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Cite this: Chem. Soc. Rev., 2012, 41, 22562282


Gold nanoparticles in biomedical applications: recent advances

and perspectives
Lev Dykmana and Nikolai Khlebtsov*ab
Received 19th June 2011
DOI: 10.1039/c1cs15166e
Gold nanoparticles (GNPs) with controlled geometrical, optical, and surface chemical properties
are the subject of intensive studies and applications in biology and medicine. To date, the ever
increasing diversity of published examples has included genomics and biosensorics, immunoassays
and clinical chemistry, photothermolysis of cancer cells and tumors, targeted delivery of drugs
and antigens, and optical bioimaging of cells and tissues with state-of-the-art nanophotonic
detection systems. This critical review is focused on the application of GNP conjugates to
biomedical diagnostics and analytics, photothermal and photodynamic therapies, and delivery
of target molecules. Distinct from other published reviews, we present a summary of the
immunological properties of GNPs. For each of the above topics, the basic principles, recent
advances, and current challenges are discussed (508 references).

1. Introduction
Gold was one of the rst metals discovered by humans, and
the history of its study and application is estimated to be a
minimum of several thousand years old. The rst information

Institute of Biochemistry and Physiology of Plants and

Microorganisms, RAS, 13 Pr. Entuziastov, Saratov 410049,
Russian Federation. E-mail:
Saratov State University, 83 Ul. Astrakhanskaya, Saratov 410012,
Russian Federation

Dr Lev A. Dykman, leading

researcher of the Immunochemistry Lab at the Institute
of Biochemistry and Physiology of Plants and Microorganisms Russian Academy
of Sciences. He has published
more than 200 scientic works
including one monograph on
colloidal gold nanoparticles.
His current scientic interests
fabrication of gold nanoparticles and their applications
to biological and medical
Lev Dykman
studies. In particular, his
research is aimed at interaction of nanoparticles and conjugates
with immune-responsible cells and at the delivery of engineered
particles to target organs, tissues, and cells.


Chem. Soc. Rev., 2012, 41, 22562282

on colloidal gold can be found in tracts by Chinese, Arabic,

and Indian scientists, who obtained colloidal gold as early as
in the fth and fourth centuries B.C. and used it, in particular,
for medical purposes (the Chinese gold solution and the
Indian liquid gold).
In the Middle Ages in Europe, colloidal gold was studied
and employed in alchemists laboratories. Specically, Paracelsus
wrote about the therapeutic properties of quinta essentia auri,
which he prepared by reducing auric chloride with alcohol or
oil plant extracts. He used potable gold to treat some mental

Professor Nikolai G. Khlebtsov,

head of the Nanobiotechnology
Lab at the Institute of Biochemistry and Physiology of
Plants and Microorganisms
Russian Academy of Sciences.
He also holds a Biophysics
Chair at the Saratov State
University. He has published
more than 300 scientic
works, including two monographs. His current scientic
interests include biophotonics
and nanobiotechnology of
Nikolai Khlebtsov
biomedical applications of
metal nanoparticles, static and dynamic light scattering by small
particles and clusters, programming and computer simulation of
light scattering and absorption by various metal and dielectric
nanostructures. Prof. Khlebtsov also serves as an Associate
Editor of the Journal of Quantitative Spectroscopy and Radiative Transfer.
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The Royal Society of Chemistry 2012

disorders and syphilis. Paracelsus once proclaimed that chemistry

is for making medicines, not for making gold out of metals.
His contemporary Giovanni Andrea applied aurum potabile to
the treatment of lepra, ulcer, epilepsy, and diarrhea. In 1583,
alchemist David de Planis-Campy, surgeon to the King of
France Louis XIII, recommended his elixir of longevityan
aqueous colloidal gold solutionas a means of life prolongation.
The rst book on colloidal gold preserved to our days was
published by philosopher and doctor of medicine Francisco
Antonii in 1618.1 It contains information on the preparation
of colloidal gold and on its medical applications, including
practical suggestions.
In 1880, a method was put forward to treat alcoholism by
intravenous injection of a colloidal gold solution (gold
cure).2 In 1927, the use of colloidal gold was proposed to
ease the suering of inoperable cancer patients.3 Colloidal
gold in color reactions toward spinal-uid and blood-serum
proteins has been taken advantage of since the rst half of the
twentieth century.4 Colloidal solutions of the 198Au gold
isotope (half-life time, 65 h) were therapeutically successful
at cancer care facilities.5 More recent examples of colloidal
gold applications include catalytic processes and electron
transport in biomacromolecules,6 transport of substances into
cells by endocytosis,7 investigation of cell motility,8 and improvement of PCR eciency.9
Despite the centuries-old history, a revolution in immunochemistry,10 associated with the use of gold particles in
biological research, took place in 1971, when British researchers
W. P. Faulk and G. M. Taylor published an article titled An
immunocolloid method for the electron microscope.11 In that
article, a technique was described to conjugate antibodies with
colloidal gold for direct electron microscopic visualization of
Salmonella surface antigens, representing the rst time that a
colloidal gold conjugate served as an immunochemical marker.
From this point on, the use of colloidal-gold biospecic
conjugates in various elds of biology and medicine became
very active. There has been a wealth of reports dealing with the
application of functionalized gold nanoparticles (GNPs; conjugates with recognizing biomacromolecules, e.g., antibodies, lectins,
enzymes, or aptamers)1214 to the studies of biochemists, microbiologists, immunologists, cytologists, physiologists, morphologists,
and many other specialist researchers.
The range of uses of GNPs in current medical and biological
research is extremely broad. In particular, it includes genomics;
biosensorics; immunoassay; clinical chemistry; detection and
photothermolysis of microorganisms and cancer cells; targeted
delivery of drugs, peptides, DNA, and antigens; and optical
bioimaging and monitoring of cells and tissues with the use of
state-of-the-art nanophotonic recording systems. GNPs have
been proposed for use in practically all medical applications,
including diagnostics, therapy, prophylaxis, and hygiene
(e.g., in water purication15). Extensive information on the most
important aspects of preparation and use of colloidal gold in
biology and medicine can be found elsewhere.1630 Such a
broad range of application is based on the unique physical and
chemical properties of GNPs. Specically, the optical properties
of GNPs are determined by their plasmon resonance, which is
associated with the collective excitation of conduction electrons
and is localized in a wide region (from visible to infrared,
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Scheme 1 Generalized scheme for the biomedical application of

GNPs. Along with basic applications in diagnostics and therapy, this
review briey discusses the immunological properties of GNPs.

depending on particle size, shape, and structure).31 Scheme 1

shows a simplied scheme for the current biomedical applications
of GNPs, which reects the structure of this review. However,
since biodistribution and toxicity have been extensively reviewed
and discussed in a number of recent publications (see, e.g., ref. 32
and references therein), we restrict ourselves to a short comment
in the Conclusions section.
Considering the great body of existing information and the
high speed of its renewal, we chose in this review to generalize
the data that have accumulated during the past few years for
the most promising directions in the use of GNPs in current
medical and biological research.

2. GNPs in diagnostics
2.1 Visualization and bioimaging methods
GNPs have been actively used in various visualization and
bioimaging methods to identify chemical and biological
agents.33,34 Historically, electron microscopy (mainly its transmission variant, TEM) has for a long time (starting in 197111)
been the principal method to detect biospecic interactions
with the help of colloidal gold particles (owing to their high
electron density). Although GNPs can intensely scatter and
emit secondary electrons, they have not received equally wide
acceptance in scanning electron microscopy.35 It is no mere
chance that the rst three-volume book on the use of colloidal
gold36 was devoted mostly to the application of GNPs in
TEM. A peculiarity of current use of the electron microscopic
technique is the application of high-resolution transmission
electron microscopes and systems for the digital recording and
processing of images.37 The major application of immunoelectron
microscopy in present-day medical and biological research is
the identication of infectious agents and their surface antigens3840
(Fig. 1a). The techniques often employed for the same purposes
include scanning atomic-force41 (Fig. 1b), scanning electron,42
and uorescence43 microscopies.
Alongside the use of classic colloidal gold with quasispherical particles (nanospheres) as labels for microscopic
studies, the past few years have seen the application of
nonspherical cylindrical particles (nanorods), nanoshells, nanocages, nanostars, and other types of particles, referred to by
Chem. Soc. Rev., 2012, 41, 22562282


Fig. 1 TEM image of a Listeria monocytogenes cell labeled with an

antibodycolloidal gold conjugate (a) and a scanning atomic-force
microscopy image of tobacco mosaic virus labeled with an antibody
colloidal gold conjugate (b). Adapted from Bunin et al.39 (a) and from
Drygin et al.41 (b) by permission from Springer.

the broad term noble-metal plasmon-resonant particles25

(Fig. 2). Table 1 illustrates some plasmonic properties and
possible biomedical applications of the gold nanomaterials
shown in Fig. 2. This is by no means a detailed description of
geometrical or optical parameters (size, shape, structure,
absorption and scattering cross sections, their spectra, etc.).
In fact, we only provide typical ranges for the major plasmon
wavelength and its sensitivity to the dielectric environment in
terms of plasmonic shift per refractive index unit (RIU).
Accordingly, Fig. 2 and Table 1 should be considered as an
attempt to show a cross section of the work in this area, rather
than a comprehensive list.
Recently, the popularity of visualization methods employing
GNPs and optical microscopy,55 in particular confocal laser
microscopy, has also been on the rise. Confocal microscopy is
a technique for detecting microobjects with the aid of an
optical system ensuring that light emission is recorded only
when it comes from objects located in the systems focal point.
This allows one to scan samples according to height and,
ultimately, to create their 3D images by superposition of
scanograms. In this method, the use of GNPs and their
antibody conjugates permits real-time detection of gold penetration
into living (e.g., cancerous) cells at the level of a single particle
and even estimation of their number.5659
Confocal images can be obtained with, e.g., detection of
uorescence emission (confocal uorescence microscopy) or
resonance elastic or two-photon (multiphoton) light scattering
by plasmonic nanoparticles (confocal resonance-scattering or
two-photon luminescence microscopy). These techniques are
based on detecting microobjects with the optical microscope, in
which the luminescence of an object is excited owing to simultaneous absorption of two (or more) photons, the energy of each of
which is lower than that needed for uorescence excitation. The
basic advantage of this method is the increase in contrast through
a strong reduction in the background signal. Specically, twophoton luminescence of GNPs enables molecular markers of
cancer to be visualized on or inside cells,6063 Bacillus spores,64
and the like. Fig. 3a shows an example of combined bioimaging
of cancer cells with the help of adsorption, uorescence, and
luminescence plasmon resonance labels.
Dark-eld microscopy remains one of the most popular GNPaided bioimaging methods. It is based on light scattering by

Chem. Soc. Rev., 2012, 41, 22562282

microscopic objects, including those whose sizes are smaller than

the resolution limit for the light microscope (Fig. 3b and c). In
dark-eld microscopy, the light entering the objective lens is solely
that scattered by the object at side lighting (similarly to the Tyndall
eect); therefore, the scattering object looks bright against a dark
Compared with uorescent labels, GNPs have greater potential
to reveal biospecic interactions with the help of dark-eld
microscopy,65 because the particle scattering cross section is three
to ve orders of magnitude greater than the uorescence cross
section for a single molecule. This principle was for the rst time
employed by Mostafa El-Sayeds group66 for their new method of
simple and reliable diagnosis of cancer with the use of GNPs. The
method is founded on the preferential binding of GNPs conjugated with tumor-antigen-specic antibodies to the surface
of cancerous cells, as compared with binding to healthy cells.
With dark-eld microscopy, therefore, it is possible to map
out a tumor with an accuracy of several cells (Fig. 3b and c).
Subsequently, gold nanorods,67 nanoshells,68 nanostars,69 and
nanocages70 were used for these purposes.
The use of self-assembled monolayers or island lms, as well
as nonspherical and/or composite particles, opens up fresh
opportunities for increased sensitivity of detection of biomolecular binding on or near the nanostructure surface. The
principle of amplication of a biomolecular binding signal
depends on the strong local electromagnetic elds arising near
nanoparticles with spiky surface sites or in narrow (on the
order of 1 nm or smaller) gaps between two nanoparticles.
This determines the increased plasmon-resonance sensitivity to
the local dielectric environment71 and the high scattering
intensity as compared to the sensitivity and intensity of
equivolume spheres. Therefore, such nanostructures show
considerable promise for use in biomedical diagnostics assisted
by dark-eld microscopy.7274
In dark-eld microscopy, GNPs are employed for detection
of microbial cells and their metabolites,75 bioimaging of
cancerous cells7678 and revelation of receptors on their surface,79,80
study of endocytosis,81 and other purposes. In most biomedical
applications, the eectiveness of conjugate labeling of cells is
assessed qualitatively. One of the few exceptions is the work of
Khanadeev et al.,82 in which a method was suggested for the
quantitative estimation of the eectiveness of GNP labeling of
cells and its use was illustrated with the specic example of
labeling of pig embryo kidney cells with gold nanoshell conjugates. Another approach was developed by Fu et al.83
Apart from the just mentioned ways to record biospecic
interactions with the help of diverse versions of optical microscopy and GNPs, the development of other state-of-the-art
detection and bioimaging methods is currently active. These are
referred to collectively as biophotonic methods.31,84 Biophotonics
combines all studies related to the interaction of light with
biological cells and tissues. Existing biophotonic methods include
optical coherence tomography,8587 X-ray and magnetic
resonance tomography,88,89 photoacoustic microscopy90 and
tomography,91 uorescence correlation microscopy,92 and other
techniques. These methods also successfully use variously sized
and shaped GNPs. In our opinion, biophotonic methods
employing nonspherical gold particles may hold particular
promise for bioimaging in vivo.34,93,94
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Fig. 2 Various types of plasmon-resonant nanoparticles: 16 nm nanospheres (a);25 gold nanorods (b);44 gold bipyramids (c);45 gold nanorods
surrounded by silver nanoshells (d);25 nanorice (gold-coated Fe2O3 nanorods) (e);46 SiO2/Au nanoshells (f)25 (the inset shows a hollow
nanoshell47); nanobowls with bottom cores (g);48 spiky SiO2/Au nanoshells (h)49 (the inset shows a gold nanostar50); gold tetrahedra, octahedra,
and cubooctahedra (i);51,52 gold nanocubes (j);51 silver nanocubes and goldsilver nanocages obtained from them (in the insets) (k);53 and gold
nanocrescents (l).54 Adapted from the data of the cited papers by permission from The Royal Society of Chemistry, Elsevier, IOP Publishing,
Springer, Wiley Interscience, and The American Chemical Society.

Table 1

Properties and possible biomedical applications of plasmonic nanoparticles


Major resonances/nm

Plasmonic shift/RIU/nm

Possible biomedical applications

Colloidal Au nanospheres (3100 nm)

Au nanorods (thickness, 1020 nm; aspect ratio, 210)
Au bipyramids
Au(core)/Ag(shell) NRs
Fe2O3 (core)/Au (shell) nanorice








Hollow Au shell
Spiky SiO2 nanoshells and Au nanostars
Au polyhedralsd
Au cubes
AuAg nanocages
Au nanocrescentse




Designations: RIUrefractive index unit, EMelectron microscopy, OIoptical imaging, HAhomogeneous assays, SAsolid phase assays,
PPTplasmonic photothermal therapy, DCdrug carriers, OAoptoacoustical applications, BSbiosensing, SERSsurface enhanced Raman
scattering. a PPT applications of clusterized Au nanospheres. b Monolayer data. c Suspension data. d Tetrahedra, octahedra, and cubooctahedra.
Nanolithography array.

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Chem. Soc. Rev., 2012, 41, 22562282


Fig. 3 Confocal image of HeLa cells in the presence of GNPs (a). Blue, the nuclei stained with Hoechst 33258; red, the actin cytoskeleton labeled
with Alexa Fluor 488 phalloidin; green, unlabeled GNPs. The image was taken by two-photon microscopy.63 Dark-eld microscopy of cancerous
(b) and healthy (c) cells by using GNPs conjugated with antibodies to epidermal growth factor.66 Adapted from the data of the cited papers by
permission from The American Chemical Society.


Analytical diagnostic methods

2.2.1 Homophase techniques. Beginning in 1980s, colloidal

gold conjugated with recognizing biomacromolecules was coming into use in various analytical methods in clinical diagnostics.
In 1980, Leuvering et al.95 put forward a new immunoanalysis
method that they called sol particle immunoassay (SPIA). A new
SPIA version was advanced by Mirkin et al.96 for the colorimetric detection of DNA. Both versions (protein and DNA
SPIA) use two principles: (1) The sol color and absorption
spectrum change little when biopolymers are adsorbed on
individual particles.25 (2) When particles move closer to each
other to distances smaller than 0.1 of their diameter, the red color
of the sol changes to purple or gray and the absorption spectrum
broadens and red-shifts.97 This change in the absorption spectrum
can easily be detected spectrophotometrically or visually
(Fig. 4a and b98,99).
The authors employed an optimized variation of SPIA (by
using larger gold particles and monoclonal antibodies to various
antigenic sites) to detect human chorionic gonadotropin.100
Subsequently, the assay was used for the immunoanalysis of
Shistosoma101 and Rubella102 antigens; estimation of immunoglobulins,103,104 thrombin (by using aptamers),105 and glucose;106
direct detection of cancerous cells;107 detection of Leptospira cells
in urine;108 detection of Alzheimers disease markers;109 determination of protease activity;110 and other purposes. The simultaneous use of antibody conjugates of gold nanorods and
nanospheres for the detection of tumor antigens was described
by Liu et al.111 Wang et al.112 provided data on the detection of
hepatitis B virus in blood with gold nanorods conjugated to
specic antibodies.
All SPIA versions proved easy to implement and were highly
sensitive and specic. However, investigators came up against the
fact that antigenantibody reactions on sol particles do not
necessarily lead to system destabilization (aggregation). Sometimes, despite the obvious complementarity of the pair, changes
in solution color (and, correspondingly, in absorption spectra)
were either absent or slight. Dykman et al.113 presented a model
for the formation of a second protein layer on gold particles
without loss of sol aggregative stability. The spectral changes
arising from biopolymer adsorption on the surface of metallic
particles are comparatively small114 (Fig. 4c and d). However, as

Chem. Soc. Rev., 2012, 41, 22562282

Fig. 4 Sol particle immunoassay. (a) Scheme for the aggregation of

conjugates as a result of binding by target molecules and (b) the
corresponding changes in the spectra and in sol color. (c) Scheme for
the formation of a secondary layer without conjugate aggregation and
(d) the corresponding dierential extinction spectra at 600 nm.
Adapted from the data of Khlebtsov et al.,97 Wu et al.,98 and
Englebienne115 by permission from The American Chemical Society
and The Royal Society of Chemistry.

shown by Englebienne,115 even such minor changes in absorption

spectra, resulting from a change in the structure of the biopolymer
layer (specically, its average refractive index) near the GNP
surface, could be recorded and used for assay in biological
For increasing the sensitivity of the analytical reaction, new
interaction-recording techniques have been proposed, including
photothermal spectroscopy,116 laser-based double beam absorption
spectroscopy,117 hyper-Rayleigh scattering,118 dierential lightscattering spectroscopy,119 and dynamic light scattering.120 In
addition, two vibrational spectroscopy methodssurface enhanced
infrared absorption spectroscopy121 and surface enhanced Raman
spectroscopy122have been suggested for use in SPIA.
The ability of gold particles interacting with proteins to
aggregate with a solution color change served as a basis for the
development of a colorimetric method for protein determination.123
A new SPIA version using microplates and an ELISA reader,
with colloidal-gold-conjugated trypsin as a specic agent for
proteins, was devised by Dykman et al.124
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Table 2

Detection of DNA with the use of gold nanoparticles




Detection method

Detection limit



Cross-linking aggregation,
UVAS, spot test

10 fmol

125, 1997

5 0 -SH(dT)10-3 0

Biotinylated PCR

2 fmol (500 copies

of PSA cDNA)

126, 2003

5 0 -N15(CH2)10SH-3 0 a;
5 0 -SH(CH2)10N15-3 0 b
5 0 -N15C-3 0 (CH2)3SH;
HS(CH2)65 0 -N18-3 0
5 0 -HSN16-3 0


500 zmol (10 copies

in 30 mL)
100250 nM;
12 nM
300 nM

127, 2004

1-round PCR

Hybridization of targets with poly-A

and streptavidin followed by a strip
test with GNSs
Bio-bar-code assay, scanometric
detection of silver-enhanced spots
Non-cross-linking aggregation in
1 M NaCl, VE, spot test
Stabilization of nanoprobes by
targets in 2M NaCl, VE, UVAS
Aggregation, DLS

128, 2006;
140, 2005
129, 2006

5 pM

130, 2008



10 zM

136, 2010


Fluorescence quenching/retaining
with nontarget/target ssDNA
Stabilization/aggregation with
nontarget/target ssDNA, UVAS
Aggregation in the case of nontarget
molecules, VE, UVAS

0.5 pM

133, 2004

2 pM

134, 2004

1 pM (DNA);
10 nM (thrombin);
10 mM (cocaine);
50 mM (Hg)
10 pM

144, 2010

1.7 nM

135, 2008

0.1 pM

145, 2010

5 -HS(CH2)6N13N15-3

(GNSs with

5 0 -N12C3SS-3 0 ;
5 0 -SSC6N15-3 0
5 0 -SH(CH2)6N15-3 0 ;
5 0 -RoxN15(CH2)3SH-3 0
Rhodamine red5 0 -N15-3 0


5 -N15-3

ssDNA, aptamers +
conjugated polyelectrolytec

ssDNA, thrombin,
cocaine, Hg

5 0 -N21-3 0 , 5 0 -N23-3 0



Aggregation of positively charged

Aggregation, UVLS

5 0 -N30-3 0


Aggregation (optimized), UVAS

5 -N21-3


PCR product

44, 2011

Designations: Nmm-bases oligonucleotide; GNSsgold nanospheres (colloidal gold nanoparticles with a roughly spherical shape); UVASUVvis
absorption spectroscopy; VEvisual evaluation; UVLSUVvis light scattering spectroscopy; PSAprostate-specic antigen; DLSdynamic light
scattering; GNRsgold nanorods. a Particle probe. b Substrate probe. c Poly [(9,9-bis (60 -N,N,N-trimethylammonium)hexyl)uorene-alt-1,4-phenylene]

At present, colorimetric DNA detection includes two strategies:

(1) The use of GNPs conjugated with thiol-modied
ssDNA96,125130 or aptamers.131 (2) The use of unmodied
GNPs132135 (Table 2).
The rst strategy is based on the aggregation of conjugates
of 1030 nm GNPs with thiol-modied ssDNA probes after
the addition of target polynucleotides to the system. A crosslinking variant of the rst strategy uses two types of probes
complementary to both terminal target sites. Hybridization of
the targets and probes leads to the formation of GNP aggregates, which is accompanied by a change in the absorption
spectrum of the solution and can readily be detected visually,
photometrically,137 or by dynamic light scattering.130,138
In contrast to the cross-linking aggregation, Maeda and
coworkers139,140 developed a non-cross-linking diagnostic system
involving GNP conjugates of only one type, with 3 0 or 5 0 thiolmodied probes. The aggregation of GNPs occurred at high
ionic strength (1 M NaCl) and only with complementary
probes and targets, whereas noncomplementary targets prevented
aggregation. Contrary to the observations of the Maeda group,139
Baptista et al.129,141 reported enhanced colloidal stability after the
addition of complementary targets to a DNA conjugate solution
even at high ionic strength (2 M NaCl), whereas noncomplementary targets did not prevent aggregation at 2 M NaCl. The
apparent contradictions between these data were explained by
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Song et al.142 through the dierence in surface functionalization

Consider now the second DNA-sensing strategy, which
utilizes unmodied GNPs. This approach is based on the
observation by Li and Rothberg133 that at high ionic strength,
ssDNA protects unmodied gold nanoparticles from aggregation,
whereas dsDNA does not. This method was employed by
Shawky et al.143 to detect hepatitis C virus. Recently, Xia et al.144
described another variant of the second strategy, which uses
ssDNA, unmodied GNPs, and a cationic polyelectrolyte.
All the above-cited reports on DNA detection used citratestabilized anionic GNPs. He et al.135 described a new version
of unmodied-particle assay employing as-prepared positively
charged CTAB-coated gold nanorods. After mixing nanorods
with the probe and target ssDNA under appropriate hybridization conditions, the authors observed particle aggregation,
as determined by absorption and scattering spectra. In contrast,
the addition of noncomplementary targets did not cause any spectral
changes. According to He et al.,135 the detection limit (DL) of
a 21-mer ssDNA was about 0.1 nM, whereas Ma et al.145
reported a 0.1 pM DL for an optimized version of He et al.s
assay135 and 30-mer ssDNA. Quite recently, Pylaev et al.44
applied CTAB-coated positively charged GNPs in combination
with spectroscopic and dynamic scattering methods for the
detection of DNA targets. Thus, there exists a more than
Chem. Soc. Rev., 2012, 41, 22562282


three-order dierence in the reported estimations of the detection

sensitivity of colorimetric methods (0.1100 pM). Further work
is called for, as the existing aggregation models are inconsistent
with the detection limits of about 0.11 pM DNA.44
The above-mentioned SPIA formats have served for the
detection of the DNA of mycobacteria,129,146,147 staphylococci,148
streptococci,149 and chlamydiae150 in clinical samples.
The sensitivity of DNA detection can be increased with
optical methods that utilize plasmonic enhancement of the
local electromagnetic eld near particle-cluster hotspots. For
example, Hu et al.136 developed a sensitive DNA biosensor
based on multilayer metalmoleculemetal nanojunctions and
the SERS technique. With regard to an HIV-1 DNA sequence,
the sensor could detect a concentration as low as 1019 M
(1023 mol) with the ability of single base mismatch discrimination.
Another way to reach PCR-like sensitivity involves a bio-bar-code
approach combined with a silver-enhanced spot test.127
2.2.2 Dot immunoassay. At the early stages of immunoassay development, preference was given to liquid-phase techniques, in which bound antibodies were precipitated or unbound
antigen was removed by adsorption with dextran-coated activated
charcoal. Currently, the most popular techniques are solidphase ones (rst used for protein radioimmunoassay), because
they permit the analysis to be considerably simplied and the
background signal to be reduced. The most widespread solid-phase
carriers are polystyrene plates and nitrocellulose membranes.
Membrane immunoassays (dot and blot assays) commonly
employ radioactive isotopes (125I, 14C, 3H) and enzymes
(peroxidase, alkaline phosphatase, etc.) as labels. In 1984, four
independent reports were published151154 in which colloidal
gold was proposed as a label in a solid-phase immunoassay.
The use of GNP conjugates in solid-phase assays is based on
the fact that the intense red coloration of a gold-containing
marker allows the results of a reaction run on a solid carrier to
be determined visually. Immunogold methods in a dotblot
assay outperform other techniques (e.g., enzyme immunoassay)
in terms of sensitivity (Table 3), rapidity, and cost.155,156 After
an appropriate immunochemical reaction is run, the sizes of
GNPs can be increased by enhancement with salts of silver157
or gold (autometallography),158 considerably expanding the
application limits of the method. An optimized solid-phase
assay using a densitometry system aorded a dynamic detection
range of 1 mM to 1 pM159 with a detection limit of 100 aM,
which was lowered to 100 zM by silver enhancement. The use
of state-of-the-art instrumental detection methods, such as
photothermal deection of a probing laser beam, caused by
heating of the local environment near absorbing particles
Table 3 Sensitivity limits for immunodot/blot methods implemented
on nitrocellulose lters by using various labels (according to ref. 155)

Sensitivity limit
(pg of protein per fraction)

Horseradish peroxidase
Alkaline phosphatase
Colloidal gold
Colloidal gold + silver
Fluorescein isothiocyanate



Chem. Soc. Rev., 2012, 41, 22562282

subjected to light pulses from a heating laser (LISNA assay),160

also ensures a very broad detection range (within three orders of
magnitude, to the extent of several individual particles on a spot).
In specic staining, a membrane with applied material under
study is incubated in a solution of antibodies (or other
biospecic probes) labeled with colloidal gold.18 As probes,
gold dot or blot assays use immunoglobulins, Fab and scFv
antibody fragments, staphylococcal protein A, lectins, enzymes,
avidin, aptamers, and other probes. Sometimes, several labels
are used simultaneously (e.g., colloidal gold and peroxidase or
alkaline phosphatase) for the detection of multiple antigens on a
Colloidal gold in membrane assays has served for the diagnosis
of parasitic,162166 viral,167170 and fungal171,172 diseases, tuberculosis,173 melioidosis,174 syphilis,175 brucellosis,176 shigellosis,177
E. coli infections,178 salmonellosis,179 and early pregnancy;180
blood group determination;181 dotblot hybridization;182 detection
of antibiotics;183 diagnosis of myocardial infarction184 and
hepatitis B;185 and other purposes.
The immunodot assay is one of the simplest methods developed
to analyze membrane-immobilized antigens. In some cases, it
permits quantitative determination of antigens. Most commonly,
the immunodot assay is employed to study soluble antigens.186
However, there have been several reports in which corpuscular
antigens (whole bacterial cells) served as a research object in dot
assays with enzyme labels.187 Bogatyrev et al.188,189 were the rst
to perform a dot assay of whole bacterial cells, with the reaction
products being visualized with immunogold markers (cellgold
immunoblotting). This technique was applied to the serotyping
of nitrogen-xing soil microorganisms of the genus Azospirillum
and subsequently to the rapid diagnosis of enteric infections.190
Gas et al.191 used a dot assay with GNPs to detect whole cells of
the toxic phytoplankton Alexandrium minutum.
Khlebtsov et al.192 rst presented experimental results for the
use of gold nanoshells as biospecic labels in dot assays. Three
types of gold nanoshells were examined that had silica core
diameters of 100, 140, and 180 nm and a gold shell thickness of
about 15 nm (Fig. 5, data for 140/15 nm nanoshells not shown).
The biospecic pair was normal rabbit serum (target molecules)
and sheep antirabbit immunoglobulins (target-recognizing
molecules). When the authors performed a standard protocol
for a nitrocellulose-membrane dot assay, with 15 nm gold
nanospheres as labels, the minimum detectable quantity of
rabbit IgG was 15 ng. Replacing colloidal gold conjugates with
nanoshells increased the assay sensitivity to 0.2 ng for 180/15 nm
gold nanoshells and to 0.4 ng for 100/15 and 140/15 nm
nanoshells. Such noticeable increases in sensitivity with nanoshells, as against nanospheres, can be explained by the dierent
optical properties of the particles.193
A very promising analytical approach is the use of colloidal gold
to analyze large arrays of antigens in micromatrices (immunochips).194,195 These enable an analyte to be detected in 384
samples simultaneously at a concentration of 6070 ng L1 or,
with account taken of the microlitre amounts of sample and
detecting immunogold marker, with a detection limit of lower
than 1 pg.
The sensitivity of protein dot-analysis can be greatly improved
with a combination of activated glass slides and a CCD
camera196,197 or a atbed scanner.198 In the case of very dilute
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Fig. 5 Dot assay of normal rabbit serum (1) by using suspensions of

conjugates of 15 nm GNPs and SiO2/Au nanoshells (100 and 180 nm
silica core diameters and 15 nm Au shell) with sheep antirabbit
antibodies. The amount of IgG in the rst square of the top row is
1 mg and decreases from left to right in accordance with twofold
dilutions. The lower rows (2) correspond to the application of 10 mg of
bovine serum albumin to each square as a negative control. The
detected analyte quantity is 15 ng for 15 nm GNPs and 0.4 and 0.2 ng
for 100/15 and 180/15 nm nanoshells, respectively. Adapted from
ref. 193 by permission from IOP Publishing.

samples, a dot-immunogold ltration assay183 has been suggested.

Finally, the sensitivity of a standard ELISA can be enhanced
up to the single-molecule detection limit199 by using GNPs in
colorimetric analysis of ELISA samples.200,201
2.2.3 Immunochromatographic assays. In 1990, several
companies began to manufacture immunochromatographic
test systems for hand-held diagnostics. Owing to their high
specicity and sensitivity, these strip tests have found a wide
utility in the detection of narcotics, toxins, highly dangerous
infections, and urogenital diseases.202208 Methods have been
developed for the diagnosis of tuberculosis,209 helicobacteriosis,210
staphylococcal infection,211 hepatitis B,212 prostatitis,213 and early
pregnancy,214 for DNA hybridization;215 for the detection of
pesticides,216 aatoxin,217 hexoestrol,218 and cephalexin219 in
environmental constituents; and for other purposes.
The immunochromatographic assay is based on eluent
movement along the membrane (lateral diusion), giving rise
to specic immune complexes at dierent membrane sites; the
complexes are visualized as colored bands.220 As labels, these
systems use enzymes, colored lattices, but mostly GNPs.221,222
The sample being examined migrates along the test strip at
the cost of capillary forces. If the sample contains the soughtfor substance or immunochemically related compounds, there
occurs a reaction with colloidal-gold-labeled specic antibodies
as the sample passes through the absorber. The reaction is
accompanied by the formation of an antigenantibody
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complex. The colloidal preparation enters into a competitive

binding reaction with the antigen immobilized in the test zone
(as a rule, the detection of low-molecular-weight compounds
employs conjugates of haptens with protein carriers for
immobilization). If the antigen concentration in the sample
exceeds the threshold level, the conjugate does not possess free
valences for interaction in the test zone and the colored band
corresponding to the formation of the complex is not revealed.
When the sample does not contain the sought-for substance or
when the concentration of that substance is lower than the
threshold level, the antigen immobilized in the test zone of the
strip reacts with the antibodies on the surface of colloidal gold,
causing a colored band to develop.
When the liquid front moves on, the gold particles with
immobilized antibodies that have not reacted with the antigen
in the strip test zone bind to antispecies antibodies in the
control zone of the test strip. The appearance of a colored
band in the control zone conrms that the test was done
correctly and that the systems components are diagnostically
active. Otherwise, the test is invalid. A negative test resultthe
appearance of two colored bands (in the test zone and in
the control zone)indicates that the antigen is absent from
the sample or that its concentration is lower than the threshold
level. A positive test resultthe appearance of one colored
band in the control zoneshows that the antigen concentration exceeds the threshold level (Fig. 6).220
Studies have shown that such assay systems are highly
stable, their results are reproducible, and they correlate with
alternative methods. Densitometric characterization of the
dissimilarity degree for detected bands yields values ranging
from 5 to 8%, allowing reliable visual determination of the
analysis results. These assays are very simple and convenient
to use.
Nowadays, GNP-assisted immunochromatographic analysis is
used actively in such elds as the rapid diagnostics of pseudorabies,223 tuberculosis,224 and botulism225 and the detection
of pesticides,226 antibiotics,227 and toxins,228 in biological
liquids and the environment.
In summary, being eective diagnostic tools, rapid tests
allow qualitative and quantitative determination, in a matter
of minutes, of antigens, antibodies, hormones, and other

Fig. 6 Results of an immunochromatographic assay: positive, negative,

and invalid determination because of the absent coloration in the
control zone. Adapted from ref. 219 by permission from The American
Chemical Society.

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diagnostically important substances in humans and animals.

Rapid tests are highly sensitive and accurate, as they can
detect more than 100 diseases (including tuberculosis, syphilis,
gonorrhea, chlamydiosis, various types of viral hepatitis, etc.)
and the whole gamut of narcotic substances used, with the
reliability of detection being high. An important advantage of
these tests is their use in diagnostics in vitro, which does not
require a patients presence.
However, immunochromatographic test strips are not devoid
of weak points, related to reliability, sensitivity, and cost-eectiveness.
Reliability and sensitivity depend, rst, on the quality of monoclonal
antibodies used in a test and, second, on the antigen concentration in a biomaterial. The quality of monoclonal antibodies
depends on the methods of their preparation, purication, and
xation on a carrier. The antigen concentration depends on
the disease state and the biomaterial quantity. For increasing
the analysis sensitivity, it has been proposed to employ the
silver enhancement procedure229 or gold nanorods as labels.230
In addition, semiquantitative and quantitative instrumental
formats of immunochromatographic analysis have been developed
that use special readers for recording the intensity of a labels
signal in the test zone of a test strip.220
2.2.4 Plasmonic biosensors. Collective oscillations of
conductive electron plasma in metals are called plasmons.231
Depending on the boundary conditions, these oscillations can be
categorized into three types:232 bulk plasmons (3D plasma);
propagating surface plasmons (PSP), or surface plasmon polaritons
(2D dielectric/metal interfaces); and localized surface plasmons
(LSP), excited in nanoparticles (Fig. 7).25 Bulk plasmons cannot
be excited by visible light, as their energy is about 10 eV for noble
The term surface plasmon polaritons designates collective
charge density oscillations at the metal/dielectric interface,
propagating in a waveguide-like fashion along the surface
(Fig. 7b). In the normal direction, the exciting electromagnetic
eld is rapidly falling o with distance. At a given photon
energy ho, the wave vector ho/c should be increased to ensure
eective coupling for the transformation of incident photons
into propagating surface plasmons.232 Therefore, direct excitation
of surface plasmon polaritons with freely propagating light is not
possible. However, this problem can be solved by two approaches:
the grating coupling method and the attenuated reection method,
which use a lattice waveguide structure or total reection inside a
prism, respectively.232
In metal nanoparticles, the electron plasma is restricted in
all three dimensions. Accordingly, localized surface plasmons
dier from propagating surface plasmons because of the

Fig. 7 Schematic representation of the bulk (a), propagating surface

(b), and localized surface (c) plasmons.


Chem. Soc. Rev., 2012, 41, 22562282

dierent boundary conditions to the Maxwell electromagnetic

equations. In a small metal nanoparticle, the incident optical
eld exerts a force on conductive electrons and displaces them
from their equilibrium positions to create uncompensated
charges at the nanoparticle surface (Fig. 7c). As the main
eect producing the restoring force is the polarization of the
particle surface, the excited nanoparticle behaves like a resonance
oscillator possessing a localized plasmon resonance (LSPR).
The principal dierence between the propagating and localized
plasmons is that the former can be directly excited by light
waves without any additional coupling set up. On the other
hand, in both LSP and PSP cases, the excited plasmons feel
the dielectric environment. It is the property that is the basis
for the LSP and the PSP.
The optical response of nanoparticles or their aggregates
(especially ordered ones) depends on particle size, shape, and
composition71,233 interparticle distance,234,235 and the properties
of the particles local dielectric environment,236,237 which
enables sensor tuning to be controlled. The theory behind
the creation of LSP biosensors and their use in practice have
been considered in ref. 238254. In general, all sensing strategies are based on the change in the intensity of the LSPR and
its spectral shift caused by a change in the local dielectric
environment owing to biospecic interactions near the particle
surface. A unique local-sensing property of the LSPR is
related to the rapid decay of its local eld, which only probes
a nanoscale volume around the particle. In particular, the local
nature of the LSPR allows one to detect single-molecular
interactions near the particle surface by using various singleparticle detecting schemes.254 Fig. 8a illustrates a sensitive
detection of about 18 streptavidin molecules after their binding
to a single biotin-functionalized gold nanorod (74  33 nm).255
Nusz et al.255 also reported a 1 nM low detection limit (Fig. 8b)
and discussed several ways to approach single-molecular detection.
In recent years, gold and silver nanoparticles and their
composites have served widely as eective optical transducers
of biospecic interactions.256 In particular, the resonant
optical properties of nanometre-sized metallic particles have been
successfully used to develop plasmonic biochips and biosensors belonging to a broad family of sensors, viz. colorimetric,
refractometric, electrochemical, and piezoelectric.220,257,258
Such devices are of much interest in biology (determination
of nucleic acids, proteins, and metabolites), medicine (screening
of drugs, analysis of antibodies and antigens, diagnosis of

Fig. 8 (a) Single-gold-nanorod light scattering spectra in water (1),

with biotin, and after binding of about 18 streptavidin molecules.
(b) LSPR peak shift as a function of the streptavidin concentration.
The low detection limit is below 1 nM. Adapted from ref. 255 by
permission from The American Chemical Society.

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infections), and chemistry (rapid environmental monitoring,

assays of solutions and disperse systems). Of particular signicance is the detection of specied nucleic acid (gene) sequences
and the construction of new materials that relies on the formation
of 3D ordered structures during hybridization in solutions of
complementary oligonucleotides attached covalently to metallic
For more than 10 years, biospecic interactions have been
studied in systems in which GNPs are represented as ordered
structures: self-assembled (thin lms)260 or as part of polymer
matrices.261 Such structures have been actively employed for
detection of biomolecules and infectious agents, development
of DNA chips, and other purposes. In this case, investigators
directly implement the possibility, in principle, of using the
strong enhancement of the optical signal from the probe
(GNPs conjugated to biospecic macromolecules) resulting
from the strengthening of the exciting local eld in the
aggregate formed from gold nanoclusters. At present, biosensors
are built with novel, unique technologies, including monolayer
self-assembly of metallic particles,262 ber-based LSPR sensing,263
and nanolithography.264 The reported types of LPR studies
of biospecic interactions include biotinstreptavidin, antigen
antibody, lectincarbohydrate, toxinreceptor, aptamerprotein,
and DNA hybridization.254 For further information about LPR
sensing, the readers are referred to a recent excellent review by
Mayer and Hafner.254
In experimental work with SPR biosensors, three stages can
be singled out:239 (1) one of the reagents (target-recognizing
molecules) is covalently attached to the sensor surface. (2) The
other reagent (target molecules) is added at a denite concentration to the sensor surface along with the ow of the buer.
The process of complex formation is then recorded. (3) The
sensor is regenerated (dissociation of the formed complexes).
As this takes place, the following conditions should be met:
 Reagent immobilization on the substrate should not lead
to a critical change in the conformation of native molecules.
 The relatively small dierence between the refractive
indices of most biological macromolecules forces one to use
a high local concentration of binding sites on the sensor
surface (10100 mM).
 The reagent being added should be vigorously agitated to
achieve eective binding to the immobilized molecules. Unbound
reagent should be promptly removed from the sensor surface to
avoid nonspecic sorption.
Apart from that, the sensitivity, stability, and resolution of a
sensor depend directly on the characteristics of the optical
system being used for recording. The most popular sensor
system of this type is BIAcoret.265,266 The measurement
principle in planar, prismatic, or mirror biosensors is similar
to the principle of the method of frustrated total internal
reection, traditionally employed to measure the thickness and
refractive index of ultrathin organic lms on metallic (reecting)
surfaces.257 The excitation of the plasmon resonance in a
planar gold layer occurs when polarized light is incident on
the surface at a certain angle. The excited electromagnetic
waves and charged density waves propagate along the metal/
dielectric interface. These propagating electromagnetic elds
are localized near the interface because of the exponential
decrease in amplitude perpendicularly to the dielectric with a
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Fig. 9 Typical setup for analyte detection in a BIAcoret-type SPR

biosensor. The instrument detects changes in the local refractive index
near a thin gold layer coated by a sensor surface with probing
molecules. SPR is observed as a minimum in the reected light
intensity at an angle dependent on the mass of captured analyte.
The minimum SPR angle shifts from A to B when the analyte binds to
the sensor surface. The sensogram is a plot of resonance angle versus
time that allows for real-time monitoring of an association/dissociation
cycle. Adapted from ref. 267 by permission from Springer.

typical attenuation length of up to 200 nm (the eect of total

internal reection, Fig. 9). The reection coecient at a
certain angle and light wavelength depends on the dielectric
properties of the thin layer at the interface, which are
ultimately determined by the mass of the captured target
molecules at the sensing surface.
Various types of GNP-aided biosensors have been developed
for the immunodiagnostics of tick-borne encephalitis,268 the
papilloma269 and HIV270 viruses, and Alzheimers disease;271,272
the detection of organophosphorus substances and pesticides,273
antibiotics,274 allergens,275 cytokines,276 carbohydrates,277 and
immunoglobulins;278 the detection of tumor279 and bacterial280
cells; the detection of brain cell activity;281 and other purposes.
GNP-based biosensors are used not only in immunoassay281284 but also for the supersensitive detection of nucleotide
sequences.96,259,285 In their pioneering works, Raschke et al.286
and McFarland et al.287 obtained record-high sensitivity of
such sensors in the zeptomolar range, and they showed the
possibility of detecting spectra of resonance scattering from
individual particles as an analytical tool. This opens up the
way to the recording of intermolecular interactions at the level
of individual molecules.288,299 To make the response stronger,
investigators often use avidinbiotin, barnasebarstar, and
other systems.290 In addition, GNPs are applied in other
analytical methods (diverse versions of chromatography, electrophoresis, and mass spectrometry).291
SPR and LSPR biosensors were compared in side-by-side
experiments by Yonzon et al.292 for concanavilin A binding to
monosaccharides and by Svedendahl et al.293 for biotinstreptavidin binding. It was found that both techniques demonstrate similar performance. As the bulk refractive index
sensitivity is known to be higher for SPR, the above similarity
was attributed to the long decay length of propagating plasmons,
as compared to localized ones. The overall comparison of SPR
and LSPR sensors can be found in ref. 251 and 254.
Future development of low-cost SPR and LSPR biomedical
sensors needs increasing the detection sensitivity and creating
substrates that can operate in biological uids and can be easy
to functionalize with probing molecules, to clean, and to reuse.
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3. GNPs in therapy

Plasmonic photothermal therapy

Photothermal damage to cells is currently one of the most

promising research avenues in the treatment of cancer and
infectious diseases.294 The essence of this phenomenon is as
follows: GNPs have an absorption maximum in the visible or
near-IR region and get very hot when irradiated with corresponding light. If, in this case, they are located inside or around
the target cells (which can be achieved by conjugating gold
particles to antibodies or other molecules), these cells die.
The thermal treatment of cancerous cells has been known in
tumor therapy since the 18th century, employing both local
heating (with microwave, ultrasonic, and radio radiation) and
general hyperthermia (heating to 4147 1C for 1 h).295 For
local heating to 70 1C, the heating time may be reduced to
34 min. Local and general hyperthermia leads to irreversible
damage to the cells, caused by disruption of cell membrane
permeability and protein denaturation. Naturally, the process
also injures healthy tissues, which imposes serious limitations
on the use of this method.
The revolution in thermal cancer therapy is associated with
laser radiation, which enabled controlled and limited injury to
tumor tissues to be achieved.296 Combining laser radiation
with ber-optic waveguides produced excellent results and was
named interstitial laser hyperthermia.297 The weak point of
laser therapy is its low selectivity, related to the need for highpowered lasers for eective stimulation of tumor cell death.298
A variant of photothermal therapy was also proposed in which
photothermal agents help to achieve selective heating of the
local environment.299 Selective photothermal therapy relies on
the principle of selective photothermolysis of a biological tissue
containing a chromophorea natural or articial substance with
a high coecient of light absorption.

GNPs were rst used in photothermal therapy in 2003.300,301

Subsequently, it was suggested to call this method plasmonic
photothermal therapy (PPTT).295 Pitsillides et al.302 rst described
a new technique for selective damage to target cells that is founded
on the use of 20 and 30 nm gold nanospheres irradiated with 20 ns
laser pulses (532 nm) to create local heating. For pulse photothermia in a model experiment, those authors were the rst to
employ a sandwich technology for labeling T-lymphocytes with
GNP conjugates.
In a particularly promising method, GNPs have found
application in the photothermal therapy of chemotherapy-resistant
cancers.303 Unlike photosensitizers (see below), GNPs are unique in
being able to keep their optical properties in cells for a long time
under certain conditions. Successive irradiation with several laser
pulses allows control of cell inactivation by a nontraumatic means.
The simultaneous use of the scattering and absorbing properties of
GNPs permits PPTT to be controlled by optical tomography.68
Fig. 10 shows an example of successful therapy of an implanted
tumor in a model experiment with mice.304
The further development of PPTT and its acceptance in
actual clinical practice305 depends on success in solving many
problems, the most important ones being (1) the choice of
nanoparticles with optimal optical properties, (2) the enhancement
of nanoparticle accumulation in tumors and the lowering of
total potential toxicity, and (3) the development of methods
for the delivery of optical radiation to the targets and the
search for alternative radiation sources combining high permeability
with a possibility of heating GNPs.
The rst requirement is determined by the concordance of the
spectral position of the absorption plasmon resonance peak with
the spectral window for biological tissues in the 700900 nm nearIR region.306 Khlebtsov et al.234 made a resumptive theoretical
analysis of the photothermal eectiveness of GNPs, depending
on their size, shape, structure, and aggregation extent. They
showed that although gold nanospheres themselves are

Fig. 10 Scheme and the results of an experiment on the photothermal destruction of an implanted tumor in a mouse (23 weeks after injection of
MDA-MB-435 human cancer cells). Laser irradiation (a, b; 810 nm, 2 W cm2, 5 min) was performed at 72 h after injection of gold nanorods
functionalized with poly(ethylene glycol) (PEG) (a, c; 20 mg Au per kg) or of buer (b, d). It can be seen that the tumor continued developing after
particle-free irradiation (control b), as it did after particle or buer administration without irradiation (controls a and d), and that complete
destruction was obtained only in the experiment (a). Designations: NIR, near-IR region; NRs, nanorods. Adapted in part from ref. 304 by
permission from The American Association for Cancer Research.


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ineective in the near-IR region, aggregates formed from them

can be very eective at suciently short interparticle distances
(shorter than 0.1 of the particle diameter). Such clusters can be
formed both on the surface of and inside cells.307 Experimental
data indicating an enhancement of PPTT through clusterization
have been presented in ref. 308310. Specically, Huang et al.308
demonstrated that small aggregates composed of 30 nm particles
enable cancerous cells to be destroyed at a radiation power
20-fold lower than that in the particle-free control.
The gold nanoshell and nanorod parameters optimal for PPTT
have also been dened.234,311 By now, there have been quite a few
publications dealing with the application in PPTT of gold nanorods;67,312314 nanoshells;301,315320 and a comparatively new particle
class, goldsilver nanocages.93,321,322 Experimental data comparing the heating eciencies of nanorods, nanoshells, and
nanocages have been reported in ref. 53, 323 and 324.
Regarding the optimization of particle parameters, one should
be aware of three matters of principle. First, the absorption cross
section is not the sole parameter determining the eectiveness of
PPTT.325 Rapid heating of nanoparticles or aggregates gives rise
to vapor bubbles,326 which can cause cavitation damage to cells
irradiated with visible309 or near-IR327 light. The eectiveness of
vapor bubble formation increases substantially when nanoparticle
aggregates are formed.301,307 Possibly, it is this eect, and not
enhanced absorption, that bears responsibility for greater damage
to cells, with all other factors being the same.325 Finally, particle
irradiation with high-power resonant nanosecond IR pulses can
lead to particle destruction as early as after the rst pulse (see, e.g.,
ref. 328 and 329 and references therein to earlier publications). In
a series of recent investigations, Lapotko et al.325,330 (see also
references therein) paid their attention to the fact that the heating
of GNPs and their destruction can sharply reduce the photothermal eectiveness of cold particles, which have been tuned to
the laser wavelength. The use of femtosecond pulses oers no
solution to this problem because of the low energy supplied, and
for this reason, it is necessary to exert close control over the
preservation of nanoparticles properties for the chosen irradiation
mode. Furthermore, bubble formation strongly depends on the
media as well as on laser intensity, thus making cellular damage
poorly controlled.
We now shift to consider the second question, associated
with targeted nanoparticle delivery to a tumor. This question
has two important aspects: increasing the particle concentration in the target and lowering the side eects caused by
GNP accumulation in other organs, primarily in the liver and
spleen (see below). Usually, there are two delivery strategies.
One is based on the conjugation of GNPs to PEG; the other,
on the conjugation with antibodies developed to specic
marker proteins of tumor cells. PEG acts to enhance the
bioavailability and stability of nanoparticles, ultimately
prolonging the time of their circulation in the blood stream.
Citrate-coated gold nanospheres, CTAB-coated nanorods,
and nanoshells are less stable in saline solutions. When
nanoparticles are conjugated to PEG, their stability is improved
considerably and salt aggregation is prevented.
In vivo PEGylated nanoparticles preferentially accumulate
in tumor tissue owing to the increased permeability of the
tumor vessels331 and are retained in it owing to the decreased
lymph outow. In addition, PEGylated nanoparticles are less
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Fig. 11 Scheme for a PPTT employing active delivery of GNPs to

cancer cells.

accessible to the immune system (stealth technologies). This

delivery method is called passive, as distinct from the active
version, which uses antibodies332,333 (Fig. 11). The active
method of delivery is more reliable and eective, employing
antibodies to specic tumor markers, most often to epidermal
growth factor receptor (EGFR) and its varieties (e.g.,
Her2),315,334,335 as well as to tumor necrosis factor (TNF).336
Particular promise is oered by the simultaneous use of
GNPantibody conjugates for both diagnosis and PPTT
(methods of what is known as theranostics).337339 In addition
to antibodies, active delivery may also use folic acid, which
serves as a ligand for the numerous folate receptors of tumor
cells,313,340,341 and hormones.342
In the very recent past, the eectiveness of targeted nanoparticle delivery to tumors has again become a subject for
detailed study and discussion.343 In experiments with liposomes
labeled with anti-Her2344 and GNPs labeled with transferrin,345
it was shown that functionalization improves nanoparticle penetration into cells but produces no appreciable increase in
particle accumulation in tumors. Huang et al.343 examined
the biodistribution and localization of gold nanorods labeled
with three types of probing molecules, including (1) an scFv
peptide that recognizes EGFR; (2) an amino terminal fragment peptide that recognizes the urokinase plasminogen activator receptor; and (3) a cyclic RGD peptide that recognizes
the avb3 integrin receptor. The authors showed that when
injected intravenously, all three ligands induce insignicant
increases in nanoparticle accumulation in cell models and in
tumors but greatly aect extracellular distribution and intracellular localization. They concluded that for PPTT, direct
administration of particles to the tumor can be more eective
than intravenous injection.
The nal important question in current PPTT concerns
eective delivery of radiation to a biological target. Because
the absorption of biological tissue chromophores in the visible
region is lower than that in the near-IR region by two orders
of magnitude,294 the use of IR radiation radically decreases the
nontarget heat load and enhances the penetration of radiation
into the tissue interior. Nevertheless, the depth of penetration
usually does not exceed 510 mm,84,311 so it is necessary to
look for alternative solutions. One approach consists in using pulse
(nanoseconds) irradiation modes in preference to continuous
ones, which enables irradiation power to be enhanced without
Chem. Soc. Rev., 2012, 41, 22562282


increasing side eects. Another approach involves ber-optic

devices for endoscopic or intratissue delivery of radiation.
The strong and weak points of such an approach are evident.
Finally, for hyperthermia, it is possible to use radiations with
greater depths of penetration, e.g., radiofrequency346349 or
nonthermal air plasma.350
GNPs conjugated to antibiotics and antibodies have also
served as photothermal agents for selective damage to protozoa
and bacteria.351354 Information on certain issues in the use of
PPTT can be found in several books and reviews.295,355359 Of
particular note is the most recent comprehensive review by
Kennedy et al.294
In summary, gold nanostructures with a plasmon resonance
oer considerable promise for selective PPTT of cancer and
other diseases. Without doubt, several questions await further
study, including the stability and biocompatibility of nanoparticle bioconjugates, their chemical interaction in physiological
environments, the period of circulation in blood, penetration
into the tumor, interaction with the immune system, and
nanoparticle excretion. We expect that the success of the initial
stages of nanoparticle use for selective PPTT can be eectively
enhanced at the clinical stage, provided that further studies are
made on the optimal procedural parameters. In particular, one
can mention the eorts of J. Feldmanns group360 related to
thermoplasmonicsa eld that is not well understood yet and
that is nowadays is a trend for hyperthermia and delivery upon
light-to-heat conversion.

Photodynamic therapy with GNPs

The photodynamic method of treating oncological diseases

and certain skin or infectious diseases is based on the application
of light-sensitizing agents called photosensitizers (including
dyes) and, as a rule, of visible light at a specic wavelength.361
Most often, sensitizers are introduced intravenously, but contact
and oral administration is also possible. The substances used
in photodynamic therapy (PDT) can selectively accumulate in
tumors or other target tissues (cells). The aected tissues are
irradiated with laser light at a wavelength corresponding to the
peak of dye absorption. In this case, apart from the usual heat
emission through absorption,21 an essential role is played by
another mechanism, related to the photochemical generation
of singlet oxygen and the formation of highly active radicals,
which induce necrosis and apoptosis in tumor cells. PDT also
disrupts the nutrition of the tumor and leads to its death by
damaging its microvessels. The major shortcoming of PDT is
that the photosensitizer remains in the organism for a long
time, leaving patient tissues highly sensitive to light. On the
other hand, the eectiveness of dye use for selective tissue
heating21 is low because of the small cross section of chromophore absorption.
It is well known362 that metallic nanoparticles are eective
uorescence quenchers. However, it has been shown recently363,364
that uorescence intensity can be enhanced by a plasmonic
particle if the molecules are placed at an optimal distance from
the metal. In principle, this idea can improve the eectiveness
of PDT.
Several investigators have proposed methods for the delivery
of drugs as part of polyelectrolyte capsules on GNPs

Chem. Soc. Rev., 2012, 41, 22562282

(which decompose when acted upon by laser radiation and

deliver the drug to the targets365,366) or by using nanoparticles
surrounded by a layer of polymeric nanogel.367,368 Apart from
that, the composition of nanoconjugates includes photoactive
substances,369peptides (e.g., CALLNN), and proteins (e.g.,
transferrin), which facilitate intracellular penetration.345,370,371
Recently, Bardhan et al.372 suggested the use of composite
nanoparticles, including, in addition to gold nanoshells, magnetic
particles, a photodynamic dye, PEG, and antibodies. Finally,
according to the data of Kuo et al.,373,374 nanoparticles
conjugated with photodynamic dyes can demonstrate a synergetic
antimicrobial eect, though the absence of such an eect has
also been reported.375
3.3 GNPs as a therapeutic agent
In addition to being used in diagnostics and cell photothermolysis,
GNPs have been increasingly applied directly for therapeutic
purposes.29 In 1997, Abraham and Himmel376 reported success
in colloidal gold treatment of rheumatoid arthritis in humans.
In 2008, Abraham published a great body of data from a
decade of clinical trials of Aurasolsan oral preparation for
the treatment of severe rheumatoid arthritis.377 Tsai et al.378
described positive results obtained when rats with collageninduced arthritis were intraarticularly injected with colloidal
gold. The authors explain the positive eect by an enhancement of antiangiogenic activity resulting from the binding of
GNPs to vascular endothelium growth factor, which brought
about a decrease in macrophage inltration and in inammation.
Similar results were obtained by Brown et al.,379,380 who
subcutaneously injected GNPs into rats with collagen- and
pristane-induced arthritis.
A series of papers by a research team from Maryland
University have described the application of a colloidal gold
vector to the delivery of TNF to solid tumors in rats.381384
When injected intravenously, GNPs conjugated to TNF accumulated rapidly in tumor cells and could not be detected in
cells of the liver, spleen, and other healthy animal organs. The
accumulation of GNPs in the tumor was proven by a recordable change in its color, because it became bright red-purple
(characteristic of colloidal gold and its aggregates), which
was coincident with the peak of the tumor-specic activity
of TNF (Fig. 12). The colloidal goldTNF vector was less
toxic and more eective in tumor reduction than was native
TNF, because the maximal antitumor reaction was attained
at lower drug doses. A medicinal preparation based on the
GNPTNF conjugate, which is called AurImmunet and
intended for intravenous injection, is already in the third stage
of clinical trials.
In experiments in vitro and in vivo, Bhattacharya et al.385
and Mukherjee et al.386 demonstrated the antiangiogenic
properties of GNPs. The particles were found to interact with
heparin-binding glycoproteins, including vascular permeability
factor/vascular endothelial growth factor and basic broblast
growth factor. These substances mediate angiogenesis, including
that in tumor tissues, and inhibit tumor activity by changing
the conformation of the molecules.387 Because intense angiogenesis (the formation of new vessels in organs or tissues) is a
major factor in the pathogenesis of tumor growth, the presence
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4. GNPs as drug carriers

4.1 Targeted delivery of anticancer drugs

Fig. 12 Accumulation of the GNPTNF conjugate in the tumor after

15 h. Diseased mice were intravenously injected with 15 mg of the
GNPTNF vector. The belly images were obtained at the indicated
times and show changes in tumor color within 5 h. The red arrow
shows vector accumulation in the tumor, and the blue arrows mark the
accumulation in the tissues around the tumor. Adapted from ref. 382
by permisson from Wiley Interscience.

of antiangiogenic properties in GNPs makes them potentially

promising in oncotherapy.388,389 The same research team has
shown that GNPs enhance the apoptosis of chronic lymphocytic leukemia cells resistant to programmed death390 and
inhibit the proliferation of multiple myeloma cells.391
Quite recently, Wang et al.392 revealed that PEG-coated
gold nanorods have an unusual property: they can induce
tumor cell death by accumulating in mitochondria and subsequently damaging them. Unexpectedly, for normal and stem
cells, such an eect is either absent or less pronounced.

Table 4

One of the most promising aspects of GNP use in medicine,

currently under intense investigation, is targeted drug
delivery.393395 The most popular objects for targeted delivery
are antitumor preparations396 and antibiotics.
GNPs have been conjugated to a variety of antitumor
substances382411 listed in Table 4.
Conjugation is done both by simple physical adsorption of
preparations on GNPs and by using alkanethiol linkers. The
action of the conjugates is evaluated both in vitro (primarily),
with tumor cell cultures, and in vivo, with mice bearing implanted
tumors of various nature and localizations (Lewis lung carcinoma,
pancreatic adenocarcinoma, etc.). For creation of a delivery
system, target molecules (e.g., cetuximab) are applied along
with the active substance so as to ensure better anchoring and
penetration of the complex into the target cells.399 It was also
suggested that multimodal delivery systems be used,412 in
which GNPs are loaded with several drugs (both hydrophilic
and hydrophobic) and with auxiliary substances (target molecules, PDT dyes, etc.;413,414 Fig. 13). Most researchers have
noted the high eectiveness of GNP-conjugated antitumor
4.2 Delivery of other substances and genes
Besides antitumor substances, other objects employed to
deliver GNPs are antibiotics and other antibacterial agents.
Gu et al.416 prepared a stable vancomycincolloidal gold

Antitumor substances conjugated with GNPs



Methods of

Auxiliary substances

Cell lines or animals


GNSs, 26 nm




GNSs, 13 nm

Physical adsorption


GNSs, 5 nm,
16 nm
GNSs, 5 nm

acid as a linker
Physical adsorption

MC-38; C57/BL6 mice implanted 382, 2006

with B16/F10; melanoma cells
LL2, ML-1, MBT-2, TSGH 8301, 397, 2007
TCC-SUP, J82, PC-3, HeLa
398, 2007



GNSs, 5 nm
36 nm
GNSs, 2 nm
c,c,t-[Pt(NH3)2Cl2(OH) GNSs, 13 nm
GNSs, 5 nm

Physical adsorption
Physical adsorption


Thiol ligand
Amide linkages


HeLa, U2OS, PC3

402, 2009
403, 2009

PEGSH as a linker

Folic acid, PEGSH

404, 2010


GNSs, 30 nm

PEGSH as a linker


Kahalalide F

406, 2009


MDA-MB-231, MCF-7, HSC-3

BT474, SKBR3, MCF-7

407, 2009
408, 2009


GNSs, 20,
Physical adsorption
40 nm
GNSs, 25 nm
PEGSH as a linker
GNRs, lmax=760 nm 11-Mercaptoundecanoic
acid as a linker
GNSs, 25 nm
Physical adsorption

OV-167, OVCAR-5,
A549, HCT116, HCT15,


GNSs, 12 nm
GNSs, 50 nm



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Physical adsorption
Physical adsorption

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PANC-1, AsPC-1, MIA Paca2

399, 2008


400, 2008
401, 2008

Cyclodextrin as a drug MCF-7

pocket, anti-EGFR,
Folate-modied PEG KB


405, 2010

409, 2009
410, 2010
411, 2010

Chem. Soc. Rev., 2012, 41, 22562282


Fig. 13 Schemes for dierent versions of drug delivery systems.

Adapted from ref. 412.

complex and showed its eectiveness toward various (including

vancomycin-resistant) enteropathogenic strains of Escherichia
coli, Enterococcus faecium, and Enterococcus faecalis. Similar
results were presented by Rosemary et al.:417 a complex
formed between ciprooxacin and gold nanoshells had high
antibacterial activity against E. coli. Selvaraj and Alagar418
reported that a colloidal gold conjugate of the antileukemic
drug 5-uorouracil exhibited noticeable antibacterial and
antifungal activities against Micrococcus luteus, Staphylococcus
aureus, Pseudomonas aeruginosa, E. coli, Aspergillus fumigatus,
and A. niger. Noteworthy is the fact that in all those cases, the
drugGNP complexes were stable, which could be judged by the
optical spectra of the conjugates.
By contrast, Saha et al.419 (antibiotics: ampicillin, streptomycin,
and kanamycin; bacteria: E. coli, M. luteus, and S. aureus) and
Grace and Pandian420,421 (aminoglycoside antibiotics: gentamicin, neomycin, and streptomycin; quinolone antibiotics:
ciprooxacin, gatioxacin, and noroxacin; bacteria: E. coli,
M. luteus, S. aureus, and P. aeruginosa) failed to make stable
complexes with GNPs. Nevertheless, those authors showed
that depending on the antibiotic used, the increase in the
activity of an antibioticcolloidal gold mixture, as compared
to that of the native drug, ranged from 12 to 40%. From these
data, it was concluded that the antibacterial activity of the
antibiotics is enhanced at the cost of GNPs. However, the
question as to the mechanisms involved in such possible
enhancement remained unclaried, which was noted by the
authors themselves. Burygin et al.422 experimentally proved
that free gentamicin and its mixture with GNPs do not
signicantly dier in antimicrobial activity in assays on solid
and in liquid nutrient media. They proposed that a necessary
condition for enhancement of antibacterial activity is the

Chem. Soc. Rev., 2012, 41, 22562282

preparation of stable conjugates of nanoparticles coated with

antibiotic molecules. Specically, Rai et al.423 suggested the
use of the antibiotic cefaclor directly in the synthesis of GNPs.
As a result, they obtained a stable conjugate that had high
antibacterial activity against E. coli and S. aureus.
Other drugs conjugated to GNPs are referred to much more
rarely in the literature. However, some of those works deserve
mention. Nie et al.424 demonstrated high antioxidant activity
of GNPs complexed with tocoferol and suggested potential
applications of the complex. Bowman et al.425 provided data
to show that a conjugate of GNPs with the preparation TAK779 exhibits more pronounced activity against HIV than the
native preparation at the cost of the high local concentration.
Joshi et al.426 described a procedure for oral and intranasal
administration of colloidal-gold-conjugated insulin to diabetic
rats, and they reported a signicant decrease in blood sugar,
which was comparable with that obtained by subcutaneous
insulin injection, Finally, Chamberland et al.427 reported a
therapeutic eect of the antirheumatism drug etanercept conjugated to gold nanorods.
In conclusion, it is necessary to mention gene therapy, which
can be seen as an ideal strategy for the treatment of genetic
and acquired diseases.428 The term gene therapy is used in
reference to a medical approach based on the administration,
for therapeutic purposes, of gene constructs to cells and the
organism.429 The desired eect is achieved either as a result of
expression of the introduced gene or through partial or
complete suppression of the function of a damaged or overexpressing gene. There have also been recent attempts at
correcting the structure and function of an improperly functioning (sick) gene. In such a case, too, GNPs can serve as
an eective means of delivery of genetic material to the
cytoplasm and the cell nucleus.430433 Furthermore, gold
glyconanoparticles have been proposed for use as a carrier
of tumor-associated antigens in the development of anticancer

5. Immunological properties of GNPs

5.1 Production of antibodies by using GNPs
Since the 1920s, the immunological properties of colloidal
metals (in particular, gold) have been attracting much research
interest. This interest was mainly due to the physicochemical
(nonspecic) theory of immunity proposed by J. Bordet, who
had postulated that immunogenicity, along with antigenic
specicity, depends predominantly on the physicochemical
properties of antigens, rst of all on their colloidal state. In
1929, Zilber and Friese successfully obtained agglutinating
sera to colloidal gold.435 (Curiously, another attempt to prepare
antisera to colloidal gold was made almost 80 years later, in
2006.436) Yet, several authors have shown that the introduction
of a complete antigen together with colloidal metals promotes
the production of antibodies.437 Moreover, some haptens may
cause antibody production when adsorbed to colloidal particles.438
Numerous data pertaining to the inuence of colloidal gold on
nonspecic immune responses are given in one of the best early
reviews.439 Specically, it was noted (with reference to the
1911 work of Gross and OConnor) that at 2 h after an
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intravenous injection of 5 mL of colloidal gold into rabbits,

there occurs a sizable increase in total leukocytes in 1 mL of
blood (from 10 000 to 19 800) against a slight decline in
mononuclear cells (from 5200 to 4900) and a considerable
increase in polynuclear cells (from 4700 to 14 900). On injection
of other colloidal metals, no such phenomena are observed.
Unfortunately, with advances in immunology and denial of
many postulates of Bordets theory, the interest in the immunological properties of colloids decreased. There is no doubt,
though, that the data obtained on the enhancement of immune
response to antigens adsorbed on colloidal particles were utilized
to develop various adjuvants.440
It is known that antibody biosynthesis is induced by substances
possessing suciently developed structures (immunogenicity).
These include proteins, polysaccharides, and some synthetic
polymers. However, many biologically active substances (vitamins, hormones, antibiotics, narcotics, etc.) have relatively small
molecular masses and, as a rule, do not elicit a pronounced
immune response. In standard methods of antibody preparation
in vivo, this limitation is overcome by chemically attaching such
substances (haptens) to high-molecular-weight carriers (most
often proteins), which makes it possible to obtain specic antisera.
However, such antisera usually contain attendant antibodies
to the carriers antigenic structures.441
In 1986, Japanese researchers442 rst reported success in
generating antibodies against glutamate by using colloidal gold
particles as a carrier. Subsequently, a number of papers were
published whose authors applied and further developed this
technique to obtain antibodies to the following haptens and
complete antigens: amino acids;443,444 platelet-activating
factor;445,446 quinolinic acid;447 biotin;448 recombinant
peptides;449,450 lysophosphatide acid;451 endostatin;452 the capsid
peptides of the hepatitis C,453 inuenza,454 and foot-and-mouth
disease455 viruses; a-amidated peptides;456 actin;457 antibiotics;458
azobenzene;459 Ab-peptide;460 clenbuterol;461 Yersinia surface
antigens;462 transmissible gastroenteritis virus;463 tuberculin;464
and the peptides of the malaria (Plasmodium) surface protein.465
In all these studies, the haptens were directly conjugated to
colloidal gold particles, mixed with complete Freunds adjuvant
or alum, and used for animal immunization. As a result, hightiter antisera were obtained that did not need further purication
from contaminant antibodies (Fig. 14).
In 1993, Pow and Crook466 suggested attaching a hapten
(g-aminobutyric acid) to a carrier protein before conjugating
this complex to colloidal gold. This suggestion was supported
in articles devoted to the raising of antibodies to some
peptides,467471 amino acids,472475 phenyl-b-D-thioglucoronide,476
and diminazene.477 The antibodies obtained in this way possessed
high specicities to the antigens under study and higher (as Pow
and Crook466 put it, extremely high) titersfrom 1 : 250 000
to 1 : 1 000 000, as compared with the antibodies produced
In 1996, Demenev et al.478 showed for the rst time the
possibility of using colloidal gold particles included in the
composition of an antiviral vaccine as carriers for the protein
antigen of the tick-borne encephalitis virus capsid. According
to the authors data, the oered experimental vaccine had
higher protective properties than its commercial analogs,
despite the fact that the vaccine did not contain adjuvants.
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Fig. 14 Schematic representation of immunogen localization on the

surface of keyhole limpet hemocyanin (KLH) and GNPs, used as
antigen carriers. (A) Antibodies toward the peptideKLH conjugate
are produced to the epitopes of both peptide and KLH. (B) Antibodies
toward the peptideGNP conjugate are produced only to the epitopes
of the peptide. Adapted from ref. 455 by permission from IOP

In 2011, Wang et al.479 suggested a new therapeutic vaccine

based on the combination of myelin-associated inhibitors and
GNPs for the treatment of rat medullispinal traumas. Also, for
GNP-assisted antigens, two groups reported new administration
ways: through the skin and mucous coats.480,481
Scheme 2 summarizes the literature data on antigens and
haptens that have been conjugated with GNP carriers and
then used for immunization of animals. The titers of the
antibodies have been increased owing to GNPs.
A considerable number of papers devoted to the use of
GNPs for creating DNA vaccines have emerged as well. The
principle of DNA immunization is as follows: gene constructs
coding for the proteins to which one needs to obtain antibodies are introduced into the organism being treated. If the
gene expression is eective, these proteins serve as antigens for
the development of an immune response. Among the nanoparticles used as DNA carriers, colloidal gold particles are
especially popular with researchers.482,483
5.2 Adjuvant properties of GNPs
Dykman et al.458,464 proposed a technology for the preparation
of antibodies to various antigens, which uses colloidal gold as
a carrier and as an adjuvant. In their method, antigens are
adsorbed directly on the GNP surface, with no cross-linking
reagents added. It was found that animal immunization with
colloidal goldantigen conjugates (with or without Freunds
complete adjuvant) yielded specic, high-titer antibodies to a
variety of antigens, with no concomitant antibodies. GNPs
can stimulate antibody synthesis in rabbits, rats, and mice, and
the required amount of antigen is reduced, as compared to
that needed with some conventional adjuvants (Table 5).
It was demonstrated that GNPs used as an antigen carrier
activate the phagocytic activity of macrophages and inuence
the functioning of lymphocytes, which apparently may be
responsible for their immunomodulating eect. It was also
revealed that GNPs and their conjugates with low- and highmolecular weight antigens stimulate the respiratory activity of
cells of the reticuloendothelial system and the activity of
macrophage mitochondrial enzymes.484 This stimulation is
possibly one of the causative factors determining the adjuvant
properties of colloidal gold. That GNPs act as both an
adjuvant and a carrier (i.e., they present haptens to T cells)
Chem. Soc. Rev., 2012, 41, 22562282


Scheme 2

Conjugates of antigens and haptens used for immunization of animals.

seems the most interesting aspect of immunogenic properties

exhibited by colloidal gold. In particular, GNPs conjugated to
antigens were found to inuence the activation of T cells,
inducing a tenfold increase in proliferation, as compared with
that observed on the addition of the native antigen. This fact
indicates that there is a fundamental possibility of targeted
activation of T cells followed by macrophage activation and
pathogen killing.
However, not a single paper available to us has reported
data on the mechanism of such properties of gold particles. In
our opinion, the reasoning given by Pow and Crook466 on the
preferable macrophage response to corpuscular antigens, as
opposed to soluble ones, is certainly valid. This fact has also
been conrmed by researchers studying the mechanism of
action of DNA vaccines and using gold particles to deliver
genetic material to cells.485,486 The role of Kuper and Langerhans cells in the development of immune response was shown
in those investigations. The inuence of dendritic cells on the
development of immune response upon injection of a GNPconjugated antigen was discussed by Vallhov et al.487 In
addition, those authors noted that when using nanoparticles
in medical practice, one has to ensure that there are no
lipopolysaccharides on their surface. The interaction of cells
of the immune system with GNPs was examined recently by
Villiers et al.488 and by Zolnik et al.489
Table 5

The penetration of GNPs complexed with peptides into the

cytoplasm of macrophages, causing their activation, was
shown electron microscopically by Bastus et al.490,491 They
established that after the conjugates have interacted with the
TLR-4 receptors of macrophages, the nanoparticles penetrate
into the cells, this being accompanied by secretion of the
proinammatory cytokines TNF-a, IL-1b, and IL-6 and by
suppression of macrophage proliferation. Dobrovolskaia and
McNeil492 do not exclude the possibility of another (noninammatory) way of GNP penetration into macrophages,
namely by means of interaction with scavenger receptors.
Franca et al.493 showed that irrespective of size, GNPs are
internalized by macrophages via multiple routes, including
both phagocytosis and pinocytosis. If either route is blocked,
the particles enter the cells via the other route. GNPs with
hydrodynamic sizes below 100 nm can be phagocytozed.
Phagocytosis of anionic gold colloids by RAW264.7 cells is
mediated by macrophage scavenger receptor A. Ma et al.494
investigated the inhibitory eect of PEG-coated GNPs on NO
production and its molecular mechanism in lipopolysaccharidestimulated macrophages. It was found that GNPs inhibited
lipopolysaccharide-induced NO production and inducible nitric
synthase expression in macrophages. Yen et al.495 noted that
on the administration of GNPs, the number of macrophages
decreases and their size increases, this being accompanied by

The antibody titers obtained during immunization of rabbits with Yersinia antigen


1st immunization

2nd immunization


Colloidal gold + antigen (1 mg)

Complete Freunds adjuvant + antigen (100 mg)
Physiological saline + antigen (100 mg)

1 : 32
1 : 32

1 : 256
1 : 256
1 : 16

1 : 10 240
1 : 10 240
1 : 512


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elevated production of IL-1, IL-6, and TNF. The inuence of

colloidal gold on immunocompetent cells was examined in vivo
also by Tian et al.496 and by Lou et al.497 They showed that
injection of nonconjugated GNPs into mice enhances the
proliferation of lymphocytes and normal killers, as well as
an increase in IL-2 production.
Recently, many articles have appeared (see above) discussing
problems in GNP use for targeted drug delivery. In our
opinion, this question should be dealt with very carefully,
with account taken of the possibility of production in animals
or humans of antibodies specic to the administered drug
adsorbed on gold particles. We believe that the discovery of
adjuvant properties in GNPs has created favorable conditions
for designing next-generation vaccines.
Alongside GNPs, other nonmetallic nanoparticles also can
serve as antigen carriers. The published examples include
liposomes, proteosomes, microcapsules, fullerenes, carbon
nanotubes, dendrimers, and paramagnetic particles.464 In
our view, especially promising carriers are synthetic and
natural polymeric biodegradable nanomaterials [polymethyl
methacrylate, poly(lactid-co-glycolid acid), chitosan, gelatin].
With the use of such nanoparticles, the immunogenicity of a
loaded substance and its representation in a host immune
system will be changed. A nanoparticle conjugate with an
absorbed or a capsulated antigen can serve as an adjuvant for
the optimization of immune response after vaccination. The
evident advantages of biodegradable nanoparticles are their
utilization in the vaccinated organism, high loading eciency
for the target substance, enhanced ability to cross various
physiological barriers, and low systemic side eects. In all
likelihood, the immune actions of biodegradable nanoparticles
and GNPs as corpuscular carriers are similar. Keeping in mind
the recent data for the low toxicity of GNPs and their ecient
excretion by the hepatobiliary system, we expect that both
nanoparticle classesGNPs and biodegradable nanoparticleswill
compete on equal footing for being used in the development of
nextgeneration vaccines.

6. Conclusions
Owing to the success of the rapid development of technologies
for the chemical synthesis of GNPs during the past decade,
investigators currently have at their disposal an enormous
diversity of available particles with required parameters in
respect of size, shape, structure, and optical properties. Moreover, the question that is now on the agenda is the primary
modeling of a nanoparticle with desired properties and the
subsequent development of a procedure for the synthesis of a
theoretically predicted nanostructure.
From the standpoint of medical applications, much signicance was held by the development of eective technologies
for the functionalization of GNPs with molecules belonging to
various classes, which ensure nanoparticle stabilization in vivo
and targeted interaction with biological targets. At this juncture,
the best stabilizers are thiolated derivatives of PEG and other
molecules. Specically, PEG-coated particles can circulate in
the blood stream for longer times and are less susceptible to
the attack of the cellular components of the immune system.
However, the creation of stealth conjugates, able to bind to
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biological targets in an eective and target-oriented way,

remains an unresolved problem.
It is now generally recognized that GNP conjugates are
excellent labels to use in bioimaging, which can be realized by
various biophotonic technologies, including dark-eld resonance
scattering microscopy, confocal laser microscopy, diverse versions
of two-photon scattering and own luminescence of GNPs, optical
coherence and acoustic tomography, and so on.
GNP conjugates have found numerous applications in
analytical research based on both state-of-the-art instrumental
methods (SERS, SEIRA, LISNA, etc.) and simple solid-phase
or homophase techniques (dot assay, immunochromatography, etc.). The following two examples are illustrative: (1)
by using GNPantibody conjugates, it is possible to detect a
prostate-specic antigen with a sensitivity that is millionfold
greater than that in the ELISA.498 (2) The sharp dependence
of the systems color on interparticle distances enables mutant
DNAs to be detected visually in a test known as the Northwestern spot test.125 Along with the literature examples of clinical
diagnostics of cancer, Alzheimers disease, AIDS, hepatitis,
tuberculosis, diabetes, and other diseases, new diagnostic applications of GNPs should be expected. Progress in this direction
will be determined by success achieved in improving the sensitivity of analytical tests with retention of the simplicity of
detection. The limitations of homophasic methods with visual
detection are due to the need to use a large number [approximately 1010 (ref. 21)] of nanoparticles. Even at the minimal ratio
between target molecules and particles (1 : 1), the detection limit
will be ca. 0.01 pMconsiderably (millionfold) higher than the
quantity of target molecules to be detected, e.g., in typical biopsy
samples.21 Thus, sensitivity can be improved either by enhancing
the signal (PCR, autometallography, etc.) or by using sensitive
instrumental methods. For instance, single-particle instrumental
methods289 have a single-molecule detection limit that is attainable in principle. Specically, the SERS is a trend technique to
detect very low concentrations of solutes (see, e.g., the recent
review and reports by Liz-Marzan and coworkers499501 regarding
SERS detection of biological molecules). However, the topical
problem is to create multiplex sensitive tests that do not
require equipment and can be performed by the end user
under nonlaboratory conditions. A prototype of such tests is
Pro Strips,TM which can simultaneously detect ve threads:
antrax, ricin toxin, botulinum toxin, Y. pestis (plague), and
staphylococcal enterotoxin B. The physical basis of the new
tests may be associated with the dependence of the plasmon
resonance wavelength on the local dielectric environment or
on the interparticle distance.
GNP-assisted PPTT of cancer, rst described in 2003, is
now in the stage of clinical trials.305 The actual clinical success
of this technology will depend on how quickly it will be
possible to solve several topical problems: (1) development
of eective methods for the delivery of radiation to tumors
inside the organism by using ber-optic technologies or nonoptical heating methods, (2) improvement of the methods of
conjugate delivery to tumors and enhancement of the contrast
and accumulation uniformity, and (3) development of methods
for controlling the process of photothermolysis in situ.
GNP-aided targeted delivery of DNA, antigens, and drugs
is one of the most promising directions in biomedicine.
Chem. Soc. Rev., 2012, 41, 22562282


Specically, research conducted by Warren Chans teams at

Toronto University502 has shown the size-dependent possibility
of delivery of GNPs complexed with herceptin to cancer cells
with much greater eectiveness than that obtained with a pure
preparation. The recent critical reexamination of the PPTT
concept, based on the intravenous targeted delivery of GNPs
conjugated with molecular probes to the molecular receptors
of cancer,343 indicates that there is a pressing need to continue
research in this direction. The discovery of adjuvant properties
in GNPs has created favorable conditions for the development
of next-generation vaccines.
Finally, there is need to continue and expand work in the
area of GNP biodistribution and toxicity. Although the past
ve years have seen signicant research activity22,32,503505
several important aspects remain to be studied. It is commonly
recognized now that the nal conclusions concerning biodistribution and toxicity can be aected by many factors, including
particle size and shape, functionalization methods, animal
types, particle administration doses and methods, and so on.
For instance, Alkilany and Murphy504 addressed the important
question of the origin of toxicity of GNP suspensions. The
well-known example of CTAB-coated gold nanorods clearly
shows that one should discriminate between the toxicity of
GNPs themselves and that of a dispersion medium in which
the GNPs are dispersed (supernatant control).504
Although bulk gold is known to be chemically inert, the
nanotoxicity of GNPs with sizes smaller than 35 nm may be
dierent from that of both organogold complexes and larger
GNPs. In particular, GNPs with diameters of 12 nm have
potentially higher toxicity because of the possibility of irreversible
binding to cell biopolymers. For example, Tsoli et al.506 and
Pan et al.507 reported that 1.4 nm GNPs were toxic to various cell
lines, whereas no toxicity was found for 15 nm GNPs. Yet,
numerous experiments with cell cultures did not reveal noticeable
toxicity of 3 to 100 nm colloidal particles, provided that the
limiting dose did not exceed a value of approximately 1012
particles per mL.
The available data on the toxicity of GNPs in vivo are rare
and somewhat controversial. One can only speculate that
noticeable toxicity does not occur during short-period
(approximately weeklong) administration of GNPs at a daily dose
not greater than 0.5 mg kg1. Perhaps this estimate may sound
too strong if we compare it with the data reported by von
Maltzahn et al.304 Those authors used gold nanorods at a total
dose of 20 mg kg1 and did not observe any toxicity in either
tumor-bearing or tumor-free mice.
Whereas there are more than 40 reports on GNP toxicity
in vitro, those studies cannot be used as good predictors of
possible toxicity in vivo. For example, Chen et al.454 observed
that the toxicity of the same GNPs was contradictory for
models in vivo and in vitro.
As pointed out by Alkilany and Murphy,504 one should
introduce standards for GNP characterization not only prior
to administration to a biological model but also after mixing
with biological media. Otherwise, there may be a change in
GNP charge, desorption of stabilizing molecules, and, eventually,
GNP aggregation.
It should be emphasized that GNPs are not biodegradable.
Therefore, the biodistribution and excretion kinetics have to

Chem. Soc. Rev., 2012, 41, 22562282

be studied comprehensively for dierent animal models. The

organs of the reticuloendothelial system are the basic primary
target for the accumulation of GNPs within the size range
10100 nm, and the uniformity of biodistribution increases
with a decrease in particle size. As the excretion of accumulated particles from the liver and spleen can take up to
34 months, the question as to the injected doses and possible
inammation processes is still of great importance. Bioaccumulated GNPs can interfere with dierent diagnostic techniques,
or accumulated GNPs can exhibit catalytic properties. All these
concerns, together with potential toxicity, are big limitations of
GNPs on a successfully clinical translation. Nowadays, despite
the huge numbers of studies regarding the synthesis and functionalization of GNPs (dierent shapes, coatings, sizes, charges,
etc.), there are very few nanomaterial-based pharmaceuticals on
the market (see Rivera Gil et al.508).
In summary, there should be a coordinated research program
to establish correlations between the particle parameters (size,
shape, and functionalization with various molecular probes),
the experimental parameters (model; doses; method and time
schedule of administration; observation time; organs, cells and
subcellular structures examined; etc.), and the observed biological


cetyltrimethylammonium bromide
epidermal growth factor receptor
gold nanoparticle(s)
poly(ethylene glycol)
photodynamic therapy
plasmonic photothermal therapy
sol particle immunoassay
surface plasmon resonance
transmission electron microscopy
tumor necrosis factor

We acknowledge support from the Russian Foundation for Basic
Research and from the Presidium of the Russian Academy of
Sciences within the program Basic Sciences for Medicine. We also
acknowledge support by grants from the Ministry of Education and
Science of the Russian Federation (no. MK-1057.2011.2, 2.1.1/
2950, 14.740.11.260, and 02.740.11.0484) and by a grant from the
Government of the Russian Federation designed to support
research projects supervised by leading scientists at Russian institutions of higher education. We thank D. N. Tychinin (IBPPM
RAS) for his help in preparation of the manuscript.

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