Quantum Dot

REVIEW

Semiconductor quantum dots and metal nanoparticles:syntheses, optical properties, and biological applications

Vasudevanpillai Biju & Tamitake Itoh & Abdulaziz Anas &

Athiyanathil Sujith & Mitsuru Ishikawa

Received: 6 December 2007 /Revised: 25 April 2008 /Accepted: 13 May 2008 / Published online: 12 June 2008# Springer-Verlag 2008

Abstract We review the syntheses, optical properties, andbiological applications of cadmium selenide (CdSe) andcadmium selenide–zinc sulfide (CdSe–ZnS) quantum dots(QDs) and gold (Au) and silver (Ag) nanoparticles (NPs).Specifically, we selected the syntheses of QDs and Au andAg NPs in aqueous and organic phases, size- and shape-dependent photoluminescence (PL) of QDs and plasmon ofmetal NPs, and their bioimaging applications. The PLproperties of QDs are discussed with reference to their bandgap structure and various electronic transitions, relations ofPL and photoactivated PL with surface defects, andblinking of single QDs. Optical properties of Ag and AuNPs are discussed with reference to their size- and shape-dependent surface plasmon bands, electron dynamics andrelaxation, and surface-enhanced Raman scattering (SERS).The bioimaging applications are discussed with reference toin vitro and in vivo imaging of live cells, and in vivoimaging of cancers, tumor vasculature, and lymph nodes.Other aspects of the review are in vivo deep tissue imaging,multiphoton excitation, NIR fluorescence and SERS imag-ing, and toxic effects of NPs and their clearance from thebody.

Keywords Quantum dots . CdSe . CdSe–ZnS .

Photoluminescence . Blinking . Nanoparticles . Au . Ag .

Plasmon . SERS . In vitro imaging . In vivo imaging

Introduction

Nanoscience, nanotechnology, and nanobiotechnology (theNanoworld) would not be as exciting as it is today ifsemiconductor quantum dots (QDs) and metal nanoparticles(NPs) did not show size-tunable optical properties. Coher-ent advancements in the synthesis and experimental andtheoretical understanding of the structural, optical, elec-tronic, and magnetic properties of semiconductor QDs andmetal NPs created and strengthened the platform of theNanoworld. Chemists, physicists, biologists, and technolo-gists then interfaced semiconductor QDs, metal NPs, andtheir properties with organic, inorganic, and biomolecules/materials. This interfacing emerged into an exciting field ofhybrid nanomaterials with potential applications in almostall the branches of science and technology today. Thedimension of matter that is important to nanoscience andnanotechnology is typically on the 0.2- to 100-nm scale(nanoscale). At this scale, the surface-to-volume ratios ofmaterials become large and their electronic energy statesbecome discrete, leading to unique electronic, optical,magnetic, and mechanical properties of the nanomaterials.While nanoscience advances with the size-dependentproperties of nanomaterials, large surface tension andfriction limit their applications in the advancement ofnanotechnology. In general, as the sizes of semiconductor,metal, and organic materials are decreased towards thenanoscale, their optical and electronic properties becomesize- and shape-dependent and largely vary from those inthe bulk and at the atomic/molecular levels. The size- and

Anal Bioanal Chem (2008) 391:2469–2495DOI 10.1007/s00216-008-2185-7

Tamitake Itoh and Abdulaziz Anas contributed equally to this article.

V. Biju (*) : T. Itoh :A. Anas :A. Sujith :M. IshikawaNano-Bioanalysis Team, Health TechnologyResearch Center, National Institute of AdvancedIndustrial Science and Technology (AIST),2217-14 Hayashi-cho,Takamatsu, Kagawa 761-0395, Japane-mail: [email protected]

V. Biju :M. IshikawaCenter for Arthropod Bioresources and Biotechnology,Kerala University,Trivandrum, India

shape-dependent properties at the nanoscale are attributedto the quantum confinement effect, i.e., strong confinementof electrons and holes when the radius of a particle is belowthe exciton Bohr radius of the material. Remarkabledevelopments in the fields of semiconductor QDs andmetal NPs emerged more recently when the fundamentalprinciples underlying the quantum confinement effect andlight–matter interactions became clearly understood.

Nanomaterials are structurally and functionally prevalentin the organic, inorganic, and biological fields. Therefore,the subject of nanomaterials is vast and a comprehensivereview is difficult. Examples of inorganic nanomaterials areQDs, quantum wires, and quantum wells of semiconduc-tors, and nanodots and nanorods of metals. Also, fullerenes,carbon nanotubes, supramolecular assemblies, proteins,and nucleic acids are examples of organic- and bio-nanomaterials. A classical example of the unique propertiesof nanomaterials is the brilliant surface plasmon color ofgold (Au) NPs involved in the coloration of glass by an-cient Romans, at a period when light–matter interactionswere unknown. Other examples of the size effects on theproperties of materials are conducting silicon NPs (insulatorin the bulk), catalytically active Au and Pt NPs (chemicallyinert in the bulk), optically transparent and super-hardcopper NPs (opaque, malleable, and ductile in the bulk),and highly luminescent metal chalcogenide NPs (non-luminescent in the bulk). Among these nanomaterials,cadmium selenide (CdSe) QDs and Au and silver (Ag)NPs are especially attractive for applications in opticaldevices, bioanalyses, and bioimaging because of their size-tunable luminescence [1, 2] and plasmon color [3].

The size-dependent photoluminescence (PL) colors ofCdSe and CdSe–ZnS QDs are distributed throughout thevisible region of the electromagnetic spectrum [1, 2, 4–8]. Atypical example of the size-dependent PL color, andabsorption and PL spectra of CdSe and CdSe–ZnS QDsare shown in Fig. 1 [7]. The size-dependent PL color of thismaterial was exploited in various applications includinglasers, light emitting diodes, and in vivo and in vitro imagingand analyses [5, 6, 9–27]. The relation between the size andelectronic band gap in semiconductor nanocrystals wasdeveloped by Luis Brus by applying the particle in a spheremodel approximation to the bulk Wannier Hamiltonian [28,29]. According to the approximation, the lowest eigenvaluein a quantum confined system is given by Eq. (1) [28].

E� � Eg þ ħ2p2

2R2

1

meþ 1

mh

� �� 1:8e2

"R

þ smaller terms ð1ÞIn simple words, as the size (R2) decreases the band gapincreases. The quantum confinement in CdSe QDs is strongwithin 5.6 nm, which is the exciton Bohr radius of CdSe.

Smaller terms including Coulomb attraction are not includedin Eq. (1). This equation was derived even before thequantum confinement was clearly observed in colloidal CdSand CdSe nanocrystals. The band edge states and the originof the PL colors in CdSe QDs are discussed below. Thesignificance of ZnS shells on core CdSe QDs is that a higherband gap material efficiently passivates surface defects inCdSe and stabilizes its optical properties by serving as aphysical barrier.

Like semiconductor QDs, metal NPs exhibit strong size-dependent optical resonance that is generally known assurface plasmon resonance (SPR). This resonance ispronounced in noble metal NPs because of the collectiveoscillations of conduction band electrons [3, 31–39]. TheSPR is especially interesting with reference to Au and AgNPs. Under photoactivation, the plasmon couples with theexcitation light and produces huge enhancement of theelectromagnetic (EM) field in metal NPs [40–51]. Inter-actions between the incident light and the oscillatingelectric fields result in the scattering and absorption oflight. Mie theory (Eq. (2) [52, 53]), which is a generalsolution of Maxwell’s equation, accounts for the scatteringof electromagnetic radiation by any homogeneous and non-magnetic spheroid particle:

Cext ¼ 24p2R3"3=2m

l"2

"1 þ 2"mð Þ2þ"22ð2Þ

The Lorenz–Mie–Debye theory is a rather complete solutionof the Maxwell’s equation for light scattering and absorptionby spheroid particles. Typical SPR bands of colloidal Au andAg NPs are shown in Fig. 2.

The enormous enhancements of the local EM field dueto the coupling of the oscillating electric fields in Au andAg NPs with the incident light make them attractive forapplications from optical devices to bioanalyses and

Fig. 1 (a) Size-dependent PL color and (b) schematic presentation ofsize, color, and PL wavelength of CdSe–ZnS QDs. (c) Absorption (solidlines) and PL (broken lines) spectra of CdSe QDs with various sizes.Reprinted with permission from Refs. [7] (a) and [30] (b). Copyright(1997, 2001) American Chemical Society

2470 Anal Bioanal Chem (2008) 391:2469–2495

bioimaging. Also, the enhanced EM field is useful foranalytical and spectroscopic characterization of differentchemical structures, and physical and chemical processesunder a broad terminology of ‘surface-enhanced spectros-copy’ [47]. Based on the type of interactions between amolecule and the enhanced EM field, the surface-enhancedspectroscopy is classified into surface-enhanced fluorescence,surface-enhanced Rayleigh scattering, surface-enhancedabsorption, and surface-enhanced Raman scattering(SERS). Among these, SERS is specifically attractivebecause of the enormous enhancement of SERS signalsby a factor of ca. 1014–1015 [42, 43, 46, 50, 55–67]. Thishuge enhancement factor involved in SERS improved thedetection limit from ensembles of molecules to single-molecule levels [58, 62, 66, 68–78].

The discussions in this review are focused on thesyntheses, optical properties, and in vivo and in vitroapplications of CdSe and CdSe–ZnS QDs, and Au and AgNPs. Specifically, we review the synthesis in organic andaqueous phases, size- and shape-dependent PL color, bandgap structure and various electronic transitions, relations ofPL and photoactivated PL with surface defects, blinking,and in vivo and in vitro applications of CdSe and CdSe–ZnS QDs. Also, this review includes synthesis, size- andshape-dependent surface plasmon bands, electron dynamicsand relaxation, SERS, and biological applications of Auand Ag NPs.

Syntheses of semiconductor QDs

Syntheses of CdSe QDs from dimethyl cadmium

In the 1980s, CdSe QDs were prepared by top-downtechniques such as lithography; however, size variations,crystal defects, poor reproducibility, and poor opticalproperties of such QDs made them unsuitable for advancedapplications. Introduction of bottom-up colloidal synthesisof CdSe QDs by Bawendi and coworkers [2] brought far-reaching changes in the properties and applications of QDs.Pyrolysis of organometallic precursors of cadmium (di-methyl cadmium, CdMe2) and selenium in a coordinatingsolvent composed of a mixture of trioctylphosphine (TOP)and trioctylphosphine oxide (TOPO) provided hydrophobi-cally capped CdS, CdSe, and CdTe QDs. In this approach,CdMe2 (13.35 mmol in 25 mL TOP) was reacted at ca.230–300 °C with TOP-selenide (10 mmol in 15 mL TOP)in the presence of TOPO. Here, TOPO was heated to ca.300 °C under vacuum for 20 min followed by injection of amixture of CdMe2 and the Se precursor in an atmosphere ofAr. The growth of CdSe nanocrystals was carried out at ca.230–260 °C. The resulting sample contained a sizedistribution (ca. 1.2–11.5 nm) of QDs, and the differentsizes were separated by size-selective purification from amixture of 1-butanol and methanol. Reproducibility, betterquantum confinement, and size-tunable PL of colloidalQDs obtained in this method attracted researchers to thecolloidal synthesis. By a simple replacement of TOP withtributylphosphine (TBP) and by avoiding Ostwald ripening,Alivisatos and coworkers considerably improved thecolloidal synthesis of CdSe QDs [79]; Ostwald ripening, agradual growth of larger QDs at a cost of gradualdissolution of smaller ones, was controlled by separatingthe spontaneous nucleation process from the relatively slownanocrystal growth process. One of the main advantages ofthis method is the preparation of size-selected QDs byselecting an injection temperature and a growth tempera-ture. By this selection of temperature, the tedious size-selective purification involved in the previous method wasavoided. These two methods reported by Bawendi andcoworkers and Alivisatos and coworkers were later im-proved by Weller and coworkers [30].

Syntheses of CdSe QDs from greener cadmium precursors

Since the above-mentioned breakthrough reports on thecolloidal synthesis of QDs from CdMe2, researchers wereinterested in replacing CdMe2 due to its toxicity, volatility,and pyrophoric nature. The use of an alternative cadmiumprecursor in the colloidal synthesis of QDs was firstintroduced by Weller and coworkers by using a non-volatilecadmium precursor (CdClO4) and aqueous phase synthesis

Fig. 2 SPR bands of Au (A) and Ag (A) NPs with different sizes.Reprinted with permission from Refs. [54] (a) and [52] (b). Copyright(1999) American Chemical Society and copyright (2005) Wiley-VCHVerlag GmbH & Co.

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using thioglycerol as a capping agent [80]. More recently,Peng and coworkers extensively investigated alternativeand greener cadmium precursors for the colloidal synthesisof QDs [81, 82]. They prepared high quality and mono-dispersed CdSe, CdS, and CdTe QDs from cadmium oxide(CdO), cadmium acetate [Cd(AcO)2], and cadmium car-bonate (CdCO3) [81–83]. A typical example is thesynthesis of CdSe QDs from Cd(AcO)2 and TOPSe [82].In this reaction, a suspension of Cd(AcO)2 in TOPO or aTOPO–phosphonic acid mixture was heated to ca. 250–360 °C under an Ar atmosphere followed by injection ofTOPSe. After the nucleation of QDs, the reaction temper-ature was decreased to ca. 200–320 °C. CdSe nanocrystalswith strong quantum confinement (size <4 nm) wereobtained in the presence of phosphonic acids and relatively

large (ca. 4–25 nm) nanocrystals were obtained in thepresence of fatty acids. Synthesis of QDs with high PLquantum efficiencies (>50%) became straightforward afterextensive investigations of various parameters such asprecursor reagents, temperature, chelating ligands, andprecursor ratios involved in the synthesis of QDs [80–85].Based on the report by Peng and coworkers, we synthesizedcore CdSe QDs with green emission by reacting Cd(AcO)2with TOPSe in the presence of a mixture of TOP and TOPO[86, 87]. Here, we employed a low temperature (75 °C)reaction and prepared size-selected QDs (Fig. 3b, inset).The PL quantum efficiency of the resulting CdSe QDs wasrelatively low (<25%) and the size-distribution was wide(PL spectral half-width>35 nm). The low PL quantumefficiency value was due to a slow growth of QDs at thelow temperature that probably induced a large number ofshallow band gap defects. These QDs were overlaid withZnS shells by following a literature method [7]. Thesyntheses of CdSe QD from various precursor reagentsare now well established [2, 30, 79, 81, 82, 88–92] and afew examples are summarized in Fig. 4.

Surface-modified QDs and water-soluble QDs

The concept of preparing protecting shells on core CdSeQDs has been widely accepted to remove surface defects,increase the PL quantum efficiencies, provide physical andphotostability to the core, and reduce toxicity by suppress-ing the dissolution of cadmium ions. ZnS shell is an idealcandidate to fulfill these requirements. Three key publi-cations appeared in 1996 and 1997 by Bawendi and co-workers [7, 93] and Hines and Guyot-Sionnest [8] on thesynthesis of core-shell QDs, with which the applications ofcolloidal QDs infiltrated into technology and biology. ZnSshells on CdSe cores were prepared from diethyl zinc andhexamethyldisilathiane at ca. 140–230 °C in a TOP–TOPOmixture. One of the major differences between the methodsreported in refs. [7] and [8] is the preparation of ZnS shellswithout isolation and purification of the core QDs in ref.[8], which thereby saves time if combined with thesynthesis of narrow size-selected QDs. Although severalmethods are reported for the synthesis of core QDs, themethods reported in refs. [7] and [8] are widely acceptedfor the synthesis of ZnS shells.

Although direct syntheses of cadmium chalcogenide NPsin aqueous phases were achieved nearly 30 years ago, thesynthesis of size-controlled and highly luminescent CdSeQDs in aqueous phases became possible only in recentyears [88–92]. A few methods for the aqueous phasesyntheses of CdSe QDs are given in Fig. 4. For example,Rogach and coworkers successfully synthesized citrate- andthiol-stabilized CdSe QDs in an aqueous phase fromsodium hydroselenide and N,N-dimethylselenourea [88,

Fig. 3 (A) Absorption and PL spectra of CdSe QDs prepared fromCdO, CdCO3, and Cd(AcO)2 in the presence of different ligands. (B)Increase in the optical density of CdSe QDs at 400 nm duringsynthesis at 75 °C; inset CdSe (a, b) and CdSe–ZnS (c) QD solutionswith (b, c) and without (a) UV illumination. Reprinted withpermission from Refs. [82] (A) and [86] (B). Copyright (2001,2005) American Chemical Society


89]. The limited availability of water-soluble QDs was oneof the limitations to utilizing their superior PL properties inbiology. In general, chemical modifications of the surfaceand ligand exchange processes were used to prepare water-soluble QDs from QDs synthesized in organic phases.Various methods for the conversion of organic QDs intoaqueous phases are systematically classified by Mattoussiand coworkers [20]. Also, the preparation of water-solubleQDs by ligand exchange followed by encapsulation insilica shells and micelles are briefly summarized by Colvinand coworkers [94]. Recently, different types of bioconju-gated and water-soluble CdSe–ZnS QDs have becomecommercially available for various in vitro and in vivoapplications. The large hydrodynamic size of QDs coatedwith polymer shells and biomolecules are not attractive forconstructing Förster resonance energy transfer (FRET) pairsand in vivo applications in human beings.

Band gap structure and relaxation processes in CdSeQDs

The strong three-dimensional confinements of excitonssurmounting coulomb interaction, and large surface-to-volume ratios are the origins of the size-tunable propertiesof QDs. Once photoactivated, excitons in a QD cool andrelax (intra-band relaxation) over 1011 s−1 before inter-bandexciton recombination [95–98]. The inter-band excitonrecombination processes include radiative relaxation at theband edge, phonon-assisted non-radiative relaxation, non-radiative Auger relaxation, and radiative and non-radiativerelaxations at the surface defects [96, 98–103]. Amongthese relaxation processes, the radiative exciton recombi-nation at the band edge is the origin of the size-tunable PLcolor of QDs; unlike in the case of bulk CdSe, the band gapin CdSe QDs is size-dependent. The origins of the PL of

CdSe QDs are (i) exciton recombination at the band edge,i.e., transitions from the lowest unoccupied state, which is acombination of the 5s orbitals of cadmium atoms, to thehighest occupied state, which is a combination of the 4porbitals of selenium atoms, and (ii) deactivation of excitedelectrons (holes) at the surface states [4, 53, 99, 100, 102,104–106]. The inter-band radiative relaxations are relative-ly slow (<109 s−1) and non-exponential [95–98, 107]. Thenon-exponentiality originates from carrier-trapping in adistribution of states including shallow surface defects.The band edge states in CdSe QDs (1Se1S3/2) aredegenerate due to asymmetric and crystal-field splittingfollowed by mixing of carrier exchange perturbations withangular momentum of the charge carriers [53, 99, 100, 102,104, 105, 108]. The splitting and mixing provide eightfolddegeneracy to the band edge and the degenerate states arecharacterized by the total angular momentum (J) values −2,−1, 0, +1, +2 for 1S3/2 and −1, 0, +1 for 1Se states. Thedegenerate band edge states and inter-band transitions inCdSe QDs are shown in Fig. 5. Among the degeneratestates, J=±2 and J=0 are spin-forbidden states. Therefore,photoactivation of QDs close to the band edge populates ±1states, and the deactivation of this population takes placevia non-radiative relaxation to the forbidden dark excitonstates (J=±2) followed by radiative or non-radiative excitonrecombination. With the characterization of the band edgestructure of CdSe QDs, detailed investigations of carrier–relaxation dynamics and PL properties became possible. Inaddition to the above degenerate states, it is necessary toconsider surface states in the relaxation of photoactivatedQDs. The surface states are considered on the basis of thelarge surface-to-volume ratios of QDs. In the case of CdSeQDs, dangling bonds of selenium atoms contribute to thesurface states; ca. 30% of the surface atoms in average-sized CdSe QDs are selenium atoms. In general, the surfacedefects can be considered as a combination of shallow and

Fig. 4 A few methods for thesynthesis of CdSe QDs. TOPOtrioctylphosphine oxide, TOPtrioctylphosphine, TOPSetrioctylphosphine selenide,TBPSe tributylphosphineselenide, HDA hexadecyl amine,HPA hexylphosphonic acid,TDPA tetradecylphosphonicacid, SA stearic acid, R–SH 2-mercaptoethanol/1-thioglycerol,DMSeU N,N-dimethylsele-nourea, NTA nitrilotriacetic acid


deep traps. Carrier relaxations through the deep trapscontribute redshifted deep-trap emission, non-radiative relax-ation and low PL quantum efficiency, and long PL lifetimes.

Activated PL of CdSe QDs

Highly luminescent CdSe QDs are promising materials foroptoelectronic devices, and in vitro and in vivo imagingand analyses. However, the PL quantum efficiencies ofCdSe QDs prepared in the classical colloidal syntheseswere low (ca. 10%). Improvement of the PL quantumefficiency of CdSe NPs has been the subject of greatresearch even before the synthesis of colloidal CdSe QDs.Recently, it has become possible to prepare highlyluminescent CdSe QDs by modified syntheses and post-synthesis surface modifications. The low PL quantumefficiency values of CdSe QDs in previous reports areattributed to the non-radiative exciton recombination at thesurface defects [2, 7, 79]. Since Weller and coworkersobtained enhanced emission from surface-passivated CdSnanocrystals [109], several strategies including surfacecapping with different band gap materials [7, 8, 110],surface treatments [101, 111–113], tuning of environmentaland dielectric factors [87, 114–118], surface passivation byoxygen and other molecules [116, 119–124], and photo-activation [103, 116, 123–130] were investigated to not

only improve the stability and PL properties of QDs butalso understand the nature and the origin of the surfacedefects in QDs. Among these investigations, surfacepassivation by oxygen molecules, ZnS shells, and photo-activation considerably improved the PL quantum efficien-cies of CdSe QDs. Photoactivation of monolayers and filmsof core CdSe QDs with low PL quantum efficiency wasindependently introduced by Buratto and coworkers [127]and Yamaguchi and coworkers [131] in 2000 and investi-gated further by Yamaguchi and coworkers [116, 128, 130],Javier and Strouse [132], and Peng and coworkers [133].According to Buratto and coworkers, the PL quantumefficiency of CdSe NPs is improved by the interactions ofwater molecules adsorbed on the surface. This proposal wasrevisited by Bard and coworkers [119] and Tani andcoworkers [134]. Although the enhancement of PL wasnot reproduced in the presence of water, Bard and cow-orkers found considerable enhancement (ca. 2–6 times,Fig. 6a) of the PL when a solution of CdSe QDs wassaturated with oxygen. Here, the PL enhancement isattributed to the formation of trace amounts of CdO andSeO2 on the surface of the QDs and the passivation of thesurface defects by the oxide layer. Similarly, Yamaguchiand coworkers found an increase in the photoactivated PLquantum efficiency of CdSe–ZnS QDs in silicon oxidematrixes with increase of oxygen composition (Fig. 6b)[116]. This effect is attributed to an increase in the numberof non-bonding oxygen hole centers. Kotov and coworkersachieved ca. 50-fold enhancement in the PL quantumefficiency of CdSe QDs by photo-assisted etching of thesurface defects [124]. All these investigations correlate theorigin of the low PL quantum efficiencies of QDs with thenon-radiative carrier recombination in the surface defects.

A detailed mechanism of the photoactivated enhance-ment of PL in QDs was proposed by Rumbles andcoworkers [123]. They found an increase in the PL lifetimeand a considerable enhancement in the PL quantum effi-ciency of a CdSe–ZnS QD sample under photoactivation.Based on steady-state and time-resolved PL measurements,they proposed a model involving ligand rearrangement toaccount for the photoactivated enhancement of PL. Theproposed model states that the PL enhancement is due to(1) the photoactivation of dark QDs into emissive ones and(2) chemical rearrangements of ligand molecules andsurface states. In other words, photoactivated rearrange-ments of ligand molecules stabilize the surface states andincrease the probability of trapped charge carriers thermal-izing into emissive states (Fig. 7). However, the rearrange-ments of ligand molecules on the ZnS shells are notexpected to affect the PL properties of the core QDs con-siderably and this aspect remains mostly unaccounted.More recently, we found that the PL quantum efficiencyof CdSe QDs are enhanced (8% to >30%) and considerably

Fig. 5 Energy level diagram (A) and splitting of 1Se1S3/2 band edgestates (B) in CdSe QDs. Reprinted with permission from Refs. [105](A) and [102] (B). Copyright (1999, 2001) American ChemicalSociety


stabilized under photoactivation in polar solvents and in thepresence of dissolved polymer molecules (Fig. 8a) [126].The enhancement and the stabilization of the PL quantumefficiency were poor in the presence or absence of polymermolecules in less-polar solvents such as toluene andchloroform. Also, we found a decrease in the PL quantumefficiency of CdSe QDs under prolonged photoactivationeven in the presence of polymers. The decrease of PLquantum efficiency indicated photobleaching of QDs or agradual formation of surface defects, i.e., defects arecontinuously formed on and removed from the surface ofQDs under photoactivation. Therefore, the stability of thephotoactivated PL depends on the stability of equilibriumbetween the photoactivated formation and removal ofdefects states (Fig. 8b) [126]. We attributed the enhanced

PL quantum efficiency to the static passivation of surfacedefects by polymers and polar molecules. This concept isbased on our previous work on the dynamic and staticinteractions of ligands on the surface of CdSe QDs [87].

In general, there are multiple ways to increase the PLquantum efficiency of CdSe QDs: (1) preparation shellsfrom ZnS and other higher band gap materials, (2) surfacepassivation by oxygen, ligands, and other molecules in theenvironment, and (3) photoactivation. It has becomepossible in recent years to synthesize highly luminescentCdSe QDs in a one-pot approach and, therefore, theinterests in the surface modifications of QDs are focusedon providing physical stability to QDs, making chemicaland bioconjugate reactions efficient, and reducing thecytotoxicity. One of the most promising aspects about thephotoactivated increase and decrease of PL is the clarifica-tion of the roles of molecules in the environment andsurface states on the carrier relaxation processes in QDs.Despite the above reports on the relations of PL enhance-ment with the surface defects, the stabilization of surfacestates, the thermalization of charge carriers, and theequilibrium between the formation and removal of surfacedefects, a definitive mechanism of the photoactivation ofPL in CdSe QDs is yet not established. After a particulartime under photoactivation the conversions of emissiveQDs into dark ones, photoactivated formation of defects,and the conversion of dark QDs into emissive ones,

Fig. 7 Decay routes of an exciton generated in CdSe–ZnS QDs.Reprinted with permission from Ref. [123]. Copyright (2003)American Chemical Society

Fig. 6 (a) Temporal behavior of PL and UV spectra (inset) of CdSenanocrystals dispersed in CHCl3. (b) The evolution curves of thefluorescence peak intensity of the QD films on four kinds of SiOx

substrates. Reprinted with permission from Refs. [119] (a) and [116](b). Copyright (2003) American Chemical Society and copyright(2006), American Institute of Physics


photoactivated removal of defects, reaches an equilibriumstate [126]. The photoactivated PL enhancement involvesthe processes before reaching the equilibrium. The conver-sion of emissive QDs into dark ones is substantiated basedon single-step photobleaching of single QDs and thegradual increase and subsequent decrease in the PLquantum efficiencies at ensemble levels. Correlations ofthe photoactivated PL with blinking and bleaching of singleQDs and the nature of the surface states are necessary tofurther understand the mechanism of the photoactivated PL.

On and off PL blinking in single QDs

Intermittent ‘on’ and ‘off’ emission of light (blinking) fromsingle CdSe and CdSe–ZnS QDs was first observed byBrus and coworkers in 1996 [135]. To date, the origin ofthe blinking is closely correlated with carrier traps

distributed on the surface and in the environment of QDs.Advancement of applications of QDs, in both technologyand fundamental science, is limited to a certain level due tothis blinking. For example, blinking of QDs is notpromising for single-photon logic devices, on-demand lightemitters, and single-QD switches. Also, the blinking ofQDs limits continuous monitoring of single biomolecules inbiochemical and biophysical experiments [136, 137].Therefore, the synthesis of non-blinking QDs or removalof blinking by post-synthesis modifications would bringinnovative changes in the above applications.

Auger ionization and transient trapping of charge carriersin band gap defects or surface defects are the origins ofblinking. An early modeling of the exciton–excitonannihilation and Auger ionization was brought by Efrosand Rosen in 1997 to account for the blinking of QDs[138]. As discussed in subsequent reports by Banin andcoworkers [139], Bawendi and coworkers [140], and Kunoand coworkers [140–143], the use of a single deep trapmodel is inadequate to account for the distributed kineticsof ‘on’ and ‘off’ times. On the other hand, a distribution ofoff times over ca. 3–5 decades, as shown in Fig. 9, isnecessary to account for the blinking of a single CdSeQD [141]. Here, the transfer of an electron between aphotoexcited state of a QD and a distribution of trap stateswas considered. The origin of the blinking is discussed indetail by Kuno and coworkers with reference to differentmodels [141–143]. More recently, Frantsuzov and Marcusrevisited the concepts proposed by Efros and coworkers andKuno and coworkers, and attributed the blinking to holetrapping associated with Auger ionization [144]. Also,based on the existence of electron traps on the surface ofQDs, Tang and Marcus proposed diffusion-controlledreaction models to account for the blinking [145–147].

Hohng and Ha observed considerable suppressionin the blinking of CdSe–ZnS QDs in the presence of β-mercaptoethanol (BME) (Fig. 10A) [137]. This observationis substantiated within the framework of the electrontransfer and Auger ionization models. More recently,Barnes and coworkers [148] made a similar observationthat blinking is suppressed when oligo(phenylene vinylene)molecules are conjugated to the surface of CdSe QDs. Also,Mulvaney and coworkers observed suppression and mod-ified blinking of QDs in the presence of electron-donoraliphatic amines [149]. Similarly, Kanemitsu and coworkerssuccessfully suppressed the blinking of single QDs placed onAu surfaces [150]. More recently, Ren and coworkersobserved non-blinking PL of single CdTe QDs synthesizedin the presence of mercaptopropionic acid [151]. Theseobservations of the reduced/suppressed blinking of singleQDs show that the rate of electron transfer (if reversible)from a surface capping agent to a surface defect state ismuch faster than the rate of resonant tunneling. If this is

Fig. 8 (A) PL spectra of CdSe QDs in chloroform recorded in thepresence of polybutadiene at different times under photoactivation at400 nm. The blue shift of the PL spectra is due to decrease in the sizeof QDs. (A) Schematic presentation of the surface passivation and theformation of surface defects in CdSe QDs under photoactivation.Reprinted with permission from Ref. [126]. Copyright (2007)American Chemical Society


valid, the non-blinking nature can be extended to sub-millisecond or even shorter timescales.

Recently, we demonstrated that the long-lived on timesin the intensity trajectories of single CdSe QDs areconsiderably decreased for a sample synthesized at a lowtemperature (75 °C) [86]. In this case, the on and off timesare rather uniformly distributed. The CdSe QDs synthe-sized at the low temperature showed relatively low PL

quantum efficiency (<25%) compared with ca. 50–80%quantum efficiencies of QDs synthesized via high temper-ature reactions. However, deep trap emission was absent inthe case of CdSe QDs synthesized at the low temperature.The low value of the quantum efficiency, the absence ofdeep trap emission, and the short-lived on states indicate ahigh density of closely distributed defects in CdSe QDs.Also, we found (i) an increase in the on times of singleCdTe QDs by changing their local environment frompolyvinyl alcohol to trehalose [153] and (ii) an increase inthe number of on events in the intensity trajectories of

Fig. 10 (A) Upper panel shows a QD intensity trace as tris-HCl (TN)buffer with 140 mM BME was injected at ca. 40 s (red dotted lines)into the sample, displacing TN buffer from the sample. Lower panelshows a time trace of a reverse case where BME buffer was washedaway using TN buffer at ca. 40 s. (B) PL intensity trace of a singleCdSe–ZnS QD present on a glass surface (a) and on a Ag NP surface(b). (C) Various relaxation paths of photoexcited QDs in the presenceof Ag NPs. Reprinted with permission from Refs. [137] (A) and [152](B and C). Copyright (2004, 2008) American Chemical Society

Fig. 9 Three successive enlargements of the off time probabilitydensity for a single CdSe QD. Reprinted with permission from Ref.[141]. Copyright (2000), American Institute of Physics


single CdSe–ZnS QDs placed on a Ag NP surface [152].The increase in the on events is similar to the PL intensityfluctuations of single CdSe–ZnS QDs placed on ITO glass,as observed by Orrit and coworkers [154], and suppressedblinking of QDs placed on a Au surface, as observed byKanemitsu and coworkers [150]. Interestingly, we observedlow PL intensities, stochastic fluctuations of PL intensi-ties, and ‘pseudo off’ states for single QDs placed on aAg NP surface. These observations are attributed to thetransient energy transfer from QDs to Ag NPs and theability of the energy transfer process to compete withelectron transfer and Auger ionization (Fig. 10B and C).To date, a common mechanism is not available to accountfor the different on and off times and the suppression ofblinking due to the difference in the distribution of defectson the surfaces, core-shell interfaces, and environment ofQDs. Possible reasons for the different blinking behaviorof single QDs are the different methods and precursorreagents involved in the syntheses, variations of cappingligands, and dielectric environments of QDs. This situa-tion demands a common source of QDs, especially, forsingle-QD-based device applications and single-moleculebioimaging.

A decade of biological applications of semiconductorQDs

Exceptional photostability and bright emission alone makeQDs powerful substitutes of organic fluorophores for avariety of biological applications. Additionally, QDs havequite a few qualities such as size-tunable color, broadabsorption and narrow emission bands, and large two-photon absorption cross section that biologists demand [1,2, 7, 17, 155]. These properties attracted not only scientistsfrom different areas of biology to QDs but also physicistsand chemists to biology. Among various QDs, CdSe andCdSe–ZnS are widely applied in biology because of theirsize-tunable PL distributed throughout the visible region ofthe electromagnetic spectrum that makes them idealsubstitutes for organic dye molecules. With the introductionof water-soluble and bioconjugated CdSe–ZnS QDs to celllabeling and imaging by Chan and Nie [6] and Alivisatosand coworkers [5], the applications of QDs and approach tobiological problems received new momentum. In thecurrent review we attempt to summarize the biologicalapplications of CdSe and CdSe–ZnS QDs with specialreference to intracellular delivery and in vivo imaging.

Biocompatible QDs and cell targeting

Despite a few recent reports on the direct synthesis of QDsin the aqueous phase, the conversion of QDs from organic

to aqueous phases continues to be the main source ofbiocompatible QDs. Chan and Nie demonstrated theconversion of CdSe–ZnS QDs from organic phase intowater by conjugating mercaptoacetic acid to the ZnS shells[6]. However, Mattoussi and coworkers found that theconjugation of bidentate ligands provides better stability towater-soluble QDs [156]. They prepared high quality, water-soluble CdSe–ZnS QDs by tethering a series of dihydroli-poic acids (DHLA) to ZnS shells. Avidin-functionalizedCdSe–ZnS QD is another attractive candidate for bioconju-gation because of its ability to bind any biotinylatedmolecule/structure. Anderson and coworkers demonstratedthe bioconjugation of QDs through an avidin–biotin bridgeby attaching avidin molecules to DHLA-capped QDsfollowed by conjugation with a recombinant fusion proteinconsisting of a maltose binding domain and a positiveleucine zipper interaction domain [157]. Different methodsto prepare biocompatible QDs are elegantly summarized ina recent review by Mattoussi and coworkers [20]. Here, wespecifically focus on cell targeting with bioconjugatedCdSe and CdSe–ZnS QDs. In general, antibodies andpeptides are promising carriers of QDs to cells. A fewexamples of bioconjugated QDs and their targets in cellsare summarized in Fig. 11 [13, 15, 25, 27, 136, 158–168].

The targeting of QDs at the cell membrane is efficientwhen they are conjugated with specific proteins, antibodies,and ligands. One classical example is the localization of aQD–secondary antibody conjugate to Her2, an overex-pressed growth factor receptor in human breast cancer cells,by Bruchez and coworkers [166]. They used streptavidin-and anti-mouse antibody-conjugated CdSe–ZnS QDs. Inanother report, Rosenthal and coworkers targeted serotonin-conjugated QDs at neurotransmitter receptors on a cellsurface [25]. Other examples are microdosimetry of heatshock proteins in mammary carcinoma cells [162], andlabeling of cell surface acceptor proteins using streptavidin-conjugated QDs [159]. These reports show that QDs areexcellent probes for multiplexed detection and extendedimaging of cells by using various combinations of antibodyand cell surface proteins. Also, there are several other QDconjugates for extracellular labeling of cells, which arerecently reviewed [20, 21, 94, 169–172].

Compared with extracellular targeting, one of the majordifficulties involved in the intracellular targeting of QDconjugates is their large size that affects membrane proteintrafficking and reduces accessibility to crowded locationsinside cells [173]. Other factors to be considered arecellular diversity, cell size, and cell viability. Differentmethods employed in the internalization of QDs in livingcells are summarized in Fig. 12 [174–176].

Electroporation and microinjection are widely employedin the transformation and transfection of recombinantgenetic materials to prokaryotic and eukaryotic cells. In


the electroporation technique, hydrophilic pores are gener-ated on cell membranes by applying high-voltage electricpulses for a short time. The generated pores allow passivetransport of genetic materials into the cells [177]. Bhatiaand coworkers [175] applied this technique and internalizedQDs in living cells (Fig. 12). However, the QDs wereaggregated inside the cells and therefore this technique maybe better applied to cell tracking and cytometry. In general,electroporation serves only as a vector for the entry of QDsinto the cytosol, and specific intracellular targeting needsbiomarkers on the surface of QDs. Microinjection is asimple mechanical process in which probe molecules arenon-specifically introduced inside the cytoplasm or nucleususing a microneedle by applying a pneumatic pressure or anelectric impulse [13, 175]. A typical example is theinternalization of QD–DNA conjugates in a Xenopusembryo by Dubertret and coworkers [13]. In this case, themicroinjected QDs were homogenously distributed insidethe embryo. However, as pointed out above, QDs labeledwith biomarkers are necessary for specific targeting. Bhatiaand coworkers [175] targeted the nucleus and mitochondriaof living cells by microinjecting conjugates of QDs with a23mer nuclear localization peptide and a 28mer mitochon-drial localization peptide (Fig. 12).

Receptor-mediated endocytosis, non-specific endocyto-sis, cell penetration, etc. are widely employed in theintracellular delivery of QDs. Natural uptake of foreignmaterials by cells is called endocytosis. In non-specificendocytosis, cells engulf foreign materials along with some

surrounding fluid in the form of a vesicle called anendosome. Receptor-mediated endocytosis takes place byspecific interactions between cell surface receptors andligands. The endocytosis of QDs was recently reviewed byParak and coworkers [22]. Hydrophilic QDs enter cells vianon-specific endocytosis [6, 160], the efficiency of which ishighly dependent on the size of QDs. Once internalized,aggregation of QDs occurs inside the cytoplasm due to theendosomal arrest [178]. Duan and Nie [176] surmountedthe endosomal arrest by a ‘proton sponge effect’ by conju-gating a polyamine grafted polymer to QDs (Fig. 12).Liposome-mediated intracellular delivery of QDs (Fig. 12)is another promising method [179, 180].

Proteins and peptides efficiently carry QDs inside livingcells. Typical examples are Tat peptides [181–186], poly-arginine [187], cholera toxin B [188], and allatostatin [174].Curtis and coworkers [181] and Mattoussi and coworkers[182] conjugated Tat peptides with QDs and successfullydelivered the conjugates inside human fibroblast cells andthe nucleus of COS 1 cells. Waggoner and coworkersdemonstrated simple and efficient intracellular delivery ofQDs by conjugating with poly (L-arginine) (9mer) [187]. Inthis case, QDs were efficiently internalized in HeLa,MG63, and 3T3 cells. Recently, Ballou and coworkersidentified that QDs coated with cholera toxin B efficientlyinternalize in different cell lines and distribute in thecytoplasm [188]. Also, we identified allatostatin, a neuro-peptide from Drosophila melanogaster, as a potentialcarrier of QDs to living cells [174]. Allatostatin was

Fig. 11 Biolabels of CdSe andCdSe–ZnS QDs and their targetsin cells


selected considering sequence similarity between its recep-tor in insects and galanin receptor in mammalian cells. Forthe preparation of the QD–allatostatin conjugate, allatosta-tin was biotinylated using biotin–NHS ester and thenconjugated to commercial streptavidin–QDs. We identifiedthat the QD–peptide conjugates are delivered inside 3T3L1and A431 cells without considerable aggregation. Interest-ingly, the QD–peptide conjugates labeled the microtubulesand delivered inside the cell nucleus (Fig. 12). Although themechanism of this intracellular and nuclear delivery isunder investigation, our work shows prospects of inverte-brate bioresources in QD/drug/gene delivery.

In vivo targeting of QDs

Despite the large size of QDs compared with organic dyemolecules, their bright PL and high photochemical endur-ance are promising for in vivo fluorescence imaging. In2002, Akerman and coworkers introduced the in vivoapplications of QDs [9]. They injected a peptide coatedCdSe–ZnS QD sample through the tail vein of a mouse anddemonstrated the specificity of the conjugate to endothelialcells in the lung blood vessels. Also, they investigated thespecificity of other peptide-coated QDs to blood vesselsand lymphatic vessels in breast cancers, both in vitro and invivo. Around the same period, Dubertret and coworkersindependently introduced the in vivo applications of CdSe–ZnS QDs coated with a micelle-forming block copolymer

composed of poly(ethylene glycol) (PEG)–phosphatidyl-ethanolamine and PEG–phosphtidylcholine [13]. Theydemonstrated the in vivo application of the micelle-coatedQDs by microinjection into a Xenopus embryo followed byfluorescence imaging. These two in vivo experimentsbrought radical changes in the biological applications ofQDs and there are many reports on the in vivo applicationssuch as locating draining lymph nodes, targeting vascula-tures, and imaging tumors [9, 15, 17, 26, 180, 189–204].The prospects and challenges in the in vivo applications ofQDs are discussed in recent articles, insightful reviews, andexpert opinions [16, 24, 111, 172, 194, 205–211]. Here, wesummarize the applications of bioconjugated CdSe–ZnSQDs and a few other NIR QDs in the in vivo visualizationof lymph nodes, cancers, and vasculature.

The fluorescence visualization of sentinel lymph node isa promising in vivo application of QDs, which helps phy-sicians to locate and dissect samples for biopsy. Since thelymph node metastasis is one of the most importantprognostic signs of cancers, QD-assisted mapping of lymphnodes is a promising technique for staging certain types ofcancers. Fluorescence imaging of lymph nodes in animalmodels using QDs was first demonstrated by Frangioni andcoworkers [196]. They intradermally injected sub-nanomolarsolutions of oligomeric phosphine-coated CdTe–CdSe QDsin mice and pig and demonstrated near-infrared (NIR)imaging of sentinel lymph nodes ca. 1 cm below the skinpre-surgically and even under bloody surgical conditions.

Fig. 12 Different methods forintracellular delivery of QDs.Reprinted with permission fromRefs [174–176]. Copyrights(2007) American ChemicalSociety and copyright (2004)Wiley-VCH Verlag GmbH &Co.


Recently, NIR QDs and their conjugates with human serumalbumin, oligomeric phosphines, and PEG derivatives wereemployed in the mapping of lymph nodes and lymph nodedrainage. The NIR imaging employing QDs is promisingfor image-guided pre-surgical and surgical oncology ofgastrointestinal tumors, metastasis of spontaneous melano-ma, breast cancer, non-small cell lung cancer, etc. [189,196, 199–202, 204]. Ballaou and coworkers injected ZnS–CdSe and ZnS–CdSe–CdTe QDs coated with negative,positive, and neutral terminated PEG in mice and demon-strated the application of the conjugates in locating lymphnodes in mice [189]. Here, the surface-coated QDs areinjected either intravenously (red QDs) or directly (NIRQDs) into subcutaneously grown tumors. The drainage ofall the three kinds of QDs towards the inguinal node wasvisualized using fluorescence imaging though the skin(Fig. 13a and b). However, the drainage of the chargedQD samples into lymph nodes was non-specific. Kobayashiand coworkers demonstrated five separate lymphatic drain-ages into different lymphatic basins in mice using multi-color CdSe and CdTe QDs [197]. Recently, the efficienciesof QDs and dye molecules to label sentinel lymph nodes

were compared by Frangioni and coworkers who concludedthat the large size of QDs limits their accessibility to thefirst draining lymph nodes, whereas dye molecules contin-ued further to distant lymph nodes [198]. Therefore, NIRQDs with accessibility to distant lymph nodes and specificityto lymph node metastasis would improve their applications inpre-surgical and post-surgical imaging and removal ofcancers.

Since the first demonstration of the in vivo imagingof breast cancer in mice using peptide-coated QDs byÅkerman and coworkers [9], conjugates of antibodies andpeptides with QDs received much attention as potentialmarkers of various cancers. For example, Nie and co-workers successfully imaged prostate cancer in nude miceusing QD conjugated with the antibody of the prostate-specific membrane antigen (PSMA) [15]. They systemati-cally modified the surface of TOPO-capped CdSe–ZnSQDs with a triblock copolymer [poly(butyl acrylate), poly(ethyl acrylate), and poly(methacrylate)], amino-functionalizedPEG, and the PSMA antibody. The triblock copolymerserved as both a protecting shell of QDs and a reactiveplatform for the conjugation of other molecules, PEG-

Fig. 13 (a, b) Mouse bearingM21 melanoma, injectedsequentially in the tail vein firstusing 655-nm QDs, followed20 min later by injection into thetumor using 800-nm QDs: avisible light image and b imagein the 655-nm window. Reprin-ted with permission from Ref.[189]. Copyright (2007) Ameri-can Chemical Society. (c) Invivo flourescence images oftumorbearing mice using QDprobes with PEG-PSMA anti-body conjugates. Reprinted bypermission from Mcmillan Pub-lishers Ltd: [Nature Biotechnol-ogy], Ref. [15], copyright(2004). (d) In vivo image of aprojection of capillary structurethrough 250um of adipose tissuein mice. From Ref. [17].Reprinted with permission fromAAAS


coating improved the blood circulation of the bioconjugatedQDs, and the antibody localized the QDs at the extracel-lular domains of PSMA on prostate cancer cells grown inmice (Fig. 13c). Both active (0.4 nmol QD–antibodyconjugate) and passive (6 nmol of control QD sample)targeting of QDs were observed in this investigation. Withthis report, the prospects of bioconjugated QDs in the non-invasive imaging of cancers received much attention. Also,in vivo imaging of QD-labeled B16 melanoma cells in micewas demonstrated by Simon and coworkers [180]. In thiswork, DHLA-labeled CdSe–ZnS QDs were incorporated inmelanoma cells using lipofectamine 2000. The QD-labeledmelanoma cells were intravenously injected in micefollowed by extraction of their lungs. Fluorescence imagesof the lungs indicated the presence of small amounts ofQDs that was released from dead melanoma cells followedby either non-specific trapping in lung tissues or internal-ization in phagocytic cells in the lungs. However, non-specific accumulation of QDs is more likely to take place inthe liver, spleen, bone marrows, and lymph nodes. Thisaspect was not investigated with reference to the release ofQDs from dead melanoma cells. This report brought to lightenormous possibilities of QDs in cancer research usingmultiphoton imaging. They detected up to five populationsof cells, each labeled with a different color QD, i.e.,multiphoton-multicolor-multicell imaging. Other reports onin vivo cancer imaging using QDs include the localization ofconjugates of QDs with epidermal growth factor (EGF)[193] and growth factor receptor antibodies [192, 203] inbreast cancer and cervical cancer.

Despite the in vivo fluorescence tagging of cancer cellsusing specific antibodies and peptides, the imaging oftumor vasculature in live animals is a primary requisite forthe early detection and treatment of cancers. With thisprospect, biocompatible CdSe–ZnS QDs and other NIRQDs are widely applied in the in vivo visualization oftumor vasculature [17, 180, 190, 191, 194, 195]. Multi-photon excitation and NIR imaging are the primaryrequirements for efficient and non-invasive imaging ofdeep tissues avoiding autofluorescence of cells and tissues.In an initial attempt along this line, Webb and coworkerscompared the two-photon fluorescence efficiency of intra-venously injected water-soluble and non-specific CdSe–ZnS QDs with that of a fluorescein isothiocyanate–dextranconjugate [17]. The large two-photon cross section of QDsallowed an efficient fluorescence visualization of deepcapillaries through the skin in living mice (Fig. 13d). Later,Bawendi and coworkers applied multiphoton excitedfluorescence imaging of QDs in the visualization of tumorvasculature in transgenic mice [26]. They successfullyimaged tumor vasculature by employing PEG–phosphati-dylethanolamine-labeled CdS–ZnS and CdSe–ZnCdS QDs.More recently, Chen and coworkers investigated the

localization of arginine–glycine–aspartic acid (RGD, anintegrin αvβ3 antagonist peptide)-conjugated NIR QDs intumor vasculature [190, 191]. The high levels of integrinαvβ3 in human glioblastoma and the specificity of thepeptide to integrin αvβ3 are the keys in this investigation.Although the glyoblastoma tumor was visualized using theQD–peptide conjugates, prominent non-specific localiza-tion of QDs in the liver, bone marrow, spleen, and lymphnodes was observed. Another emerging in vivo applicationof QDs is the imaging of inflammation associated withvarious disease conditions [212–214]. Inflammation is acomplex process involving numerous cell types and surfaceproteins, characterized by leukocyte rolling and tetheringalong endothelial cells followed by transmigration intotissues, where their immuno-defensive functions such asphagocytosis are lost. Undesirable provocation of theinflammatory response is an unfavorable feature of numer-ous diseases such as diabetes, atherosclerosis, colitis, andasthma. Haselton and coworkers investigated the in vivoimaging of inflammation in retinal endothelium of strepto-zotocin-treated diabetic rats [195]. They conjugated variousQDs with monoclonal antibodies specific to leukocytes andneutrophils and the conjugates were intravenously injectedinto rats. Using this technique, they not only imageddifferent color-coded biomarkers simultaneously but alsoidentified increased retinal vasculature in diabetic animals.In another investigation, Murthy and coworkers usedantibody-conjugated QDs and demonstrated the targetedmeasurement of myeloperoxidase, interleukin 1α (IL-1α),and tumor necrosis factor-α (TNF-α) as inflammatorymarkers in the dextran sodium sulfate (DSS) experimentalmodel of colitis in mice [213].

All the above investigations show the potentials of QDsas in vivo biomarkers of various disease conditionsincluding cancer. Yet, the major concerns in the applica-tions of QDs in human beings are pharmacokinetics andtoxicity of QDs, autofluorescence of tissues under visiblelight excitation, and the penetration depth of excitation lightand emitted fluorescence light. The last two of thesedifficulties were removed by applying multiphoton excita-tion and NIR imaging of various QDs [15, 17, 155, 180,196, 215, 216]. The large two-photon cross section of QDsis advantageous for NIR in vivo imaging. The toxicity ofCdSe QDs from cadmium can be avoided by coatingprotecting layers such as ZnS shells [12, 217–219]. On theother hand, the pharmacokinetics of QDs is largelyunknown. The major reasons for the toxicity of QDs arethe metallic components and their nanodimensions. Cad-mium has a half-life of about 20 years in human beings.Also, cadmium is a potential carcinogen that accumulates inkidney, liver, and many other tissues inducing DNA andprotein damages [220]. Although the leakage of cadmiumcan be avoided for a limited period of time by overlaying


protecting shells such as ZnS and polymers, concerns forgradual deterioration of the shells by reacting withbiomolecules such as enzymes remain. The possibility ofshell deterioration and leakage of metal ions holds outbased on the observations of a decrease of PL intensity anda blueshift of PL spectra of QDs inside living cells [221].The toxicity aspects are of great concern, especially whenQDs stay inside the body for unpredictable periods of time.Therefore, time-correlated investigations of the clearance ofinjected QDs from the body became necessary. In a recentstudy, Chan and coworkers identified the localization ofQDs in sinusoid edges of liver, red pulp in spleen, sub-capsular sinus in the lymph nodes, and in the vascular sinusperiphery in the bone marrow of rats [222]. More recently,Frangioni and coworkers systematically investigated the

size-dependent renal clearance of CdSe–ZnS QDs in mice[223]. They selected zwitterionic cystein-coated QDs withhydrodynamic dimensions in the ca. 4.3- to 8.65-nmrange. Charged QDs are not selected due to the formationof large aggregates with serum proteins, and polymer-coated neutral QDs are not selected due to their largehydrodynamic size. The zwitterionic QDs were taggedwith radioactive 99Tc for radioscintigraphy imaging andadministered in mice via intravenous injection. Interesting-ly, analyses of blood, urine, and carcass samples (Fig. 14aand b) revealed that QDs with ca. 5.5-nm diameter werecleared from the body within 4 h of injection, whereaslarger QDs showed higher uptake in the liver, kidneys,lungs, and spleen. The renal clearance of smaller QDs with4.36-nm diameter into the urinary bladder and the distribu-

Fig. 14 (A) Blood concentra-tion (%ID/g) of 99mTc-labeledQDs after intravenous injectioninto CD-1 mice. (B) Urine ex-cretion and carcass retention of99mTc-QDs of various hydrody-namic diameters 4 h after intra-venous injection into CD-1mice. Radioscintigraphic imagesof whole animals and resectedorgans after injecting (C) 99mTc-QD515 (4.36 nm) and (D)99mTc-QD574 (8.65 nm).Reprinted by permission fromMcmillan Publishers Ltd:[Nature Biotechnology], Ref.[15], copyright (2007)


tion of larger QDs with 8.65-nm diameter in the vitalorgans are characterized by radioimaging of whole animalsand resected organs (Fig. 14C and D). The large hydrody-namic size of QDs, small pore sizes of the mammalianvasculature, and the glomerular barrier are the limitingfactors in the clearance of NPs from the body. Therefore, invivo administration of QDs and other NPs with ahydrodynamic diameter >5 nm is not safe, especially whenthey contain toxic materials such as heavy metals. AlthoughQDs, magnetic NPs, and hybrid NPs of metal and semi-conductors are promising for various combinations ofmultimodal imaging among NIR fluorescence, magneticresonance (MRI), computed tomography (CT) and other X-ray, and positron emission tomography (PET) imaging, thesize effects on the clearance of these materials from thebody remain unresolved. Therefore, NPs are less promisingfor in vivo applications in human beings.

Syntheses of colloidal Au and Ag NPs

Although the brilliant color of colloidal Au NPs has beenknown since the ancient Roman times, a systematicsynthesis of pure and nanosize Au (activated Au) was onlyintroduced by Michael Faraday in 1857. In his classicalwork, inspired by the preparation of potable Au byParacelsus, Faraday reduced AuCl with phosphorous intoAu NPs. Unlike in the case of colloidal Au NPs, thesynthesis of colloidal Ag NPs is a rather new concept,except that the antibacterial activity of Ag has been knownfor hundreds of years. Currently, a large number of methodsare available for the syntheses of colloidal Au and Ag NPs.Here, a few widely used methods for the synthesis of Auand Ag NPs are summarized and a few additional methodsare included in Fig. 15 [63, 224–233].

Syntheses of colloidal Au NPs

A simple, systematic, and bottom-up synthesis of colloidalAu NPs was introduced by Turkevitch in 1951[232]. In thissynthesis, narrow size-dispersed (ca. 20 nm) Au NPs wereobtained by reducing AuCl�4 ions with sodium citrate. Thismethods was subsequently modified in different ways,among which a phase-transfer and reduction methodintroduced by Brust and coworkers [224] is widelyaccepted. In this method, an aqueous solution of HAuCl4(30 mL, 30 mM) was mixed with a solution of tetraocty-lammonium bromide (80 mL, 50 mM) in toluene. Thisorganic-aqueous two-phase mixture was vigorously stirreduntil the yellowish-orange color was transferred from theaqueous to organic phase, which indicates the transfer ofAuCl�4 from the water to toluene layer by tetraoctylammo-nium ions. This was followed by the addition of 170 mg

dodecane thiol and a freshly prepared aqueous solution ofsodium borohydride (NaBH4, 25 mL, 0.4 M) that resultedin the reduction of AuCl�4 into Au NPs and the formation ofdodecane thiol-capped NPs. This method has been modi-fied over the years and different physical and chemicalmethods have been introduced for the size- and shape-controlled synthesis of Au NPs. Various methods forsynthesis of Au NPs are summarized by Daniel and Astruc[33]. A few general methods for Au and Ag NP synthesesare summarized in Fig. 15.

Synthesis of colloidal Ag NPs

Like the syntheses of Au NPs, different chemical andphysical methods are available for the syntheses ofcolloidal Ag NPs. Among these, the Lee–Meisel methods[63], modifications of Au NP synthesis introduced byTurkevitch [232], are widely accepted. Lee and Meiselreported the reduction of AgNO3 by NaBH4 and sodiumcitrate (Fig. 15). In the borohydride reduction reaction, anaqueous solution of AgNO3 (5 mM, 100 mL) was graduallyadded to an ice-cold aqueous solution of NaBH4 (2 mM,300 mL). Also, an aqueous solution of polyvinyl alcohol(1%, 50 mL) was added as a stabilizing agent during thereaction. This was followed by decomposing any unreactedNaBH4 by boiling the solution for 1 h. In a second methodin this report, Ag NPs were synthesized by adding an

Fig. 15 A few methods for colloidal Au and Ag NP syntheses


aqueous solution of sodium citrate (1%, 10 mL) to a boilingaqueous solution of AgNO3 (90 mg in 500 mL water). Afew other widely accepted methods for the synthesis of AgNPs are photochemical reduction of AgClO4 [234],sonoelectrochemical reduction of AgNO3 [233], thermalreduction of AgNO3 by ethylene glycol [230], andreduction in micelles [231] (Fig. 15).

Size and shape of Au and Ag NPs and plasmon-sensitivedetections

The coupling of oscillating electric fields in Ag and Au NPswith light provides huge enhancement of local electromag-netic fields and makes these noble metal NPs powerful foroptical sensing. Therefore, the availability of SPR withdifferent bandwidths and energies has been investigated fora long time. These aspects are combined with sensitivitiesof optical sensing in the vicinity of Ag and Au NPs andwere reviewed recently [235–237]. One of the importantparameters of metal NPs for optical sensing is the lineshapeof their plasmon resonance [31, 238–242]. Link and El-Sayed demonstrated correlations between plasmon reso-nance bandwidth and spectral behavior that are predictedusing a two-level model for Au NPs with intrinsic (<25 nm)and extrinsic sizes (>25 nm) [241]. Based on thiscorrelation, they identified a homogeneous broadening ofthe plasmon band of Au NPs with their size. Also, Whettenand coworkers observed broadening of the surface plasmonband with decrease in the size of highly size-selected AuNPs (0.2 nm apart in the ca. 1.4- to 3.2-nm range) andcorrelated their observations with the surface plasmonbands calculated using Mie theory (Fig. 16a) [31].

Feldman and coworkers examined the lineshape ofplasmon resonance of Au NPs at single-particle level(Fig. 16b) [35]. They observed that the dephasing timesof the plasmon resonance spectra of single NPs vary around8 fs. This observation is in good agreement with thecalculations based on Mie theory. This investigation showslarge local field enhancement factors and relatively highlight-scattering efficiencies, making rod-like NPs promisingfor optical applications [243]. Although the Mie theory oflight scattering was extended for different sizes and shapesof metal NPs, a more recent and simple analytical formuladeveloped by Miyano and coworkers [244, 245] quantita-tively predicted the plasmon resonance from Ag NPs witharbitrary shapes. Recently, Okamoto and coworkers inves-tigated the optical properties of single rod-like Au NPsusing scanning near-field optical microscopy and identifiedseveral longitudinal and oscillating plasmon patterns in thenear-field transmission and PL spectra [246].

Masuhara and coworkers applied plasmon resonance ofsingle Au NPs (ca. 80-nm diameter) embedded in polymer

films with different thicknesses as a tool to sense environ-mental dielectric conditions [247]. Interestingly, they ob-served ca. 30-nm redshift of the plasmon band with increasein the thickness of the polymer films. This effect is attributedto an increase in the effective refractive index of thenanoenvironment of single Au NPs. A similar observationwas made by Barnes and coworkers when the thickness ofLangmuir–Blodgett films was increased on metal NPs [248].These observations indicate that the plasmon resonancebands are sensitive to the local environments even for agiven size/shape of metal NPs.

Fig. 16 (A) Experimental (solid line) and calculated surface plasmonbands of a 1.7, b 1.9, c 2.0, d 2.1, e 2.3, f 2.5 nm Au NPs [31]. (B)Near-field transmission spectra of individual NPs:(a) particle #1(squares); solid line calculated Mie scattering efficiency Qsca; dottedline far-field optical density of the composite film in arbitrary units.(b) Particles #2 (circles) and #3 (diamonds); dashed and solid linesLorentzian fits to the data. (c) Object #4; solid line theory. Reprintedwith permission from Refs. [31] (A) and [35] (B). Copyright (1997)American Chemical Society and copyright (1998) by the AmericanPhysical Society


Dynamic optical properties of Au and Ag NPs

As discussed above, one of the most important parametersof metal NPs for the sensitivity of sensing is the lineshapeof their plasmon resonance. Electron dynamics of metalNPs and the related phenomena including enhancedfluorescence and nonlinear scattering were reviewed re-cently [37, 249, 250]. Therefore, only the dynamic opticalproperties of Au and Ag NPs related to SERS and plasmonresonance are summarized here.

The plasmon resonance due to oscillation modes of theconduction band electrons is the source of EM enhance-ment for sensitive detection using metal NPs. Thus, variousproperties of the conduction band electrons, especially theirultrafast dynamics that defines the quality of the plasmonresonance, are extensively studied to clarify the decayprocesses of the energy of the EM enhancement. Based onultrafast absorption measurements of metal NPs, the decayprocesses are classified into electron–electron (e–e) scatter-ing, electron–phonon (e–ph) scattering, and phonon–pho-non (ph–ph) scattering [37, 249, 250]. Link and El-Sayedstudied in detail the ultrafast electron dynamics in Ag–AuNP alloys and revealed that the relaxation processes due toe–ph scattering in Au NPs are independent of the shape/sizeand surface nature of NPs and the mode of the plasmon[54]. However, some uncertainty related to ensembleaveraging effect involved in conventional ultrafast absorp-tion spectroscopic investigation remained in their work.Therefore, it became important to define the electrondynamics at single-particle levels [251, 252]. For this,Masuhara and coworkers developed an ultrafast light-scattering spectroscopy system by combining a conven-tional microscope with a pump–probe setup and measuredthe plasmon resonance bands of individual Au NPs withmean radius of 40 nm [253]. They demonstrated thatphotoexcited electrons relax through an e–ph scattering inca. 4 ps and a ph–ph scattering with solvent matrix in>25 ps, excluding inhomogeneous broadening of theplasmon resonance bands. Vallee and coworkers investigat-ed the ultrafast optical responses of single metal NPs bycombining a highly sensitive femtosecond pump–probesetup with a spatial modulation microscope and demon-strated that the e–ph energy transfer time in a single NPdepends on its electronic temperature [254]. They quanti-tatively interpreted the relation between energy transfer andtemperature using a two-temperature (lattice temperatureand electron temperature) model. Guyot-Sionnest and cow-orkers measured the ultrafast optical responses fromindividual rod-like Au NPs and avoided inhomogeneousbroadening by exciting with ultrafast laser pulses inresonance with longitudinal plasmon modes of the NPs[252]. They investigated correlations among the ultrafastoptical responses, heating of the conduction band electrons,

and coherent plasmon oscillations. From this correlatedinvestigation, an unanticipated damping of strongly drivenplasmon was identified. Recently, the ultrafast opticalresponses of individual Au and Ag NPs were studied usingnear-field spectroscopy. One typical example is the obser-vation of differences in the collective motion of electrons atthe center and edges of rod-like NPs [246].

Surface-enhanced Raman scattering (SERS) of metalNPs

SERS became an interesting and a promising spectroscopytechnique in ultrasensitive, non-isotopic, non-fluorescentdetection of molecules since the breakthrough discovery ofstrong Raman signals by McQuillan and coworkers frompyridine molecules adsorbed on rough Ag electrodes [59].The great popularity of SERS as an analytical techniquewas the result of the enormous enhancement of SERSsignal by a factor of ca. 1014–1015 [42, 43, 62, 65, 66, 74,255, 256]. This huge enhancement factor improved thedetection limit from ensembles of molecules to single-molecule levels, as observed by Nie and Emory, Kneippand coworkers, and others [42, 43, 58, 62, 65, 66, 68–78,255, 256]. The huge enhancement factor is achieved bypreparing SERS-active aggregates and nanostructured filmsof Au and Ag NPs (ca. 10–150 nm). Theoretical andexperimental investigations of the origin of SERS and itsrelations to light–matter interactions, noble metal nano-structures, and the chemistry of analyte molecules suggestedthe involvement of multiple factors in the SERS enhance-ment. The exact mechanism of the SERS enhancement is stillthe subject of debate in the literature. Broadly speaking, theSERS enhancement is attributed to electromagnetic enhance-ment [42, 56] and chemical enhancement [55, 56]. Accord-ing to the electromagnetic enhancement, the incident andscattered electromagnetic fields around a noble metalnanostructure are considerably enhanced by coupling withplasmon, a collective oscillation of an electron gas, of noblemetal nanostructures. The electromagnetic enhancementfactor in SERS depends on the excitation field and Ramanscattering field. Therefore, the intensity of SERS signal isproportional to the fourth power of the electromagneticenhancement factor. Additionally, nanojuctions and sharpedges of metal NPs considerably contributed to theelectromagnetic enhancement of SERS. The origin of thechemical enhancement of SERS was hypothesized on thebasis of different SERS intensities of N2 and CO moleculesunder identical experimental conditions. The chemicalenhancement originates from electronic coupling betweenadsorbed molecules and a metal surface, i.e., a resonanceRaman effect as a result of charge-transfer interactionsbetween metal and adsorbed molecules. Fundamental


aspects of SERS, SERS active nanostructures, and themechanism of SERS were summarized recently [48, 51].

Borjesson and coworkers identified that SERS activity issignificant at inter-particle junctions or hot spots [257].This aspect is known since extensive investigations ofSERS on the surface of spherical metal NPs [47]. Morerecently, Katajima and coworkers used scanning near-fieldoptical microscopy and identified that the SERS enhance-ment is related to NP junctions in aggregates [258]. Also,calculations of EM field at inter-particle junctions in metalNP aggregates supported the enormous enhancement ofSERS by coupling with plasmon resonance [259]. There-fore, characterization of relationships between SERS andplasmon resonance of metal NP aggregates is valuable toidentify ideal conditions of SERS. Experimental investiga-tions of relationships between SERS and plasmon reso-nance are difficult at ensemble levels due to size- andshape-dependent plasmon resonance energy of metal NPs,i.e., plasmon resonance bands become averaged in collec-tive ensemble measurements and it is difficult to discuss therelationships due to inhomogeneous broadening of plasmonresonance bands. This difficulty was resolved by develop-ing dark-field microspectroscopy systems for correlatedmeasurements of plasmon resonance and SERS.

Relations of excitation laser polarization with plasmonresonance of single Ag NP aggregates and SERS ofrhodamine 6G (R6G) were examined by Ozaki and cow-orkers who identified that selective enhancement of SERS

bands is correlated with polarization-dependent plasmonresonance bands [260]. This result supports the notion thatamplified EM field at NP junctions contributes to SERS[48, 51, 258, 260]. Also, Masuhara and coworkersidentified that SERS of R6G molecules adsorbed on singleAg NP aggregates was quenched when the plasmonresonance was changed [247]. This was followed byobserving variations of excitation profiles of SERS withchanges in the plasmon resonance bands. This observationhelped to identify the contribution of EM enhancement tothe total SERS enhancement. In this case, on the basis ofbroadening of envelopes of SERS excitation profiles andshifts of plasmon resonance bands towards low energy,Ozaki and coworkers identified that molecular electronicresonance and plasmon resonance contribute to SERS[261]. We identified evidence of twofold EM enhancementin SERS by comparing SERS spectra with plasmonresonance spectra [262]; spectral variations are induced byselective enhancement of SERS bands close to the plasmonresonance maxima (Fig. 17).

In another investigation of optical properties of singleAg NP aggregates–R6G system, we identified correlations

Fig. 17 Selective enhancement of SERS bands depending on theplasmon bands of Ag NP aggregates. The blue traces are experimentalspectra and the red traces are calculated spectra. SERS spectra in theleft panel were calculated based on plasmon bands and those in theright panel were calculated based on Lorentzian bands. Reprinted withpermission from Ref. [262]. Copyright (2007) by the AmericanPhysical Society

Fig. 18 (a) Polarization angle dependence of the plasmon resonanceRayleigh scattering spectra; (b) polarization angle dependence ofbackground emission plus SERS spectra; (c) intensities of the lowestenergy and the highest-energy plasmon resonance maxima vs.polarization angles; and (d) the intensities of the lowest-energy andmedium-energy plasmon resonance maxima vs. the backgroundemission, and SERS bands vs. the polarization angle (θ). Reprintedwith permission from Ref. [263]. Copyright (2006), AmericanInstitute of Physics


among plasmon resonance, SERS, and background lightemission; the background light emission is the fluorescenceof R6B, as its maximum was not correlated with theexcitation energy [263]. However, the polarization depen-dence of both SERS and the background light emission wasthe same as that of the lowest-energy plasmon resonancemaximum associated with a longitudinal plasmon (Fig. 18).From the common polarization dependence, we identifiedthat the lowest-energy plasmon is coupled with both SERSand the background light emission. These relations betweenSERS and plasmon resonance show that conditions ofSERS enhancement can be optimized by controlling theplasmon resonance of Ag/Au NP aggregates.

Biological applications of Au and Ag NPs

Recently, the SPR of Au and Ag NPs has attracted muchattention in bioanalyses and bioimaging. Bioconjugated Auand Ag NPs are tested for quantitative detections of avariety of biomolecules and chemical species [33, 43, 264–270]. Among different sizes and shapes of Au and Ag NPs,nanorods with NIR plasmon bands are promising for celland tissue imaging. This is an advantage of the shape-dependent energy of the longitudinal plasmon: as the aspectratio of nanorods increases, the longitudinal plasmon bandshifts towards the NIR region. El-Sayed and coworkersidentified that the scattering coefficients of nanorods are anorder of magnitude larger than that of nanoshells andnanospheres with comparable diameter [271]. Mirkin andcoworkers demonstrated the detection of specific DNAsequences by tethering oligonucleotides on the surface ofAu NPs [272]. A few other bioanalytical applications of AuNPs are cell labeling and immunosensing using antibodyconjugates [273–275].

Although bioconjugation of Ag NPs are not straightfor-ward like Au NPs, they are promising for bioanalyses andSERS imaging with reference to their higher extinctioncoefficients. Recently, Mirkin and coworkers identified astrategy to prepare Ag NP–oligonucleotide conjugates basedon a cyclic disulfide-anchoring approach [276]. Unlike singleNPs, aggregates of bioconjugated Ag NPs are promising forultrasensitive bioanalyses using SERS. For example,Borjesson and coworkers attached Ag NPs to hemoglobinand demonstrated the detection of single hemoglobinmolecules using SERS [257]. Additionally, a large numberof conjugates of Au and Ag NPs are known in bioanalyses,which are summarized elsewhere [33, 43, 264–270].

Application of Au and Ag NPs in cell imaging is an areaof recent research interest. Non-isotopic and non-toxiclabeling of cell surface receptors and cell organellesincluding nucleus with bioconjugated Au and Ag NPs ispromising for imaging of structures and functions of cell

and tissues. Specificity and efficiency of conjugation of Auand Ag NPs with antibodies, peptides, sugars, folic acid,and other ligands to label normal and cancer cells haverecently been identified. Typical examples of cell imagingusing bioconjugated Au and Ag NPs are shown in Fig. 19.Figure 19a and b shows dark-field light-scattering imagesof human karatinocytes labeled with Au nanorods and theconjugates of Au nanorods with a nuclear signaling peptide.Here, El-Sayed and coworkers identified that the internal-ization of Au nanorods in cells was considerably improvedby the peptide conjugation [277]. A typical example of Ag

Fig. 19 Dark-field light-scattering images of HaCat normal cells afterincubation with (a) Au nanorods and (b) peptide-conjugated Aunanorods. (c) Near-field scanning optical microscopy image offibroblast cells labeled with IgG-Ag NP conjugates. The translocationdomains in the cells were labeled with protein A prior to IgG-Aglabeling. Reprinted with permission from Refs. [277] (a and b) and[278] (c). Copyrights (2007) American Chemical Society


NPs in cell labeling was demonstrated by Xu and co-workers [278]. In this report, Ag NPs were conjugated withIgG followed by targeting protein A-labeled translocationdomains in fibroblast cells. They demonstrated the sensi-tivity and the selectivity of the Ag–IgG conjugates in cellimaging (Fig. 19c). Various bioconjugated Au and Ag NPsand their applications in cell imaging are summarized inFig. 20 [277–286].

SERS in bioimaging

SERS is a nondestructive analytical technique providinggreat potential for high-throughput assays and non-toxicimaging of cells and tissues [287–292]. Recent reports onSERS in bioimaging show the potential of this technique todifferentiate cells. For example, Oh and coworkers suc-cessfully differentiated living normal and cancer cells(HEK293) by SERS imaging [290]. Here, they used afunctional double-probe based on Ag/Au core/shell NPs.The double-probe consists of rhodamine 6G and amonoclonal antibody specific to PLCγ1 marker that is overexpressed in HEK293 cancer cells. The double-probes werelocalized on the surface of cancer cells using the cancermarkers and the labeled cancer cells were distinguishedfrom normal cells by combined fluorescence and SERSimaging (Fig. 21). Cho and coworkers reported a similardouble-probe strategy by conjugating Ag NPs with silicaparticles followed by attaching fluorescent dyes [292]. Inthis case, 4-aminothiophenol and 4-mercaptotoluene wereused as SERS reporters and fluorescein isothiocyanate andalexa647 dyes were used as fluorescence reporters. Theyconjugated annexin V with the double-probe, targeted

phosphatidylserine, and detected apoptosis. We used SERSanalyses and Raman imaging to understand the presence ofcytochrome c-rich domains on the surfaces of ShewanellaOneidensis, a gram-negative bacterium, under anaerobicconditions [287]. For this, SERS spectra of single bacterialcells were compared with SERS bands (ν2, ν3, ν4, and ν10vibrational modes of porphyrin) of bovine heart cyto-chrome c. Kneipp and coworkers used p-mercaptobenzoicacid (p-MBA)-conjugated Au nanoaggregates for SERSimaging of pH in living NIH 3T3 cells [289]. Here, the pHsensitivity of carboxylic acid group, CO�

2 at higher pH andCO2H at lower pH, of p-MBA was exploited as a SERSreporter. This technique can be extended to different SERSsensors for live cell imaging if experimental conditions do

Fig. 21 Bright-field (left) and dark-field (right) images of a normalHEK293 cells (A) and a PCLγ1-expressing HEK293 cancer cells (B).Reprinted with permission from Ref. [290]. Copyrights (2007)American Chemical Society

Fig. 20 Bioconjugates of Auand Ag NPs, their targets incells, and applications in cellbiology


not alter aggregate size/structure and SERS activity.Pezacki and coworkers demonstrated the imaging oftransmembrane domain of platelet-derived growth factorreceptors in HeLa cells using a cyano-labeled Ag NPs as aSERS reporter [288]. They measured the characteristic CNstretching band at 2,230 cm−1 in the Raman spectra fromintense SERS-active spots on the cell membrane. All thesereports on the bioanalyses and cell imaging applications ofSERS show future possibilities of SERS in biomedicalfields for non-isotopic and non-toxic in vivo imaging.However, in addition to targeting Au and Ag NPs tobiological samples/systems, it is necessary to considerpossible difficulties related to making and stabilizingSERS-active structures around biological structures andbiomolecules inside cells and tissues.

Recently, Nie and coworkers introduced surface-enhancedRaman reporter Au NPs for in vitro and in vivo applications[293]. For this, malachite green molecules were adsorbedon the surface of ca. 60-nm-sized Au NPs followed byoverlaying with a PEG shell. This was followed bytethering ScFv antibodies, cancer reporters specific toEGF receptors, on the PEG layer. Thus, the Au NPs arebifunctional with a SERS reporter molecule and a cancermarker antibody. The specificity of the antibody-coatedSERS reporter Au NPs to cancer cells and their ability toreport SERS signals were characterized both in vitro and invivo. In the in vitro experiments, they selected EGFreceptor-positive human head and neck cancer cells andEGF receptor-negative human non-small cell lung carcino-ma cells. In the in vivo investigations, the Au NPs weresystemically administered in mice bearing human head andneck tumor. The NPs were efficiently localized at the tumorsite and detected by specific SERS bands of malachitegreen. Figure 22 shows NIR illumination of the tumor andliver sites and SERS signals from the tumor and liver sites.This method has several advantages such as the absence oftoxic heavy metals, the use of NIR laser, and non-invasivedetection. However, as discussed under In vivo applicationsof QDs, clearance of the large-sized NPs (ca. 60 nm) fromthe body is a matter of concern. Retention of such largeNPs in the body may interfere with other imagingtechniques such as MRI, CT, PET, echo, etc. at later stages.Also, blockage of capillaries and glomerular space by largeNPs needs attention. A gradual accumulation of theparticles in the liver and elimination through the bile acidinto the intestine is a possible clearance route of such largeNPs. However, in vivo SERS imaging has a bright future innon-toxic imaging of disease conditions such as cancers if(1) large SERS-active structures are prepared by aggregat-ing small (<5 nm) Au or Ag NPs in the presence of reportermolecules, (2) biodegradable cages/shells are prepared forthe aggregates, and (3) SERS reporters are introduced atSERS-active sites in the aggregates and disease markers are

tethered on the biodegradable cages. This would allow agradual degradation of the protecting shells and the releaseof smaller Au/Ag NPs. If the size of Au/Ag NPs in SERS-active aggregates is selected <5 nm, they will be cleared fromthe body without affecting the capillaries and glumerularfiltration, as observed by Frangioni and coworkers for QDswith >5.5 nm diameter.

Summary and perspectives

The influence of semiconductor QDs and metal NPs on thepresent status of nanotechnology and nanobiotechnology isimmeasurable. Advancements in the synthesis of semicon-ductor QDs and metal NPs and experimental and theoreticalunderstanding of their optical properties at ensemble andsingle-particle levels accelerated their practical applicationsin different fields from optical devices to biomedicaldetection and imaging. Introduction of different methodsfor the synthesis of QDs and metal NPs and investigationsof various conditions involved in the synthesis considerablycontributed to their quality. Also, investigations of thefundamental electronic and optical properties of QDs andmetal NPs advanced their applications in biology andtechnology. In vivo detection and imaging of diseaseconditions such as cancers using QDs and metal NPstagged with cancer markers and other reporter moleculesare already established in animal models.

The advantages of QDs are easy to understand bycomparing their optical properties with that of fluorescent

Fig. 22 In vivo cancer targeting and surface-enhanced Ramandetection by using ScFvantibody- conjugated Au NPs that recognizethe tumor biomarker EGF receptor: (A) and (B) SERS spectraobtained from the tumor and the liver locations by using targetedand nontargeted NPs. (C) Photographs showing a laser beam focusingon the tumor site and on the anatomical location of the liver. Reprintedby permission from Macmillan Publishers Ltd: [Nature Biotechnolo-gy], Ref. [293], copyright (2008)


dye molecules. Simply, QDs are brighter and more stableand therefore they are better than dye molecules, exceptwhen their broad absorption and large size are notlimitations. An unresolved limitation of QDs is theintermittent blinking that hampers their applications in theanalyses/imaging of single biomolecules. At the same time,Ag and Au NPs offer bright plasmon color for dark-fieldimaging and enhanced Raman signals for SERS imagingand detection. Also, bioconjugated QDs and metal NPs arepromising for multimodal imaging in vivo. Initial impedi-ments related to the autofluorescence of tissues and poorpenetration depth of excitation light during in vivoapplications are resolved considerably by using QDs withNIR fluorescence, Au and Ag NPs with NIR plasmon, andmultiphoton/NIR excitation. However, there are seriousconcerns about their toxicity for in vivo applications inhuman beings. In particular, the toxicity of heavy metals inCdSe and other QDs, poor clearance of NPs >5 nm fromthe body, possible deterioration of their surface coating,interference of NPs retained in vital organs in the body withother imaging techniques, aggregation of NPs and deteri-orated NPs in capillaries and glomarular space, interactionsof NPs and their components with genetic materials, etc.need significant considerations. Although the toxicity of Auand Ag metal is less, their clearance from the body is amatter of concern. A possible way to overcome thisdifficulty, as proposed in the previous section, is the useof SERS-active aggregates of Au and Ag NPs in biode-gradable cages/shells. This ‘caged aggregate’ strategywould allow in vivo SERS/plasmon analyses/imaging,immediate biodegradation of cages, and clearance fromthe body. Therefore, another breakthrough in the applica-tions of NPs in biology is anticipated with the introductionof (1) non-blinking and non-toxic QDs and (2) Au and AgNP aggregates within biodegradable cages. This would notonly bring radical changes to nanotechnology and biomed-ical imaging but also introduce nanodiagnosis and nano-therapy for the early detection and curing of potential heathproblems.

At present, research and developments in the semicon-ductor QD and metal NP fields are advancing in parallel,and far-reaching implications are not too far away if thesetwo classes of nanomaterials are combined for solar energyconversion, nanoelectronics, multiplexed detection/imagingin analytical and biomedical fields, and therapeutic inter-ventions such as NIR photothermal and multiphotonphotodynamic therapy of cancer.

Acknowledgement We thank a Seed Research Fund and a SpecialCoordination Fund for Promoting Science and Technology of theMinistry of Education, Culture, Sports, Science, and Technology,the Japanese Government. Also, we thank Professor John V.Frangioni, Professor Shuming Nie, and Dr. Ximei Qian for helpfulsuggestions.

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