2256 Chem. Soc. Rev., 2012, 41, 22562282 This journal is c The Royal Society of Chemistry 2012

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

Gold nanoparticles in biomedical applications: recent advancesand 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

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

a Institute of Biochemistry and Physiology of Plants andMicroorganisms, RAS, 13 Pr. Entuziastov, Saratov 410049,Russian Federation. E-mail: khlebtsov@ibppm.sgu.ru

b Saratov State University, 83 Ul. Astrakhanskaya, Saratov 410012,Russian Federation

Lev Dykman

Dr Lev A. Dykman, leadingresearcher of the Immuno-chemistry Lab at the Instituteof Biochemistry and Physiol-ogy of Plants and Micro-organisms Russian Academyof Sciences. He has publishedmore than 200 scientic worksincluding one monograph oncolloidal gold nanoparticles.His current scientic interestsinclude immunochemistry,fabrication of gold nano-particles and their applicationsto biological and medicalstudies. In particular, his

research is aimed at interaction of nanoparticles and conjugateswith immune-responsible cells and at the delivery of engineeredparticles to target organs, tissues, and cells.

Nikolai Khlebtsov

Professor Nikolai G. Khlebtsov,head of the NanobiotechnologyLab at the Institute of Bio-chemistry and Physiology ofPlants and MicroorganismsRussian Academy of Sciences.He also holds a BiophysicsChair at the Saratov StateUniversity. He has publishedmore than 300 scienticworks, including two mono-graphs. His current scienticinterests include biophotonicsand nanobiotechnology ofplasmon-resonant particles,biomedical applications of

metal nanoparticles, static and dynamic light scattering by smallparticles and clusters, programming and computer simulation oflight scattering and absorption by various metal and dielectricnanostructures. Prof. Khlebtsov also serves as an AssociateEditor of the Journal of Quantitative Spectroscopy and Radia-tive Transfer.

Chem Soc Rev Dynamic Article Links

www.rsc.org/csr CRITICAL REVIEW

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This journal is c The Royal Society of Chemistry 2012 Chem. Soc. Rev., 2012, 41, 22562282 2257

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 improve-

ment of PCR eciency.9

Despite the centuries-old history, a revolution in immuno-

chemistry,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; conju-

gates with recognizing biomacromolecules, e.g., antibodies, lectins,

enzymes, or aptamers)1214 to the studies of biochemists, micro-

biologists, 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,

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 trans-

mission 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 quasi-

spherical particles (nanospheres) as labels for microscopic

studies, the past few years have seen the application of

nonspherical cylindrical particles (nanorods), nanoshells, nano-

cages, nanostars, and other types of particles, referred to by

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.

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2258 Chem. Soc. Rev., 2012, 41, 22562282 This journal is c The Royal Society of Chemistry 2012

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 simulta-

neous 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, two-

photon 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 GNP-

aided bioimaging methods. It is based on light scattering by

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

background.

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 byMostafa 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 con-

jugated 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 bio-

molecular 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 con-

jugates. 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 micro-

scopy 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

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.

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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

Particle Major resonances/nm Plasmonic shift/RIU/nm Possible biomedical applications

Colloidal Au nanospheres (3100 nm) 510570 4590 EM, OI, HA, SA, PPT,a DCAu nanorods (thickness, 1020 nm; aspect ratio, 210) 6501200 150290 OI, OA, PPT, HIA,Au bipyramids 6501100 150540 OI, BSAu(core)/Ag(shell) NRs 5501000 OI, SERSFe2O3 (core)/Au (shell) nanorice 11001300 790810 SERS, BSSiO2(core)/Au(shell) 6001100 160

b

315c OI, SA, PPTHollow Au shell 125 OI, PPTNanobowls 5601000 SERSSpiky SiO2 nanoshells and Au nanostars 600850,

675770 240665 OI, SERSAu polyhedralsd 550750 EM, OIAu cubes 550700 83 EM, OIAuAg nanocages 4501000 410620 OIA, BS, SERS, PPTAu nanocrescentse 9802600 240880 BS

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.e Nanolithography array.

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2.2 Analytical diagnostic methods

2.2.1 Homophase techniques. Beginning in 1980s, colloidal

gold conjugated with recognizing biomacromolecules was com-

ing 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 colori-

metric 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 immuno-

globulins,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 determi-

nation of protease activity;110 and other purposes. The simulta-

neous 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). Some-

times, 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

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

applications.

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 light-

scattering spectroscopy,119 and dynamic light scattering.120 In

addition, two vibrational spectroscopymethodssurface 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

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.

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.

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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 cross-

linking 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 aggre-

gates, 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 30 or 50 thiol-modied 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 noncomple-

mentary targets did not prevent aggregation at 2 M NaCl. The

apparent contradictions between these data were explained by

Song et al.142 through the dierence in surface functionalization

density.

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 citrate-

stabilized 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 hybridi-

zation 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

Table 2 Detection of DNA with the use of gold nanoparticles

Particles Probe Target Detection method Detection limit Ref.

ModiedGNSs(GNSs withchemicallyattachedprobes)

50-HS(CH2)6N13N15-30 ssDNA Cross-linking aggregation,UVAS, spot test

10 fmol 125, 1997

50-SH(dT)10-30 Biotinylated PCRproduct

Hybridization of targets with poly-Aand streptavidin followed by a striptest with GNSs

2 fmol (500 copiesof PSA cDNA)

126, 2003

50-N15(CH2)10SH-30a;

50-SH(CH2)10N15-30b

ssDNA Bio-bar-code assay, scanometricdetection of silver-enhanced spots

500 zmol (10 copiesin 30 mL)

127, 2004

50-N15C-30(CH2)3SH;HS(CH2)65

0-N18-30PCR product Non-cross-linking aggregation in

1 M NaCl, VE, spot test100250 nM;12 nM

128, 2006;140, 2005

50-HSN16-30 1-round PCRproduct

Stabilization of nanoprobes bytargets in 2M NaCl, VE, UVAS

300 nM 129, 2006

50-N12C3SS-30;50-SSC6N15-30

ssDNA Aggregation, DLS 5 pM 130, 2008

50-SH(CH2)6N15-30;50-RoxN15(CH2)3SH-30

ssDNA SERS 10 zM 136, 2010

UnmodiedGNSs

Rhodamine red50-N15-30 ssDNA Fluorescence quenching/retainingwith nontarget/target ssDNA

0.5 pM 133, 2004

50-N15-30 ssDNA, PCRproduct

Stabilization/aggregation withnontarget/target ssDNA, UVAS

2 pM 134, 2004

ssDNA, aptamers +conjugated polyelectrolytec

ssDNA, thrombin,cocaine, Hg

Aggregation in the case of nontargetmolecules, VE, UVAS

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

144, 2010

50-N21-30, 50-N23-30 ssDNA Aggregation of positively chargedGNSs, UVAS, DLS

10 pM 44, 2011

UnmodiedGNRs

50-N21-30 ssDNA Aggregation, UVLS 1.7 nM 135, 2008

50-N30-30 ssDNA Aggregation (optimized), UVAS 0.1 pM 145, 2010

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]bromide.

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2262 Chem. Soc. Rev., 2012, 41, 22562282 This journal is c The Royal Society of Chemistry 2012

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 immuno-

assay development, preference was given to liquid-phase techni-

ques, in which bound antibodies were precipitated or unbound

antigen was removed by adsorption with dextran-coated activated

charcoal. Currently, the most popular techniques are solid-

phase ones (rst used for protein radioimmunoassay), because

they permit the analysis to be considerably simplied and the

background signal to be reduced. Themost 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

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

membrane.161

Colloidal gold in membrane assays has served for the diagnosis

of parasitic,162166 viral,167170 and fungal171,172 diseases, tuber-

culosis,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 nano-

shells, 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 (immuno-

chips).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

Table 3 Sensitivity limits for immunodot/blot methods implementedon nitrocellulose lters by using various labels (according to ref. 155)

LabelSensitivity limit(pg of protein per fraction)

125I 5Horseradish peroxidase 10Alkaline phosphatase 1Colloidal gold 1Colloidal gold + silver 0.1Fluorescein isothiocyanate 1000

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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 sought-

for 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

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 concen-

tration 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 pseu-

dorabies,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. 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 twofolddilutions. The lower rows (2) correspond to the application of 10 mg ofbovine 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.

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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2264 Chem. Soc. Rev., 2012, 41, 22562282 This journal is c The Royal Society of Chemistry 2012

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 concen-

tration 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

metals.231

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 ensureeective 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 couplingmethod and the attenuated reectionmethod,

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

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 strate-

gies 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 single-

particle 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).255Nusz 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 bio-

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

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

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 signi-

cance 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

nanoparticles.259

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 concen-

tration 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 leadto a critical change in the conformation of native molecules.

The relatively small dierence between the refractiveindices 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

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 immuno-

assay281284 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, electro-

phoresis, 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 biotinstrep-

tavidin binding. It was found that both techniques demon-

strate 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.

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.

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2266 Chem. Soc. Rev., 2012, 41, 22562282 This journal is c The Royal Society of Chemistry 2012

3. GNPs in therapy

3.1 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 corres-

ponding 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 Forlocal heating to 70 1C, the heating time may be reduced to34 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 high-

powered 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 photo-

thermia 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 near-

IR 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 nanorodsfunctionalized 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 nano-

rods;67,312314 nanoshells;301,315320 and a comparatively new particle

class, goldsilver nanocages.93,321,322 Experimental data com-

paring 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 photo-

thermal 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 concen-

tration 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

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 nano-

particle 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 pene-

tration 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 frag-

ment peptide that recognizes the urokinase plasminogen acti-

vator receptor; and (3) a cyclic RGD peptide that recognizes

the avb3 integrin receptor. The authors showed that wheninjected intravenously, all three ligands induce insignicant

increases in nanoparticle accumulation in cell models and in

tumors but greatly aect extracellular distribution and intra-

cellular 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

Fig. 11 Scheme for a PPTT employing active delivery of GNPs to

cancer cells.

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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 nano-

particle 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.

3.2 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 chromo-

phore 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

(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 collagen-

induced arthritis were intraarticularly injected with colloidal

gold. The authors explain the positive eect by an enhance-

ment 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 accu-

mulated 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 record-

able 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 angio-

genesis (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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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 lympho-

cytic 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 sub-

sequently damaging them. Unexpectedly, for normal and stem

cells, such an eect is either absent or less pronounced.

4. GNPs as drug carriers

4.1 Targeted delivery of anticancer drugs

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 mole-

cules, PDT dyes, etc.;413,414 Fig. 13). Most researchers have

noted the high eectiveness of GNP-conjugated antitumor

preparations.415

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

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

15 h. Diseased mice were intravenously injected with 15 mg of theGNPTNF 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.

Table 4 Antitumor substances conjugated with GNPs

Drugs ParticlesMethods offunctionalization Auxiliary substances Cell lines or animals Ref.

Paclitaxel GNSs, 26 nm PaclitaxelSH PEGSH, TNF MC-38; C57/BL6 mice implantedwith B16/F10; melanoma cells

382, 2006

Methotrexate GNSs, 13 nm Physical adsorption LL2, ML-1, MBT-2, TSGH 8301,TCC-SUP, J82, PC-3, HeLa

397, 2007

Daunorubicin GNSs, 5 nm,16 nm

3-Mercaptopropionicacid as a linker

K562/ADM 398, 2007

Gemcitabine GNSs, 5 nm Physical adsorption Cetuximab(monoclonalantibodies)

PANC-1, AsPC-1, MIA Paca2 399, 2008

6-Mercaptopurine GNSs, 5 nm Physical adsorption K-562 400, 2008Dodecylcysteine GNSs,

36 nmPhysical adsorption EAC 401, 2008

5-Fluorouracil GNSs, 2 nm Thiol ligand MCF-7 402, 2009c,c,t-[Pt(NH3)2Cl2(OH)(O2CCH2CH2CO2H)]

GNSs, 13 nm Amide linkages DNA HeLa, U2OS, PC3 403, 2009

Cisplatin GNSs, 5 nm PEGSH as a linker Folic acid, PEGSH OV-167, OVCAR-5,HUVEC, OSE

404, 2010

Oxaliplatin GNSs, 30 nm PEGSH as a linker PEGSH A549, HCT116, HCT15,HT29, RKO

405, 2010

Kahalalide F GNSs, 20,40 nm

Physical adsorption HeLa 406, 2009

Tamoxifen GNSs, 25 nm PEGSH as a linker PEGSH MDA-MB-231, MCF-7, HSC-3 407, 2009Herceptin GNRs, lmax=760 nm 11-Mercaptoundecanoic

acid as a linker BT474, SKBR3, MCF-7 408, 2009

b-Lapachon GNSs, 25 nm Physical adsorption Cyclodextrin as a drugpocket, anti-EGFR,PEGSH

MCF-7 409, 2009

Doxorubicin GNSs, 12 nm Physical adsorption Folate-modied PEG KB 410, 2010Prospidin GNSs, 50 nm Physical adsorption HeLa 411, 2010

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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: genta-

micin, 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

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 TAK-

779 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 con-

jugated 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 over-

expressing gene. There have also been recent attempts at

correcting the structure and function of an improperly func-

tioning (sick) gene. In such a case, too, GNPs can serve as

an eective means