial gold
surfaces results in mixed monolayers, where the monolayer
composition is driven by the solvent’s selectivity for one ligand
over another, and the intermolecular interactions between all
molecular species in the monolayer. In the course-grained sta-
tistical mechanical model they developed, following the work
by Folkers et al.,[135] the molar change in the Gibbs free energy
of monolayer formation at constant temperature and pressure
is defined in Equation (2), where k is Boltzmann’s constant,
T is temperature in Kelvin, µiSAM is the chemical potential of
ligand i in the 2D SAM, µisol is the chemical potential of species
i in solution, and xi is the mole fraction of ligand i within the
system. The intermolecular interactions within this model are
defined by exchange parameters, which utilize the Hildebrand
solubility parameters to define each molecule’s hydrophobic
interactions. Furthermore, each molecule within the model is
considered structureless to maintain simplicity, and steric inter-
actions between molecules are not considered.
Δf = ⎡⎣( μ ASAM − μ Asol ) x A + ( μBSAM − μBsol ) xB ⎤⎦ (2)
In considering ligand exchange on Au NRs, or any nanopar-
ticle morphology, for biological applications, the model devel-
oped by Oyerokun et al.[133,134] cannot be directly applied, given
that Hildebrand solubility parameters become inaccurate in
systems where intermolecular interactions such as hydrogen
bonding are present. Moreover, in cases where larger molecules
such as polymers and proteins are utilized, or in cases where
large size disparity exists between competing ligands, steric
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factors relating to the molecular structure of each ligand, and
entropic considerations related to the conformational freedom
of the adsorbed molecules must also be made for each ligand
to determine the monolayer surface composition.[133,134] How-
ever, Equation (2) illustrates the importance of considering
how the concentration of each ligand, the solvent employed,
as well as the intermolecular interactions between all species
will influence ligand exchange and the equilibrium molecular
composition of the SAM. For example, in cases where the sol-
vent is selective for one ligand, the ratio of ligands with respect
to each other in solution will not be equal to their ratio in a
mixed monolayer. In a practical sense, one can generally expect
the SAM composition to change over time if the functional-
ized nanoparticles are stored within a solution having different
ligand concentrations than those employed during the ligand
exchange reaction.
The nanoparticle morphology also plays a role in deter-
mining the likelihood of ligand adsorption and the ligand
grafting density. Oyerokun and Vaia[134] investigated the
dependence of end-functionalized homopolymer adsorption to
curved nanoparticle surfaces, and showed that the grafting den-
sity increases with nanoparticle curvature. They also showed
that the polydispersity in the grafting density increases as the
particle size becomes smaller than the unperturbed radius of
gyration of a single polymer chain in solution. Thus, alterations
in the nanoparticle curvature would produce different molec-
ular monolayers if one employed an identical ligand exchange
procedure. Similarly, for large molecules such as proteins, the
altered curvature is also expected to change the proclivity of
each species to bind to the nanoparticle surface.
The above works describe ligand exchage on spherical gold
nanoparticles, as well as competitive adsorption of ligands
onto a planar gold surface, and while not directly applicable to
ligand exchange on Au NRs, illustrate areas of research, which
will have to be undertaken to realize the application of AuNRs
bearing multifunctional SAMs. Beyond the disparate reactivity
between Au NR tips and longitudinal facets, studies to charac-
terize the dependence on reaction conditions of the SAM com-
position at equilibrium are needed (e.g., solvent and ligand con-
centration). Moreover, understanding the role of each ligand’s
molecular structure and interactions with other desired ligands
is necessary, whether treated through theoretical or heuristic
means, in order to answer questions such as: what reaction
conditions are necessary to provide the desired surface compo-
sition of multifunctional SAMs, or to make CTAB removal ther-
modynamically favorable? Similarly, what storage conditions
are necessary to achieve the desired bioactivity? Such studies
would also establish the time scales that define multicompo-
nent SAM formation, and thus the ligand exchange methods
that can be utilized.
This discussion on ligand exchange is by no means exhaus-
tive of the many exotic organic molecules and proteins
employed to functionalize gold NRs, but is meant to review
important works in ligand exchange on Au nanoparticles and
surfaces, as well as to suggest that detailed investigations into
the energetics of Au NR functionalization are lacking. As the
fields of application of gold NRs grow in number, so does the
necessity to fully understand the mechanisms at the basis of
ligand exchange. As we have described, some data have been
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made available by elegant studies on spherical gold particles
and flat surfaces, and without a doubt this information can be
extrapolated to study gold NRs. However, because of their fun-
damental structural differences both in shape and monolayer
density, a wholly independent treatment should be carried out.
6. Characterization Techniques used to Study
CTAB Removal
CTAB-capped NRs exhibit significant cytotoxicity and colloidal
instability in biological media.[92,95,96,136] These limitations
can be overcome to a certain extent by replacing CTAB with
thiolated PEG, alkanethiols, and also by surface coating with
polyelectrolytes and phospholipids.[56,97,101,108] These surface
modifications not only alleviate the NR’s cytotoxicity but also
facilitate further functionalization for specific cell targeting,
bio- and chemical sensing, and self-assembly.[40,56,137] However,
these types of reactions follow dynamic equilibria that may lead
to situations where the full removal or encapsulation of CTAB
cannot be achieved. In order to evaluate the successful removal
and for protocol optimization, a thorough materials characteri-
zation is necessary.
Both qualitative and quantitative analysis can be conducted
to confirm the degree of ligand exchange. The NR surface
ligand composition, surface packing density, and more impor-
tantly strong evidences of relative surface ligand coverage must
be presented to confirm the extent of CTAB replacement with
the incoming ligands. Direct and indirect quantitative charac-
terization techniques such as zeta-potential measurements, gel
electrophoresis, and Raman and FTIR spectroscopies have been
extensively used to confirm the presence of incoming ligands
after ligand exchange reactions but not the relative composition
of CTAB and the incoming ligand on the NR surface. However,
quantitative analytical tools such as XPS, NMR, and mass spec-
trometry (MS) can provide useful evidence of ligand exchange.
Few commendable studies of NR ligand exchange processes
that have employed quantitative techniques along with theo-
retical calculations can be found in the literature.[138] In this
section, we will highlight some of the characterization tools that
have been employed in published work to analyze the NR inter-
face after ligand exchange.
The measurement of the zeta potential is the most widely
used characterization technique to confirm the surface modi-
fications of CTAB-capped NRs.[93,104,139] It is an indirect
technique that only provides information of the charged sur-
rounding of the NR but not any conclusive evidence on com-
plete CTAB removal or on the percentage of ligand surface cov-
erage. It also limits its applicability to the ligand exchange of
cationic CTAB with either anionic or neutral incoming ligands.
For instance, Jain et al.[39] have used zeta potential measure-
ments to map the positive to negative charge reversal in gold
NRs upon ligand exchange of CTAB with polystyrenesulfonate
(PSS) and claimed it as a sufficient evidence of successful
ligand exchange. However, this method is unable to distinguish
the replacement of CTAB with another cationic ligand such as
polyallylamine hydrochloride, and to provide any useful infor-
mation on the chemical identity or the composition of the NR
surface ligands. For example, it is not able to identify whether
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CTAB has been completely or only partially removed from the

NR surface.
Another indirect qualitative method that has often been used
to characterize ligand exchange and NR surface chemistry is
agarose gel electrophoresis.[117,139–141] In gel electrophoresis,
the electrophoretic mobility shifts depend on the net surface
charge and the total molecular weight of the samples. Upon
ligand exchange, the total molecular weight of the NR and the
surface ligand chemistry are changed, which are evidenced by
the degree of electrophoretic mobility shift and the direction of
mobility, respectively. The round-trip ligand exchange method
developed by Hamad-Schifferli and co-workers[117,141] to ligand
exchange CTAB with thiolated-PEG and also with polyelectro-
lytes uses gel electrophoresis to study the reaction products.
Based on the gel electrophoretic mobility shifts, successful
ligand exchange of CTAB and the saturation of NR surfaces
with the incoming ligand were confirmed. Shi et al.[140] have
recently presented an efficient method of NR surface modifica-
tion with thiolated ligands for core-satellite assembly. They have
used agarose gel electrophoresis to confirm the conjugation of
different oligonucleotides to NRs. Due to their instability in
tris-borate-EDTA, the buffer used in gel electrophoresis, CTAB-
capped NRs aggregate in the wells and do not show any elec-
trophoretic mobility. But NRs that have attained stability upon
DNA conjugation do exhibit distinct electrophoretic mobilities
that can be directly correlated to the size of the conjugated oli-
gonucleotides. The authors have also claimed that the presence
of smear bands in the gel is an evidence of incomplete ligand
exchange, which becomes less prominent upon near to com-
plete surface coverage of NRs by oligonucleotides. A similar
study by Pekcevick et al.[142] has also used agarose gel electro-
phoresis to determine the relative surface coverage of single-
stranded DNA on NRs based on their relative positions on the Figure 6. FTIR spectra of pure CTAB, pure PEG, AuNR@PEG (containing
gel in comparison to the original NR sample. However, gel CTAB), pure disulfide initiator, AuNR@initiator, pure PVP, and AuNR@
electrophoresis cannot be used for accurate quantitative estima- PVP samples (from top to bottom). The peaks around 2300–2400 cm−1
tions of the surface coverage of ligand on NRs, and quantitative in some of these samples are due to the absorbance of CO2 in air. Repro-
analytical tools must be employed. duced with permission.[143] Copyright 2011, Royal Society of Chemistry.
Qualitative characterization of the ligand exchange in CTAB-
capped NRs has also been evidenced by FTIR and Raman spec- appearance of characteristic GSSG bands for the GSSG-capped
troscopy.[117,143–145] FTIR provides information on the NR sur- NRs, which was used as a strong evidence to support complete
face chemistry by means of characteristic vibrational modes ligand exchange of the CTAB bilayer with GSSG. The authors
of surface ligands. For instance, Li et al.[143] have showed the have assumed that the residual CTAB on the NR surface is neg-
coexistence of PEG-CTAB and polyvinylpyrrolidone (PVP)- ligible. In comparison to other characterization techniques, it
CTAB after ligand exchange reactions by the presence of their is fair to state that Raman or IR spectroscopy do provide strong
characteristic peaks on FTIR spectra. For the CTAB-NR sam- qualitative evidence of ligand exchange reactions but, because
ples ligand exchanged with thiolated PEG, the peaks centered of their reduced sensitivity, they are still not sufficient to draw
at 1466 cm−1 correspond to δasym (C–H) and δsym (C–H) of quantitative conclusions with a high degree of accuracy. For
the CH3–N+ moiety in CTAB, and the peak at 1086 cm−1 cor- example, the presence of a relatively higher percentage of one
responds to the characteristic ether (C–O–C) bond stretching ligand could mask the spectroscopic peaks of another ligand
of PEG, supporting the partial ligand exchange of CTAB with with a lower percentage that appears in the same region thereby
PEG (see Figure 6). Gomez et al.[144] have also demonstrated misleading to conclude that the complete CTAB removal has
the complete removal of CTAB followed by NR surface capping takn place.
with PSS and PEG by FTIR analysis. Wang et al.[145] have used XPS has been used in many instances to confirm the com-
Raman spectroscopy to characterize the surface modification pleteness of CTAB ligand exchange processes.[130,131,146] XPS is
of CTAB-NRs with zwitterionic oxidized glutathione (GSSG) a direct, quantitative analytical method that provides detailed
to improve their biocompatibility and their application in tar- information on the ligand composition on the NR surface,
geted tumor imaging and therapy. The Raman spectrum of and therefore strong evidence of ligand exchange. The peaks
the GSSG-capped NRs shows the disappearance of the strong corresponding to the kinetic energies of the elements can be
characteristic CTAB peaks at 763, 1061, and 1460 cm−1, and the used to directly identify the ligands, while quantitative data (the

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relative concentration of the elements and hence the relative
percentage of ligands) can be extracted from the peak height
or the peak area. The complete displacement of CTAB by an
incoming ligand can be confirmed by the absence of the bro-
mine (Br3d) signal (67.4 eV) and the nitrogen signal originating
from CTAB, and the presence of characteristic elemental sig-
nals typical of the incoming ligand (e.g., thiol signal for thi-
olated ligands).[131,146] Pekcevick et al.[142] have used the high-
resolution N1s spectra to differentiate and quantify the free
CTAB molecules in the solution and the NR-bound CTAB.
They have reported a 3.1 eV shift between the N1s peaks for NR-
bound and unbound CTAB, which could be a useful measure
of the purity of the NR sample. In addition, a comparison of
the characteristic N1s spectra of the incoming ligand and that
of CTAB have been used to evaluate the success of ligand
exchange processes.[142] In many studies, XPS has been used
merely as a qualitative characterization tool to confirm ligand
exchange by elemental analysis, but its full potential in quanti-
tative evaluations has not been appreciated well enough, which
could be successfully implemented to achieve complete NR sur-
face analysis.
1H NMR spectroscopy has also been used to qualitatively
and quantitatively confirm NP ligand exchange processes. For
qualitative purposes, the disappearance of peaks corresponding
to CTAB, and the appearance of the incoming ligand-specific
peaks can be taken into consideration. The disappearance of
peaks at 1.3 and 3.1 ppm for –CH2– and –N(CH3)3 of CTAB
can be used as a strong qualitative evidence of the successful
replacement of CTAB with thiolated molecules such as long-
chain alkanethiols and thiolated-PEG molecules.[130,140,141] For
instance, Kim et al.[147] have confirmed the successful ligand
exchange of CTAB with Pluronic 127 by the disappearance
of the –N(CH3)3 peak corresponding to CTAB along with the
appearance of characteristic peaks for Pluronic –CH2– groups
at 3.7 ppm. However, the 1H NMR peak assignments could be
limited when the incoming ligand has functional similarities
with the molecule of CTAB, which leads to less accurate results.
In some cases, the peak assignment for ligands could also be
impacted by changes in peak shift, splitting, and broadening
upon binding of ligands to the NRs, and thus careful analysis
will be required prior to quantitative analysis. Despite its lim-
ited sensitivity compared with other techniques, 1H NMR can
be used for quantitative purposes by comparing the relative
peak areas of the ligand-specific peaks, even though it should
be remembered that the identification of surfactants directly
bound to the NR surface could prove challenging because of
the reduced molecular tumbling time and hence the resulting
broadened peaks.[126,148,149] This notwithstanding, the quan-
tification of ligands by 1H NMR has been successfully car-
ried out with many different types of inorganic nanoparticles
including Janus gold nanoparticles and spherical gold nano-
particles, and has proven extremely valid when the identifica-
tion of ligands is carried out after nanoparticle digestion.[149] An
excellent example is provided by a recent study by Vigderman
et al.,[127,148] who have used quantitative 1H NMR spectroscopy
to prove the complete replacement of CTAB by its thiolated
analogue MTAB. In that work, CTAB- and MTAB-capped NRs
were dissolved using KCN to remove surface-bound ligands
before NMR analysis. The presence of CTAB in the as-prepared
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solutions was confirmed by the signal from the terminal methyl
group at 0.91 ppm, which is absent in the MTAB spectrum.
No signal corresponding to the terminal methyl group was
detected in MTAB-capped NRs, confirming the complete CTAB
removal. The authors have also claimed that given the fact that
the concentration of the test solutions (10−2 M) is above the min-
imum detection limit of 1H NMR (10−5 M), there is only less
than 0.1% possibility that residual CTAB has gone undetected
by NMR spectroscopy. Thus, high-resolution 1H NMR could
also be applicable as a suitable analytical tool for quantitative
characterization of NR surface ligand exchange processes.
Mass spectrometry is one of the most powerful analytical
techniques that has been successfully used to identify and to
determine the surface density and coverage of ligands of the
mixed ligand monolayers on gold nanoparticles.[150–152] An
increased use of inductively coupled plasma mass spectrom-
etry (ICP-MS) and ion mobility mass spectrometry (IM-MS) for
rapid, facile, and relative quantification of thiolated PEG-capped
gold nanoparticles can be found in the recent literature.[151,153]
Mass spectrometry is applicable to ligands of similar function-
alities since their identity is based on the their mass and unique
fragmentation patterns. Therefore, mass spectrometry would
be the best analytical tool to employ to achieve precise and accu-
rate quantitative characterization of NR surfaces upon ligand
exchange processes. In a typical mass spectrometry analysis,
prior to data acquisition, gold nanoparticles are subjected to a
digestion process followed by an extensive purification protocol
to isolate the surface ligands from the gold core. However, only
a handful of studies can be found in the literature that employ
this technique for the analysis of CTAB-capped NRs. For CTAB,
the peak for the CTA+ ion at m/z = 284.33 is considered for
analytical comparisons. A recent study by Chen et al.[154] has
reported almost complete CTAB removal due to the absence of
the CTA+ signal from a ligand-exchanged NR sample at 0.06 ×
10−3 M concentration, which is well above the minimum detec-
tion limit of 10 × 10−9 M for CTAB by ICP-MS. Alkilany et al.[151]
have used liquid chromatography–mass spectrometry (LC-MS)
to quantify the percentages of CTAB (23 ± 1%) and 11-(acryloy-
loxy)undecyl trimethylammonium bromide (77 ± 3%), an anal-
ogous polymerizable surfactant, after ion exchange processes.
Quantification of the NR surface ligand coverage has also
been achieved by a combination of experimental analysis (e.g.,
colorimetric assays, thermogravimetric analysis) with some
theoretical calculations. Bogliotti et al.[155] have used a UV
spectroscopy-based colorimetric analysis to indirectly quantify
the replacement of CTAB with HS-PEG-NH2. Here, NR-bound
NH2-PEG moieties are allowed to react with N-succinimidyl-
3-(2-pyridyldithio)propionate followed by dithiothreitol, which
releases thiopyridine as a product. The released thiopyridine
can be quantified by its absorbance at 345 nm, and was used
as an indirect measure of the NH2-PEG moieties, which in turn
can translate to the NR surface coverage. These experimental
results have shown a good agreement with the calculations
based on PEG footprint and the surface area of NRs for com-
plete ligand exchange. In a similar approach, Xia et al.[156] have
used a ninhydrin-based assay to quantify the surface coverage
of PEG-NH2 on NRs by means of UV-vis spectroscopy. They
have calculated the number of PEG-NH2 on NRs by subtracting
that for the supernatant from the number of HS-PEG-NH2
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added to the NR suspension. Petri-Fink and coworkers[128]
have employed thermogravimetric analysis (TGA) to quantify
the amount of PEG, and thereby to calculate the PEG grafting
density on the NR surface.Joshi et al.[138] have also performed
similar calculations where methoxy-PEG and residual CTAB are
determined by XPS and compared with the footprint evaluation
for CTAB and methoxy-PEG upon ligand exchange.
In summary, both qualitative and quantitative analytical
tools can be useful to characterize the surface of NRs. But
understanding the NR interfaces and concluding that complete
ligand exchange has successfully taken place requires stronger
and more convincing experimental data beyond preliminary
qualitative evidence that is only able to provide the chemical
identity/overall composition of the NR surface ligands. Instru-
mentation such as XPS, NMR, and mass spectrometry should
be employed (up to the maximum potentials that they can offer)
in order to provide sensitive, precise, and accurate quantitative
data relative to the ligands on the surface of the NRs, which
could be used to quantify the extent of ligand exchange. The
quantitative measurements and theoretical calculations could
be used in synergy to reveal more useful information for a
better understanding of the NR surface chemistry and thus the
ligand exchange processes.
7. The Interface
The interactions between the capping ligands and the gold sur-
face determine many of the properties of gold NRs, namely
their stability, their solvation, and their miscibility with an
external matrix. As a consequence, only a thorough under-
standing of the surface properties and of the ligand exchange
reactions that enable us to tune them, will give us the ability to
utilize gold NRs in many of their envisioned applications. It is
therefore fundamental to be able to characterize the gold NR
interface, so that we can understand its properties and predict
how they will vary depending on the conditions in which the
NRs are employed. In this section, we will examine the con-
cept of interface in gold NRs. It is generally agreed that in gold
NRs two different interfaces can be encountered, the first being
identified as the one that separates the NR as a whole (i.e., core
and ligand shell) from the outer environment, hence deter-
mining solvation and dispersibility, and the other being the one
between the gold surface and the bound or adsorbed capping
ligand. To understand which of the two is the relevant interface
in our specific application and to identify its relevant proper-
ties, we should devise, on a case-by-case basis, a set of accu-
rate and quantitative experiments to characterize it. As we hope
to have sufficiently stressed in this review, indirect evidence is
not sufficient to precisely describe the properties of a complex
system such as gold NRs, and a quantitative approach to study
the interface, before and after ligand exchange, should always
be employed.
Some very thorough reviews have been published in the last
few years that focus on the notion of interface in the context
of gold nanoparticles.[157–160] These reviews target gold nano-
particles as a general category, and the majority of them focus
on the biological interface. This interface is considered as the
one between a nanoparticle as a whole (i.e., the core and the
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capping layer) and a biological environment surrounding it
(such as a protein, a cell, a tissue). The bio-nano interface has
for several reasons gained a position of primary interest in the
scientific community, for the obvious important consequences
that its control, or lack thereof, can bring to all the biological
applications in which gold nanoparticles find a key role. In fact,
a lack of control of the properties of the interface can lead to
coalescence and precipitation, opsonization, lack of uptake,
endocytosis and enzymatic digestion, and more than anything,
to toxicity. On the other hand, the better we control the way
we manipulate the interface between the nanoparticle and the
biological environment, the more likely we are to succeed in
bringing nanotechnology to meet the expectations that society
has been posing on this field for the last decade or so.
The second type of interface, as described above, is the one
that separates the metallic surface from the chemisorbed or
physisorbed monolayer. Covalently bound monolayers impart
increased stability to the surface compared with physisorbed
ones, and their behavior has been studied in quite some depth
for spherical gold nanoparticles.[126,133,134] On the other hand,
the behavior of physisorbed monolayers such as CTAB is more
difficult to quantify and, as a consequence, a thorough under-
standing of how gold NRs can be functionalized by ligand
exchange is still lacking. An excellent example of how the inter-
face between the protein corona and the surface of Au NRs can
be examined was reported by Chen and coworkers,[110] who, by
combining X-ray absorption near-edge structures (XANES), cir-
cular dichroism (CD), and molecular dynamics (MD) simula-
tions, were able to identify the binding motifs of bovine serum
albumin (BSA), an abundant sulfur rich protein commonly
used to render the nanoparticles biocompatible and as a surface
blocking agent (Figure 7). By looking at the k-edge XANES of
sulfur, the authors were able to identify that, when BSA inter-
acts with the surface of gold NRs, its 17 disulfides, 5 methio-
nine S-CH3, and one free thiol decrease in number to give rise
to Au–S species, with the disulfides giving the largest contribu-
tion. The authors were also able to determine that the Au–S
bond formation is accompanied by a protein morphology evolu-
tion, that has been proven via CD. Although the authors were
not able to observe a change in the Au XANES patterns due to
the resolution limits of the instrument (the modified surface Au
atoms are outnumbered by the inner ones), and even though
their study applies only to mixed monolayers of CTAB and BSA
on gold NRs, this work is an example of how a quantitative anal-
ysis of the interface on gold NRs can successfully be carried out.
The characterization of the interface and of the phenomena
that happen at the interface has recently become a buzzword in
science. Notable fundamental studies are being proposed and
executed, but the importance of a clear understanding of the
interface on the research outcomes has not yet been recognized.
Despite the fact that two types of interface have been identified
for gold nanoparticles, the experimental works reported in the
literature often focus on either one or the other, with a demar-
cation that becomes blurry in the case of gold NRs. A study to
bridge these two definitions has been published by Hamad-
Schifferli and coworkers,[161] who using a sensitive optical
pump-probe technique have examined the thermal conductance
of the ligand layer in gold NRs, determining that its properties
also depend on the amount of free ligand in solution. Despite
Part. Part. Syst. Charact. 2014, 31, 819–838
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PROGRESS REPORT
Figure 7. Characterization of the interaction between AuNRs and sulfur-containing molecules (BSA, cysteine, cystine, methionine, and 11-mercap-
toundecanoic acid (MUA, thiol)). TEM images of: A) CTAB/AuNRs and B) BSA corona-coated AuNRs. C) Normalized S K-edge XANES spectra of
various sulfur species in reference samples: Au–S, R–S (cysteine, thiol, Met), and R–S–S–R′ (cystine). D) Chemical species of sulfur after incubation
with AuNRs. E) Experimental and least-square fitting S K-edge XANES data for free BSA and for BSA on AuNRs. F) Disulfides of BSA (yellow) binding
to the Au (111) surface of AuNRs. BSA is rendered as a cartoon with the three domains in cyan, red, and blue. G) Chemical species of gold in various
samples incubated with BSA, cysteine, cystine, and thiol, respectively. The chemical species of gold are shown as normalized gold L-III edge XANES. H)
Changes in the secondary structures of BSA adsorbed on AuNRs, determined by CD spectroscopy. Reproduced with permission.[110] Copyright 2013,
American Chemical Society.

its relevance however, this work is only an isolated example and
evidences once more the need for further studies covering the
fundamentals of the interface in gold NRs.
8. Conclusions
In this Progress Report, we have critically analyzed the most
recent literature on gold NRs trying to extract the current knowl-
edge on ligand exchange. We have also attempted to provide a
curated list of techniques that are fundamental for the characteri-
zation of the surface composition of gold NRs before and after
ligand exchange, while highlighting the limits of some of those
currently employed in the field. What is important to remember
is the fact that a purely qualitative analysis of the properties of
gold NRs is no longer sufficient if we want to move these mate-
rials to the next stage of technological development, and that it
should be replaced by a quantitative evaluation of the materials
properties. In addition to this, it is important to keep in mind
the fact that CTAB cannot be considered a mere surfactant, as its
presence influences the synthesis of the nanorods and the crys-
tallographic properties of their thermodynamically stable facets,
and that its replacement via ligand exchange should be studied
with quantitative experiments and possibly modeled via theoret-
ical calculations to draw a much clearer picture than currently
available. Throughout this Progress Report, we have also stressed
that the toxicity of CTAB is to attribute primarily to the pres-
ence of free CTAB in the nanorod suspension, but also that the
available studies have been carried out using a wide range of NR
concentrations, aspect ratios, incubation times, and most impor-
tantly different cell lines and cell models, which significantly
impact the reported results. The knowledge we currently have on
gold NRs has been a fundamental basis for the development of
the current nanorod-based technologies, but it is not sufficient to
bring these materials to the next level unless we implement a set
of quantitative characterization protocols and theoretical models
capable of overcoming the limitations of the current approaches.
Because of the limits of the analytical techniques and proto-
cols currently employed, it is nowadays difficult to understand
and to predictably carry out ligand exchange on gold NRs and to
fully characterize the reaction products. As scientists we know
that to understand a phenomenon, or to find the solution to a
problem, we have to clearly define it. If we do not know how to
define it, we cannot pose the right questions and find the right
experimental approaches that could help us answer them. The
future for the technological applications of gold NRs is bright.
On a daily basis, we can find interesting new results being
published in the scientific literature that bring the promise of
technological breakthroughs. But perhaps these breakthroughs
would be even more important, if we were able to study the
fundamental properties of these materials. The scientific com-
munity should perhaps turn back and look again at the funda-
mental studies, focusing on the mechanisms, understanding
the kinetics and thermodynamics, and try to clearly define the
properties of these materials using quantitative characteriza-
tion techniques. For many reasons, we are often pushed toward

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experiments that could bring a certain degree of “wow factor.”
But at this day and time, it would certainly be more appropriate
to look at the chemistry and physics of gold NRs with an old
school philosophy. In order for these materials to move forward
to future technological applications, the scientific community
should reapply well-established scientific approaches (however
not the techniques) and fundamentally understand them. All in
all, we should just go back to the future.
Acknowledgements
This work was supported by start-up funds from Rutgers University.
R.C.W. acknowledges the National Science Foundation grant 0903661
(“Nanotechnology for Clean Energy IGERT”).
Received: January 9, 2014
Revised: February 23, 2014
Published online: April 1, 2014
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