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Hindawi Publishing Corporation Journal of Nanomaterials Volume 2011, Article ID 834139, 13 pages doi:10.1155/2011/834139 Review Article Application of Quantum Dots in Biological Imaging Shan Jin, Yanxi Hu, Zhanjun Gu, Lei Liu, and Hai-Chen Wu Key Laboratory for Biomedical Eects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences, Beijing 100049, China Correspondence should be addressed to Lei Liu, [email protected] Received 15 May 2011; Accepted 2 June 2011 Academic Editor: Xing J. Liang Copyright © 2011 Shan Jin et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Quantum dots (QDs) are a group of semiconducting nanomaterials with unique optical and electronic properties. They have distinct advantages over traditional fluorescent organic dyes in chemical and biological studies in terms of tunable emission spectra, signal brightness, photostability, and so forth. Currently, the major type of QDs is the heavy metal-containing II-IV, IV-VI, or III-V QDs. Silicon QDs and conjugated polymer dots have also been developed in order to lower the potential toxicity of the fluorescent probes for biological applications. Aqueous solubility is the common problem for all types of QDs when they are employed in the biological researches, such as in vitro and in vivo imaging. To circumvent this problem, ligand exchange and polymer coating are proven to be eective, besides synthesizing QDs in aqueous solutions directly. However, toxicity is another big concern especially for in vivo studies. Ligand protection and core/shell structure can partly solve this problem. With the rapid development of QDs research, new elements and new morphologies have been introduced to this area to fabricate more safe and ecient QDs for biological applications. 1. Introduction Semiconductor nanocrystals, or so-called quantum dots (QDs), show unique optical and electronic properties, including size-tunable light emission, simultaneous excita- tion of multiple fluorescence colors, high signal brightness, long-term photostability, and multiplex capabilities [14]. Such QDs have significant advantages in chemical and biological researches in contrast to traditional fluorescent organic dyes and green fluorescent proteins on account of their photobleaching, low signal intensity, and spectral over- lapping [57]. These properties of QDs have attracted great interest in biology and medicine in recent years. At present QDs are considered to be potential candidates as luminescent probes and labels in biological applications, ranging from molecular histopathology, disease diagnosis, to biological imaging [810]. Numerous studies have reported the use of QDs for in vitro or in vivo imaging of sentinel lymph nodes [1117], tumor-specific receptors [1820], malignant tumor detectors [21], and tumor immune responses [22]. However, the major concerns about potential toxicity of II-IV QDs (such as CdTe and CdSe) have cast doubts on their practical use in biology and medicine. Indeed, several studies have reported that size, charge, coating ligands, and oxidative, photolytic, and mechanical stability, each can contribute to the cytotoxicity of cadmium-containing QDs. Another critical factor that determines the cytotoxicity of QDs is the leakage of heavy metal ions from the core caused by photolysis and oxidation [2325]. The long-term eects of these ions enrichment and durations within the body raise concerns about the biocompatibility and safety of QDs [24, 26]. On the other hand, the rapid clearance of QDs from the circulation by the reticuloendothelial system (RES) or entrapment of QDs in the spleen and liver [27, 28] can result in the degradation of the imaging quality of the objective and the enhancement of background noise. One promising solution to these problems, including QDs sequestered in the liver/spleen and long-term retention in the body, is to evade the recognition and uptake by the reticuloendothelial system thus extending the circulation time of the particles in the body. In addition, for biological studies, QDs should be aqueous soluble in order to adapt the biological environment. Therefore, when QDs are employed in biological imaging, many factors should be considered and

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Hindawi Publishing CorporationJournal of NanomaterialsVolume 2011, Article ID 834139, 13 pagesdoi:10.1155/2011/834139

Review Article

Application of Quantum Dots in Biological Imaging

Shan Jin, Yanxi Hu, Zhanjun Gu, Lei Liu, and Hai-Chen Wu

Key Laboratory for Biomedical Effects of Nanomaterials and Nanosafety, Institute of High Energy Physics, Chinese Academy of Sciences,Beijing 100049, China

Correspondence should be addressed to Lei Liu, [email protected]

Received 15 May 2011; Accepted 2 June 2011

Academic Editor: Xing J. Liang

Copyright © 2011 Shan Jin et al. This is an open access article distributed under the Creative Commons Attribution License, whichpermits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Quantum dots (QDs) are a group of semiconducting nanomaterials with unique optical and electronic properties. They havedistinct advantages over traditional fluorescent organic dyes in chemical and biological studies in terms of tunable emission spectra,signal brightness, photostability, and so forth. Currently, the major type of QDs is the heavy metal-containing II-IV, IV-VI, or III-VQDs. Silicon QDs and conjugated polymer dots have also been developed in order to lower the potential toxicity of the fluorescentprobes for biological applications. Aqueous solubility is the common problem for all types of QDs when they are employed in thebiological researches, such as in vitro and in vivo imaging. To circumvent this problem, ligand exchange and polymer coating areproven to be effective, besides synthesizing QDs in aqueous solutions directly. However, toxicity is another big concern especiallyfor in vivo studies. Ligand protection and core/shell structure can partly solve this problem. With the rapid development of QDsresearch, new elements and new morphologies have been introduced to this area to fabricate more safe and efficient QDs forbiological applications.

1. Introduction

Semiconductor nanocrystals, or so-called quantum dots(QDs), show unique optical and electronic properties,including size-tunable light emission, simultaneous excita-tion of multiple fluorescence colors, high signal brightness,long-term photostability, and multiplex capabilities [1–4].Such QDs have significant advantages in chemical andbiological researches in contrast to traditional fluorescentorganic dyes and green fluorescent proteins on account oftheir photobleaching, low signal intensity, and spectral over-lapping [5–7]. These properties of QDs have attracted greatinterest in biology and medicine in recent years. At presentQDs are considered to be potential candidates as luminescentprobes and labels in biological applications, ranging frommolecular histopathology, disease diagnosis, to biologicalimaging [8–10]. Numerous studies have reported the use ofQDs for in vitro or in vivo imaging of sentinel lymph nodes[11–17], tumor-specific receptors [18–20], malignant tumordetectors [21], and tumor immune responses [22].

However, the major concerns about potential toxicity ofII-IV QDs (such as CdTe and CdSe) have cast doubts on

their practical use in biology and medicine. Indeed, severalstudies have reported that size, charge, coating ligands, andoxidative, photolytic, and mechanical stability, each cancontribute to the cytotoxicity of cadmium-containing QDs.Another critical factor that determines the cytotoxicity ofQDs is the leakage of heavy metal ions from the core causedby photolysis and oxidation [23–25]. The long-term effectsof these ions enrichment and durations within the bodyraise concerns about the biocompatibility and safety of QDs[24, 26]. On the other hand, the rapid clearance of QDs fromthe circulation by the reticuloendothelial system (RES) orentrapment of QDs in the spleen and liver [27, 28] can resultin the degradation of the imaging quality of the objective andthe enhancement of background noise.

One promising solution to these problems, includingQDs sequestered in the liver/spleen and long-term retentionin the body, is to evade the recognition and uptake bythe reticuloendothelial system thus extending the circulationtime of the particles in the body. In addition, for biologicalstudies, QDs should be aqueous soluble in order to adapt thebiological environment. Therefore, when QDs are employedin biological imaging, many factors should be considered and

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2 Journal of Nanomaterials

CdSCdSe InPCdTeCdHgTe/ZnS

CdTe/CdSe

InAsPbSe

10

9

8

7

6

5

4

3

2

1400 600 800 1000 1200 1400

Emission wavelength (nm)

Qdo

tdi

amet

er(n

m)

0

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0.4

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1

Wavelength (nm)

600 800 1000 1200 1400

(a)

480 520 560 600 640 680 720

480 520 560 600 640 680 720

Abs

orpt

ion

Wavelength (nm)

Lum

ines

cen

ce(b)

GFP qdot qrod SAV MBP IgG

20 nmbead

FITC(x 5)

5 nm

(c)

Figure 1: Emission maxima and sizes of quantum dots of different composition (with permission from [3]).

the system should be properly designed in order to meet a setof requirements [29–31].

In this report, we first briefly review different types ofQDs that have been fabricated so far and their synthesismethods, including surface functionalization. Next, we focuson their applications in biological imaging. Finally, the newtrends of QDs imaging are discussed.

2. Types and Characteristics of QDs

Traditionally, the typical QDs consist of a II-IV, IV-VI, or III-V semiconductor core (e.g., CdTe, CdSe, PbSe, GaAs, GaN,InP, and InAs; Figure 1) [3, 32, 33], which is surroundedby a covering of wide bandgap semiconductor shell such asZnS in order to minimize the surface deficiency and enhancethe quantum yield [5, 34].The unique optical and elec-tronic properties of QDs, including narrow light emission,wideband excitation, and photostability, provide them withsignificant advantages in the applications of multiplexedmolecular targets detection [21] and as optical bioimagingprobes [8–10]. One major drawback which severely hindersthe application of II-IV, IV-VI, or III-V QDs for in vivo bio-logical research is the concern about the toxicity associatedwith the cadmium, lead, or arsenic containing QDs. The

toxic heavy metal ions can be easily leaked out into biologicalsystems if the surfaces are not properly covered by the shellsor protected by ligands.

The limitation of heavy metal-containing QDs stimulatesextensive research interests in exploring alternative strategiesfor the design of fluorescent nanocrystals with high biocom-patibility. In this case, the practical strategy is to develophighly fluorescent nanoparticles based on nontoxic elementsor explore luminescent π-conjugated polymer dots.

Silicon nanoparticle is another type of QDs which possessunique properties when their sizes are reduced to below10 nm. Similar to their predecessors, silicon QDs also havemany advantages over traditional fluorescent organic dyes[35], including resistance to photobleaching and wide emis-sion range from visible to infrared region with relatively highquantum yields. Moreover, owing to silicon’s nontoxic andenvironment-friendly nature, Si QDs are used as fluorescentprobes for bioimaging [35]. It has been reported that forin vivo applications, Si QDs mainly degrade to silicic acidwhich can be excreted through urine [36], and for in vitrouse, Si QDs are considered 10-times safer than Cd-containingquantum dots (Figures 2 and 3) [37]. However, the chal-lenges present in employing Si QDs in bioimaging arise fromtheir water solubility and biocompatibility. Usually, Si QDs

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Journal of Nanomaterials 3

are produced in nonpolar solvents with hydrophobic ligands(such as styrene and octene) on their surface in order toprotect the Si cores. Therefore, it is a common problem thatSi QDs show poor solubility and unstable photolumines-cence (PL) in aqueous solutions [38–40]. PL degradation canoccur gradually after modified Si QDs are transferred fromorganic solvents to aqueous solutions. Thus, fabricating goodwater-disperse Si QDs with stable optical characters is vital totheir applications in bioimaging studies. Recently, Tilley andYamamoto reported that water-soluble Si QDs covered byallylamine [41, 42] and poly(acrylic acid), respectively, hadbeen successfully obtained and showed good performance infixed-cell labeling [40].

The semiconducting properties of conjugate polymersderive from their π-electron delocalization along the poly-mer chain. Mobile charges are allowed to move between πand π∗ orbitals. Highly bright fluorescence can be stableunder one-photon or two-photon excitation. Polymers withsuch structures are promising in the fields of LED and bi-osensors [43, 44]. Burroughes and coworkers first reportedthe luminescent conjugated polymers in 1990 [45]. Later on,Friend and So successfully applied π-conjugated polymersto light-emitting devices [46, 47]. Recently, related worksfocused on fluorescent polymer encapsulation and func-tionalization to yield conjugated polymer dots (CP dots)for bioimaging and probes (Figure 4) [48, 49]. Comparedwith other fluorescent probes, CP dots are convenient forfluorescence microscopy and laser excitation because of theirabsorption ranging from 350 to 550 nm. Also, fluorescencequantum yields can be slightly variable around 40%. More-over, CP dots can exhibit higher brightness than any othernanoparticles of the same size under certain conditions [50].

3. Synthesis Methods of QDs

3.1. Organometallic Method. Organometallic chemistry pro-vides an efficient pathway to produce monodisperse QDs innonpolar organic solvents, such as CdSe nanocrystals cov-ered by tri-n-octylphosphine oxide (TOPO) ligand. In atypical preparation, Me2Cd and Bis(trimethylsilyl)selenium((TMS)2Se) can be used as organometallic precursors. Mon-odisperse CdSe with uniform surface derivatization and reg-ularity in core structure can be obtained based on the pyroly-sis of organometallic reagents by injection into a hot coordi-nating solvent between 250◦C to 300◦C [51]. The adsorptionof ligands results in slow growth and annealing of thecores in the coordinating solvents. QDs with different sizescan be successfully obtained at different temperatures. Theadvantage of this method is obvious because this method canproduce a variety of QDs with high quantum yields and thesize distribution can be easily controlled by altering temper-ature or reaction time. Organometallic method is currentlyconsidered as the most important means to synthesize QDs.

3.2. QDs Synthesized in Aqueous Solution. Since Rajh andcoworkers first reported the synthesis of thiol-capped CdTeQDs in aqueous solution, a great deal of efforts havefocused on synthesizing water-dispersed QDs directly [52].

50 μm

(a)

(b)

Figure 2: Fluorescence images of mesoporous silica beads (5-μmdiameter, 32 nm pore size) doped with QDs emitting light at 488(blue), 520 (green), 550 (yellow), 580 (orange), or 610 nm (red; withpermission from [37]).

In this process, ionic perchlorates such as Cd(ClO)4·6H2Oand Al2Te3 are used as the precursors. In the presence ofligands such as 3-mercaptopropionic acid (3-MPA), glutathi-one (GSH), or other hydrosulfyl-containing materials, CdTeQDs can be successfully obtained in aqueous medium. Theadvantage of this method lies in its environment-friendlysynthesis procedure and relatively low cost. QDs produced bythis method can be directly applied in biological researcheswithout ligand exchange procedure. However, comparedwith the QDs produced by organometallic method, thiol-capped QDs show low quantum yields, and broad full widthof half-maximum (FWHM), which can be attributed to itswide size distribution and poor stability in aqueous solution.

3.3. Hydrothermal Method/Microwave-Assisted Method. Inorder to reduce QDs surface defects generated during thegrowth process, these two methods are put forward to syn-thesize QDs with narrow size distribution and high quantumyields. According to hydrothermal method, all reaction re-gents are loaded in hermetic container and then heated tosupercritical temperature; high pressure produced by thetemperature can efficiently reduce reaction time and surfacedefects of QDs [53]. Moreover, because high reaction tem-perature can separate the processes of nucleus formation andcrystal growth, this method has clear advantages over tradi-tional aqueous synthesis, affording narrow size distributionQDs.

Microwave-assisted irradiation is another attractivemethod for QDs synthesis. This method was first introducedby Kotov group [54] and followed by Qian and coworkers todemonstrate a fast and simple synthesis of CdTe and CdSe-CdS QDs by microwave-assisted irradiation in 2006 [55].Microwave irradiation can be considered as heating source

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4 Journal of Nanomaterials

1 10 100

140

120

100

80

60

40

20

0

24 hr48 hr

Concentration of Si (μg/mL)

Cel

lvia

bilit

y(%

)

(a)

1 10

100

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60

40

20

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24 hr

Concentration of CdHgTe (μg/mL)

Cel

lvia

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)(b)

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100

80

60

40

20

0

24 hr

Concentration of CdTe (μg/mL)

Cel

lvia

bilit

y(%

)

10

(c)

Figure 3: Cytotoxicity of (a) Si, (b) CdTe, and (c) HgCdTe quantum dots toward Panc-1 cells. Error bars represent standard deviation(N = 3). Note that the concentration range is much higher in (a) than in (b) and (c) (with permission from [38]).

n

n

n

MEH-PPV

PFPV

OCH8

PDHFH13C6

C8H17H17C8

C6H13 MeO

MeO

OC8H17

H17

(a) (b)

25

20

15

10

5

0

200 nm

(nm

)

(c)

(GM

)

102

103

104

105

PFPV

MEH-PPVPDHF

Rhodamine B

770 790 810 850 870

Wavelength (nm)

8 03

σ2pϕ

(d)

Figure 4: (a) Chemical structures of the conjugated polymers. (b) Photograph of the fluorescence from aqueous CP dot dispersions undertwo-photon excitation of an 800 nm mode-locked Ti : sapphire laser. (c) A typical AFM images of the PFPV dots on a silicon substrate. (d)Semilog plot of two-photon action cross-sections (σ2pϕ) versus the excitation wavelength for CP dots and rhodamine B (reference compound;with permission from [49]).

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Journal of Nanomaterials 5

Functionalization

QDs

Aqueous solubility

Ligand

Coatin

Peptides

Antibodies

Proteins

A

B

C

D

exchangePMAL

COOH

NH

O =P

O = PO = P

Organic phase:chloroFrom

InP/ZnSInP/ZnS

+

NH

+

MSA coated

MSA

Overnightstirring

Hydrophobic ligands

Aqueous phase

QD705-RGD C(RGDyK)

Tissuesection

Cellmembrane

Nucleus

HO

HO

HOHO

O O

Sialicase

COO−

ZnS

CdTe

QD

Scheme 1: Strategies to deal with QDs aqueous solubility problems and fabricate functionalized QDs for biological applications.

to optimize synthesis conditions, and in this system, wateris an excellent solvent for its tan δ is equal to 0.127. Thewhole solution can be quickly heated to above 100◦C to yieldhomogeneous QDs and the quantum yields can reach as highas 17%.

4. Toxicity, Solubilization, andFunctionalization of Quantum Dots

In order to apply QDs to biological studies, safety, and bio-compatibility are the issues that must be concerned. Toxico-logical studies have suggested that certain potential adversehuman health effects may be resulted from their exposureto some novel nanomaterials, such as gold nanoparticlesand QDs, but the fundamental cause-effect relationships arerelatively obscure [23]. Thus, it is necessary to figure outthe interaction of QDs with biological systems and theirpotentially harmful side effects in cells. Several studies revealthat the toxicity of QDs depends on many factors whichcan summarized as inherent physicochemical properties andenvironmental conditions [23, 24, 26]. Not all QDs are alike,each type of QDs should be characterized individually as toits potential toxicity. QD sizes, charges, concentrations, outercoatings materials and functional groups, oxidation, andmechanical stability all have been implicated as contributingfactors to QD toxicity [24]. Derfus et al. studied the cytotox-icity of CdSe QDs and pointed that without coating, the cy-totoxicity of CdSe cores correlated with the liberation of freeCd2+ ions due to deterioration of QDs lattice. And in vivo, theliver is the primary site of acute injury caused by cadmiumions even though the concentration of Cd2+ is reduced to100–400 μM [23]. In order to eliminate QD toxicity re-sulted from heavy metal ions releasing, surface coating andcore/shell structure are considered to be efficient solutionsto this problem. Many new types of QDs such as core/shell(CdSe/ZnS) and core/shell/shell (CdSe/ZnS/TEOS) QDswith high luminescence are developed [34, 35]. Some re-searchers also found that the quantum size effect whichresulted from the minute size of nanomaterials could lead

to cytotoxicity. Since nanoparticles within certain diameters(this value may be different according to biological environ-ment) are of similar size to certain cellular components andproteins, they may bypass natural mechanical barriers, thusleading to adverse tissue reactions [26].

The application of QDs in biological imaging also re-quires QDs with good water solubility. Several strategies havebeen developed to circumvent this problem, which are sum-marized as follows.

The first one is to synthesize QDs in aqueous solutiondirectly, which has been depicted in this review (vide supra).The second is to obtain QDs in organic solvents, and thentransfer them to aqueous solutions (Scheme 1). Nie andWeiss groups pioneered the preparation of water-dispersedQDs by ligand exchange method (Figure 4) [5, 56]. In ad-dition, polymer and lipid coating is another efficient way torender QDs with good water solubility [57, 58].

To improve in vitro and in vivo imaging quality anddetection sensitivity in biological fields, multiplex capabili-ties QDs have been developed. Recent efforts mainly focus onthe functionalization of QDs to accommodate the demandsof imaging sensitivity and specificity [59]. Nie reportedmulticolor QD-antibody conjugates for the rapid detectionof rare Hodgkin’s and Reed-Sternberg (HRS) cells frominfiltrating immune cells such as T and B lymphocytes [60];Shi combined Fe3O4 with amine-terminated QDs to yieldfluorescent superparamagnetic QDs for in vivo and in vitroimaging (Figure 5) [61].

5. QDs in Biological Imaging

In order to apply QDs in biological imaging, recent studieshave focused on developing near-IR luminescent QDs whichexhibit an emission wavelength ranging from 700 to 900 nm.Light within this range has its maximum depth of penetra-tion in tissue and the interference of tissue autofluorescence(emission between 400 nm and 600 nm) is minimal [62, 63].In addition, for the purpose of gathering more informationduring specific cellular processes in real time, QDs must be

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6 Journal of Nanomaterials

QDs

NH

PEG NH2

5 nm 5 nm

Fe3O4

Carboxylate-functionalized MNS

HO

QDs-conjugated MNSs

Amine-functionalizedquantum dot (QD)

Polyethyleneoxide

O

O

C

++++−−

−−Polystyrene

(a)

+

5 nm

5 nm

PLGA

Paclitaxel (PTX)

PLGA + PTX

HO

HOHO

O

O

O

O

O

O

O

OOO

OO

O

OO

CH3

CH3

CH3CH3

CH3

NH H

H

H3CH3C

m n

QDsQDs

PTX

PLGA

QDs-conjugated MNSs

PTX-PLGA- -MNSsQD

(b)

Figure 5: Schematic diagrams illustrating surface functionalization of magnetic nanospheres (MNSs): (a) conjugation of amine-functionalized quantum dots (QDs) to the surface of carboxylate-functionalized MNSs using conventional NHS/EDC coupling method;(b) PTX loading using a thin PLGA coat on the surface of QD-MNSs (with permission from [61]).

conjugated with molecules which have the capabilities of rec-ognizing the target. These surface modifications can also helpprevent aggregation, reduce the nonspecific binding, andare critical to achieving specific target imaging in biologicalstudies. Further functionalization of QDs can be achievedby the modification of protecting ligands [64–66], introduc-ing targeting groups such as apolipoproteins and peptides(Scheme 1) [64]. In this part, we mainly introduce the recentdevelopment of conjugated QDs for in vitro and in vivo imag-ing; some new QDs related to bioimaging are also included.

5.1. In Vitro Imaging. Early studies of in vitro imaging usedQDs to label cells. For instance, PbS and PbSe capped withcarboxylic groups had been obtained in aqueous solution byligands exchange to label cells [67]; CdTe capped with 3-mercaptopropionic acid (3-MPA) was used as imaging toolto label Salmonella typhimurium cells [68]. Jaiswal et al.reported labelling HeLa cells with acid-capped CdSe/ZnSQDs [69]. It is found that the cells can store QDs in vesicles

through endocytosis after washing away the excess QDs. Fur-ther, they also demonstrated the potential application of QDsin cell tracking by using avidin-conjugated QDs to label cells.

With the development of biomarkers in cell biology, thetracking of some specific cells (such as cancer cells) becomespossible. Gac et al. have successfully detected apoptotic cellsby conjugating QDs with biotinylated Annexin V, whichenables the functionalized QDs to bind to phosphatidylserine(PS) moieties present on the membrane of apoptotic cells butnot on healthy or necrotic cells (Figure 6) [70]. The detectionand imaging of apoptotic cells makes it possible to moni-tor specific photostable apoptosis. Wolcott reported silica-coated CdTe QDs decorated with functionalized groupsnot only for labelling proteins, but also for preventingtoxic Cd2+ leaking from the core [71]. Nie developed cell-penetrating QDs coated with polyethylene glycol (PEG)grafted polyethylenimine (PEI), which were capable of pene-trating cell membranes and disrupting endosomal organellesin cells [72]. Recently, in the demand of using biocompatible

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Journal of Nanomaterials 7

Lightmicroscope

FITC FITC FITC FITC

Qdots Qdots Qdots Qdots

Alex fluor 647 Alex fluor 647 Alex fluor 647 Alex fluor 647

t = 0 s t = 500 s t = 1000 s t = 1500 s

(a)

0

20

40

80

100

120

0 200 400 600 800 1000 1200 1400 1600 1800

I/I1

0(%

)

I/I10 (%) AlexaI/I10 (%) QdotsI/I10 (%) FITC

Time (s)

(b)

Figure 6: Comparison of the staining efficiency of Annexin V-functionalized Qdots (staining achieved in one step using 2 nM ofQdots and 4 nM of biotinylated Annexin V, red), FITC-AnnexinV (0.5% v/v, green) and Alexa Fluor 647-Annexin V (0.5% v/v,pink). (a) Light microscope pictures and pictures taken at t =0, 500, 1000, 1500 s. (b) Intensity variation as a function of timefor the three staining agents conjugated to Annexin V, FITC, AlexaFluor 647 and Qdots. Intensity values expressed as relative valuesI/I0, where I0 is the intensity measured at the first irradiation of thesample (with permission from [70]).

and nontoxic QDs as nanoprobes, rare earth (RE) elementsare used to fabricate a new type of QDs, such as Gd-dopedZnO QDs. RE-doped QDs have distinct advantages overheavy metal-containing QDs, not only because of avoidingthe increase of particle size by polymer or silica coatingin synthesis procedure, but also providing a simple, greensynthesis method. Liu et al. reported the development ofGd-doped ZnO QDs with enhanced yellow fluorescence, andthese QDs can be used as nanoprobes for quick cell detec-tion with very low toxicity [73]. Other progresses includeCdSe/ZnS with PEG coating and CdSe/ZnS-peptide conju-gation for cytosol localization and nucleus targeting [74–77],CdS passivated by DNA to yield nontoxic QDs as new bio-logical imaging agent [78], CdSe/L-cysteine QDs designedto label serum albumin (BSA) and cells [79], polymer QDs

Dox

QD

QD QD

QD-APt: “ON” QD-APt(DOX): “OFF”

(a)

Target cancer cell

Drug release

Nucleus QD: “ON”Dox: “ON”

QD

QD QD

ysosomeL

“OFF”

(b)

Figure 7: (a) Schematic illustration of QD-Apt(Dox) Bi-FRETsystem. (b) Schematic illustration of specific uptake of QD-Apt(Dox) conjugates into target cancer cell through PSMA mediateendocytosis. The release of Dox from the QD-Apt(Dox) conjugatesinduces the recovery of fluorescence from both QD and Dox (“ON”state), thereby sensing the intracellular delivery of Dox and enablingthe synchronous fluorescent localization and killing of cancer cells(with permission from [85]).

(PS-PEG-COOH) with conjugation to biomolecules, suchas streptavidin and immunoglobulin G (IgG) to label cellsurface receptors and subcellular structures in fixed cells [21,80], and other non-Cd-containing QDs such as Ge-QDs andnear-IR InAs for imaging of specific cellular proteins [81, 82].In order to broaden QDs applications, MRI detectable QDswere demanded. Many studies have set out to explore para-magnetic QDs. Sun reported a new kind of magnetic fluores-cent multifunctional nanocomposite which contained silica-coated Fe3O4 and TGA-capped CdTe QDs. This nanocom-posite was successfully employed for HeLa cell labelling andimaging, as well as in magnetic separation [83]. Mulder etal. have successfully modified CdSe/ZnS with paramagneticcoating and arginine-glycine-aspartic acid (RGD) conjuga-tion in order to combine the functions of specific proteinbinding and contrast agent for molecular imaging [84].

The challenges encountered in cancer therapy stimulatecomprehensive studies. Many efforts focus on the trackingand diagnosis of cancer cells. Cao et al. used near-IR QDswith an emission wavelength of 800 nm (QD800) to labelsquamous cell carcinoma cell line U14 (U14/QD800). Thefluorescent images of U14 cells can be clearly obtained after6 h by cell endocytosis [62]. In 2007, Bagalkot et al. reporteda more complex QDs-aptamer- (Apt-) doxorubicin (Dox)conjugate system [QD-Apt(Dox)] to endow QDs with thecapability of targeting, imaging, therapy, and sensing theprostate cancer cells that express the prostate-specific mem-brane antigen (PSMA) protein (Figure 7) [85]. Other typesof QDs that have been applied in pancreatic cancer cells

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8 Journal of Nanomaterials

Cumulative histogram of tumor (left) and the mouse body (right)

Count: 19116

Mean: 62.164

StdDev: 16.051

Min: 10

Max: 106

Mode: 61 (763)

Count: 501534

Mean: 42.799

StdDev : 23.450

Min: 4

Max: 107

Mode: 10 (29749)

Count: 535446

Mean: 127.063

Min: 7

Max: 255

Mode: 44 (7691)

Count: 25448

Mean: 176.818

StdDev: 37.301

Min: 37

255

Tumor

0 2550 2550 2550

Mode: 197 (1034) StdDev: 63.694

Tumor

Max: 2 62

Composite image of mouse, colored NIR on top of field

(a)

260.29 292.43 324.57 181 184.21 187.43 190.64 193.86 176.26 182.35 188.44 194.63 200.62

224.98 227.26 229.55 231.83 234.11

196 228.14

260.29 292.43 324.57 181 184.21 187.43 190.64 193.86196 228.14

(b)

Figure 8: Targeting of lung melanoma tumor by injecting 2C5 QD-Mic in two mice. (a) Composite images (white field image superimposedwith the fluorescence intensity), and cumulative histograms for the tumor region and the whole body of two mice; (b) composite ex vivoimages (white field image superimposed with the fluorescence intensity) of lungs from mice bearing metastatic B16F-10 lung melanomatumor (with permission from [94]).

imaging include CdSe/CdS/ZnS using transferrin and anti-Claudin-4 as targeting ligands, InP QDs conjugated withcancer antibodies, and water-soluble Si-QDs micelles encap-sulated with polymer chains [86–88]. Another importantrole played by QDs in biological application is transfection.Many studies reported fluorescence imaging and nucleus

targeting of living cells by transfection and RNA delivery. Bijuidentified an insect neuropeptide, also-called allatostatin,which can transfects living NIH 3T3 and A431 humanepidermoid carcinoma cells and transports quantum dots(QDs) inside the cytoplasm and even the nucleus of thecells [89]. This method showed the conjugation of QDs with

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Journal of Nanomaterials 9

allatostatin could achieve high transection efficiency, whichwas promising for DNA gene delivery and cell labelling.Also, with specific conjugation with allatostatin, the QDs hadanother potential application in cancer, called photodynamictherapy [90].

5.2. In Vivo Imaging. In addition to their usage as nanop-robes and labels for in vitro imaging, QDs have also beenwidely used as in vivo imaging agents (Figure 8) [91–94].Cancer-specific antibody, coupled to near-IR QDs with pol-ymer coatings is the most popular QDs agent for tumor-targeted imaging. Progress in this field has been reviewed inone literature [94, 95].

However, the depth of targets makes the QDs in vivoimaging very challenging. This special application of QDsrequires them with low toxicity, high contrast, high sensitiv-ity, and photostability. Dubertret et al. fabricated one typeof CdSe/ZnS QDs which were encapsulated individually inphospholipid block-copolymer micelles. After further modi-fication by DNA, this multifunctionalized DNA-QD-micellescan be developed to obtain Xenopus embryo fluorescentimages in PBS. Moreover, after injection into embryos, theQD micelles showed high stability and nontoxicity (<5× 109

nanocrystals per cell) [96]. Another study reported byMichalet showed that CdSe/ZnS QDs can exhibit high con-trast and imaging depth in two-photon excitation confocalmicroscopy by visualizing blood vessels in live mice [3, 97].They confirmed that coatings such as PEG on the surfacesof QDs could further reduce their toxicity and accumulationin liver. Some studies also pointed out that the increaseof the length of PEG coating polymer on the surface ofQDs was able to slow down their extraction toward theliver, so coating polymer on the surfaces was another factorworthy of considering when QDs are employed for in vivoimaging [98]. Other reports that used low-toxicity QDsinclude the development of non-Cd-containing QDs suchas CuInS2/ZnS core/shell nanocrystals for in vivo imaging[95]. In addition, sensitivity is another important factor thatmust be considered in QDs-related in vivo imaging. Onestudy used nude mice for in vivo imaging after near-IRQD800-labeled BcaCD885 cells (BcaCD885/QD800) beingimplanted [99]. Fluorescence signals of QDs accumulated inthe tumor could be detected after 16 days of incubation atcertain concentrations. It suggested that, compared with CTand MRI, QD800-based imaging could efficiently increasethe sensitivity of early diagnosis of cancer cells.

With the development of QDs in recent years, manystudies tried to explore the potential of QDs in widerfields. Kobayashi reported fluorescence lymphangiographyby injecting five QDs with different emission spectra [100].Through simultaneous injection of five QDs into differentsites in the middle of phalanges, the upper extremity, theears, and the chin, different parts of the mouse bodycan be identified by certain fluorescence color. This is thefirst demonstration of simultaneous imaging of traffickinglymph nodes with QDs having different emission spectra.As biological fluorescent markers, no matter in vitro orin vivo, much attention has been concentrated on developingspherical QDs conjugations, but there are few reports on how

other morphologies of QDs will interact with the biologicalsystems. Yong et al. reported CdSe/CdS/ZnS quantum rods(QRs) coated with PEGylated phospholipids and RGDpeptide for tumor targeting [101]. This conjugation wasdemonstrated to be a bright, photostable, and biocompatibleluminescent probe for the early diagnosis of cancer and wasbelieved to offer new opportunities for imaging early tumorgrowth.

6. Prospects

Owing to the initial success of QDs-biomolecule conjugatesemployed for in vitro and in vivo imaging, it is conceivablethat future researches will continue to focus on the devel-opment of QDs conjugations for cell labelling and cancerdiagnoses. However, for bioapplications, biocompatibility ofQDs remains the major challenge which must be carefullydealt with. Many studies have showed the short-term stabilityand long-term breakdown of Cd-containing QDs in in vivoimaging, which raise concerns about their chronic toxicity.Some studies showed that even robust coatings on thesurfaces of QDs cannot prevent their releasing of Cd2+ ions.The emerging picture of Cd-containing QDs toxicity notonly suggests that QDs are required to improve safety beforetheir wide biological applications as fluorescent nanoprobes,but also a variety of noncadmium alternatives are in urgentdemand. At present, many efforts have focused on exploringnew types of fluorescent nanomaterials such as upconver-sion materials NaYF4 and NaGaF4. In addition, with thedevelopment of new techniques and detection methods, QDsare shown to have applications in wider fields. Zhang et al.successfully introduced Kelvin force microscopy (KFM) as amethod to examine the binding of QDs with DNA in vitroand in vivo [102]; Huang et al. demonstrated a QDs sensorfor quantitative visualization of surface charges on singleliving cell [103]. We believe that with the development of newQDs with low toxicity and multiplex functionalities, QDs willfind more versatile applications in biological fields other thanbioimaging.

Acknowledgment

The authors are grateful to the “100 Talents” programof the Chinese Academy of Sciences, and 973 Program(2010CB933600) for funding support.

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