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Colloids and Surfaces B: Biointerfaces 87 (2011) 346– 352

Contents lists available at ScienceDirect

Colloids and Surfaces B: Biointerfaces

j our na l ho me p age: www.elsev ier .com/ locate /co lsur fb

fficient assembly of multi-walled carbon nanotube-CdSe/ZnS quantum dotybrids with high biocompatibility and fluorescence property

ingying Zhanga, Weiling Qina, Hao Tanga,∗, Feng Yanb, Liang Tana, Qingji Xiea,ing Maa, Youyu Zhanga, Shouzhuo Yaoa

Key Laboratory of Chemical Biology and Traditional Chinese Medicine Research, Ministry of Education, College of Chemistry and Chemical Engineering, Hunan Normal University,hangsha 410081, PR ChinaDepartment of Applied Physics and Materials Research Center, The Hong Kong Polytechnic University, Hung Hom, Kowloon, Hong Kong, China

r t i c l e i n f o

rticle history:eceived 21 February 2011eceived in revised form 8 May 2011ccepted 21 May 2011vailable online 30 May 2011

a b s t r a c t

CdSe/ZnS core–shell quantum dots (QDs) were efficiently tethered onto polyamidoamine dendrimer-modified multi-walled carbon nanotubes (MWCNTs) by covalent linkage and mercapto-mediatedassembly. The obtained MWCNT-QD hybrids were both photophysically and morphologically charac-terized. The QDs are well-distributed on single nanotube surface in high density and the assembly of QDsonto MWCNTs does not change the fluorescence emission wavelength of QDs but significantly decreases

eywords:dSe/ZnS quantum dotsulti-walled carbon nanotubesybridsytotoxicity

the emission density. Cytotoxicity of MWCNT-QD hybrids to HeLa cells and their fluorescence propertyin living cell system were evaluated in detail. The hybrids show a little effect on cell viability even at veryhigh concentration (100 �g mL−1). Moreover, they possess intense red fluorescence signal under opticalfluorescence microscopy and good fluorescence stability over 72-h exposure in living cell system.

© 2011 Elsevier B.V. All rights reserved.

luorescence property in living cell system

. Introduction

Carbon nanotubes (CNTs) are among the most promising nano-aterials because their extraordinary physical, chemical, andechanical properties [1–4]. Moreover, CNTs have the ability to be

unctionalized and good biocompatibility [5], which has enhancedheir potential applications in biosensing, molecule delivery, andissue engineering including cellular tracking or imaging [6–10]. Toxtend and optimize applications of CNTs in these fields, it is highlyesirable to understand the interaction between CNTs and cells, andlso need to develop functionalized CNTs that easy to be detected,or example, visualized in biological environments using simple flu-rescence microscopy. Although CNT cellular tracking or imagingan be characterized by transmission electron microscopy (TEM),t has some disadvantages such as relatively high cost and not suit-ble for real-time analysis. Therefore, CNTs functionalized withuorophores were developed for CNT visualization. For example,ristine multi-walled CNTs (MWCNTs) [11] and single-walled CNTsSWCNTs) [12] have been coupled to conventional organic small-

olecule fluorophores. Unfortunately, the tethered fluorophoresave exhibited low quantum yields and short lifetimes in aqueousnvironment.

∗ Corresponding author. Tel.: +86 731 88872046; fax: +86 731 88872046.E-mail address: tanghao@hunnu.edu.cn (H. Tang).

927-7765/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.colsurfb.2011.05.038

Semiconductor quantum dots (QDs), a new type of fluores-cent labeling material superior to conventional fluorophores inmany respects including narrow emission bandwidth, photochem-ical stability and high quantum yield, have obtained tremendousattentions in high throughput biodetection and cellular imaging[13–16]. Recently, many works have reported the synthesis of CNT-QD nanohybrids through covalent or noncovalent approaches andtheir potential employments as ideal candidates for multifunc-tional architectures [17–22], involving in vivo or intro fluorescentnanoprobes [23–25]. However, some issues are remained in thereported works. It has been reported that the fluorescence emis-sion from QDs is strong suppressed when they are attached toCNTs [26–28], which may be attributed to the charge or energytransfer between CNTs and QDs. On the other hand, the amountof QDs functionalized onto CNT surface is small, usually result-ing in weak fluorescence signal and consequently limiting CNTvisualization in biological environments by optical fluorescencemicroscopy. Although the nondestructive decoration of full-lengthSWCNTs with QDs were accomplished using a surfactant [29] orstreptavidin [30], obtaining CNT-QD nanohybrids with high QDloading is still quite challenging. Moreover, to the best of ourknowledge, cytotoxicity of the CNT-QD hybrids and their fluo-

rescence stable property in living cell system are still not fullyexplored.

Herein, with the above goals, we reported the efficient func-tionalization of CdSe/ZnS QDs on polyamidoamine (PAMAM)

Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 346– 352 347

he pre

dmdodsstobaoas[sotduQct(oti2o

Fig. 1. Schematic demonstrating t

endrimer-modified MWCNTs by covalent linkage and mercapto-ediated assembly (Fig. 1). Due to the chemical inertness of CNTs,

ifferent physical and chemical strategies have been developed inrder to activate the nanotube surface. For instance, the acid oxi-ation of CNTs, resulting in the carboxyl groups at the nanotubeurface, is one of the usually used approaches [31]. However, ituffers from the small amount of functional groups, limiting effec-ive QD-loading on CNTs. PAMAM dendrimers possess high densityf functional groups on periphery, rendering numerous activeinding sites for anchoring the QDs [32,33]. On the other hand, self-ssembly is one of the most important approaches for the synthesisf nanostructures with different chemical composition, shapesnd functionalities. The rational assembly of QDs in layer-by-layertructures were achieved to fabricate so-called “nanorainbows”34] and tubular QD-nanostructures using nanoporous aluminaubstrate [35]. As shown in Fig. 1, by combining the advantagesf PAMAM-modified MWCNTs with mercapto-mediated assembly,he uniform functionalization of CdSe/ZnS QDs on MWCNTs in highensity may be realized, resulting in intense fluorescence signalnder optical fluorescence microscopy. The obtained MWCNT-D hybrids were both photophysically and morphologicallyharacterized by ultraviolet–visible (UV–vis) spectrophotome-ry, fluorescence spectrophotometry, and high resolution TEMHRTEM). Moreover, in order to evaluate the potential utilizationsf the MWCNT-QD hybrids in CNT cellular tracking or imaging,

he cytotoxicity to HeLa cells, fluorescence property and stabilityn living cell system were investigated by 3-(4,5-dimethylthiazol--yl)-2,5-diphenyltetrazolium bromide (MTT) viability assay andptical fluorescence microscopy.

paration of MWCNT-QD hybrids.

2. Materials and methods

2.1. Chemicals and materials

The MWCNTs of over 95 wt.%, 5–15 �m length and 10–20 nmdiameter were purchased from Shenzhen Nano-Technologies PortCo., Ltd. (China). 1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide(EDC, hydrochloride form) and N-hydroxy-succinimide (NHS) wereobtained from Bio Basic Inc. Co., Ltd. Amine group terminatedPAMAM dendrimers (G4.0) and carboxyl group functionalizedCdSe/ZnS core–shell QDs (emission wavelength, 620 nm; 4 �M)were purchased from Sigma–Aldrich and Ocean NanoTech LLC,respectively. HeLa cells were provided by Cancer Research Insti-tute of Xiangya Medical College. RPMI-1640 medium (Gibco),heat-inactivated newborn calf serum (Invitrogen Corporation),and trypsinase (0.25% + 0.02% EDTA), and MTT were purchasedfrom Amresco. Thioglycollic acid (TGA, analytical grade, >90%) wasobtained from Tianjin Guangfu Fine Chemical Research Institute.Phosphate buffered saline (PBS, pH 7.4) solutions were used in theexperiments. All other reagents were of analytical grade or bet-ter. Milli-Q ultrapure water (>18 M� cm, Milli-pore Co., Ltd.) andfreshly prepared solutions were used for all chemical procedures.

2.2. Preparation of MWCNT-QD hybrids

The preparation of MWCNT-QD hybrids is schematically shownin Fig. 1. Firstly, PAMAM dendrimer-functionalized MWCNTs(MWCNT-PAMAM) were prepared by covalent binding betweenamine group terminated PAMAM dendrimers and the carboxylated

348 Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 346– 352

WCNT

Mtatasochf

a2sfsT1tcaT

Fig. 2. HRTEM images of QDs (A–C), M

WCNTs (MWCNT-COOH) using EDC and NHS as coupling reac-ants (Fig. 1A) [36]. The details about preparation of MWCNT-COOHnd MWCNT-PAMAM were presented in Supporting Informa-ion. The MWCNT-QD hybrids were synthesized by two differentpproaches (Fig. 1B and C). N2-saturated solutions or suspen-ions were used throughout in order to prevent the oxidationf CdSe/ZnS QDs [37]. Additionally, all of the synthesis pro-esses were performed at room temperature under stirring andeliophobe environment. The detail experiments are described as

ollows.For the first approach (Fig. 1B), 8 mg EDC and 8 mg NHS were

dded into 0.2 mL of TGA solution and the mixture were reacted for h. Then, 0.2 mL of 0.5 mg mL−1 MWCNT-PAMAM aqueous suspen-ions were added slowly into the above activated TGA solution andollowed by 2-h reaction. TGA was tethered onto MWCNT-PAMAMurface by amidation between TGA and PAMAM dendrimers. TheGA-modified MWCNT-PAMAM were purified by centrifugation at3,000 rpm for 30 min and careful rinse with ultrapure water three

imes, and redispersed in 0.2 mL ultrapure water. The QDs wereoupled to MWCNT-PAMAM-TGA through the mercapto-mediatedssembly between QDs and mercapto-groups of the functionalizedGA. Briefly, 50 �L of 4 �M QDs aqueous solution was added into

-QD-1 (D–F) and MWCNT-QD-2 (G–I).

the above suspension and allowed to react for 2 h. After purificationby centrifugation at 13,000 rpm for 30 min and rinse with ultrapurewater three times, the MWCNT-QD-1 hybrid aqueous suspensions(0.5 mg mL−1) were obtained for future use.

For the second approach (Fig. 1C), 50 �L of 4 �M QDs weremixed with 50 �L of TGA solution for 2 h, and then 8 mg EDCand 8 mg NHS were added, followed by 2-h reaction to obtainactivated TGA and activated TGA-modified QDs. The mixture wasdiluted with ultrapure water to 0.2 mL. Thereafter, 0.2 mL of0.5 mg mL−1 MWCNT-PAMAM were added into the above mixtureand allowed to react for 2 h. The activated TGA and activated TGA-modified QDs were simultaneously grafted onto MWCNT-PAMAMby amidation between TGA and PAMAM dendrimers. This mix-ture was purified by centrifugation and rinse as described aboveand the obtained products were redispersed in 0.2 mL ultrapurewater. QDs were again tethered onto MWCNT-PAMAM through themercapto-mediated assembly between QDs and mercapto-groupsof the functionalized TGA. Briefly, 50 �L of 4 �M QDs were added

into the above suspension and allowed to react for 2 h, followedby centrifugation at 13,000 for 30 min and rinse with ultrapurewater three times. The MWCNT-QD-2 hybrid aqueous suspensions(0.5 mg mL−1) were prepared for future use.

Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 346– 352 349

Fig. 3. UV–vis spectra of QDs (black line) and MWCNT-QD hybrids (red line). Insertpto

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Fig. 4. Photoluminescence spectra of PAMAM (pink line), MWCNT-PAMAM (black

lot: the enlarged UV–vis curves in the range of 425–700 nm. (For interpretation ofhe references to color in this figure legend, the reader is referred to the web versionf the article.)

.3. Incubation of HeLa cells in MWCNT-QD hybrid suspensionsnd MTT assay

All the cell cultures were conducted in a flask in RPMI-1640edium containing 10% heat-inactivated newborn calf serum at

7 ◦C in a humidified atmosphere containing 5% CO2. 30 �L of × 105 cells mL−1 HeLa cells were seeded into 96-well culturelates and allowed to grow over 24 h (the cells reached 70–80%onfluence). The cells were washed with PBS three times. Theedium was then replaced with 0.2 mL fresh RPMI-1640 contain-

ng 10 �g MWCNT-QD hybrids (without heat-inactivated newbornalf serum) and the cells were allowed to grow for 6 h. After 6 h,he cells were washed with PBS and cultured for different time (0,6, 24, 48, and 72 h, respectively) with fresh RPMI-1640 containing0% heat-inactivated newborn calf serum at 37 ◦C in a humidi-ed atmosphere containing 5% CO2. The fluorescence property ofWCNT-QD hybrids that interacted with HeLa cells was investi-

ated by fluorescence microscopy.The metabolic activity of HeLa cells was evaluated by MTT

ssay. When the cells seeded in 96-well culture plates had reached0–80% confluence, the medium was replaced with 0.2 mL freshPMI-1640 containing different amount of MWCNT-QD hybridsnd the cells were allowed to grow over 24 h. Thereafter, HeLaells were washed with PBS and 0.2 mL RPMI-1640 medium and0 �L MTT reagents (5 mg mL−1) were added into each well. Theells were incubated for another 4 h until a purple precipitate wasisible. The medium was removed and 0.1 mL dimethyl sulphox-de was added to completely liberate the crystals. The absorbancet 570 nm was measured [38]. The cells without treatment ofWCNT-QD hybrids were taken as control group and three parallel

amples were carried out in each group. Additionally, the biochem-cal assay of cellular viability was also confirmed by bright-fieldptical microscopy.

.4. Instruments and measurements

The morphologies of QDs and MWCNT-QD hybrids were char-cterized by HRTEM (JEOL 3010; accelerating voltage, 300 kV). For

RTEM sample preparation, one drop of QDs or MWCNT-QD hybridqueous suspension was placed on a lacey support film and thexcess suspension was removed. The samples were used after dry-ng at room temperature. Fourier transform infrared (FTIR) spectra

line), MWCNT-QD hybrids (red line), and free QDs (blue line). (For interpretation ofthe references to color in this figure legend, the reader is referred to the web versionof the article.)

(KBr tablet of the solid material of interest, transmission mode)were recorded on a Nicolet Nexus 670 FTIR spectrophotometer(Nicolet Instrument Co., Madison, WI) with the Ominic software.UV–vis spectra were recorded on a UV-2450 UV-vis spectropho-tometer (Shimadzu Co., Kyoto, Japan). Photoluminescence spectraof the MWCNT-PAMAM, MWCNT-QD hybrids, and free QDs (hav-ing the same concentration of the QDs bound to MWCNT-PAMAMsurface) were collected on F-4500 (Hitachi, Japan). The prolifer-ation morphology of HeLa cells were observed with an invertedoptical microscope (Olympus IM). The fluorescence property ofMWCNT-QD hybrids interacted with HeLa cells was investigatedby fluorescence microscope (Nikon, Eclipse Ti-S) and the obtainedfluorescent images were analyzed using Image-pro Plus 6.0 imageanalysis software.

3. Results and discussion

3.1. Characterization of MWCNT-QD hybrids

Due to the chemical inertness of MWCNTs, it is nec-essary to activate the nanotube surface or introduce activebinding sites for anchoring the QDs. In our experiments,the received MWCNTs were firstly treated with mixed acidsand then PAMAM dendrimers were covalently functional-ized on the carboxylated MWCNTs (Fig. 1A). FTIR spectrumanalysis results have shown the successful coupling of PAMAM toMWCNTs (Supporting information, Fig. S1).

Fig. 2 shows the typical HRTEM images depicting QDs andMWCNT-QD hybrids prepared by the two approaches. QDs of about5 nm diameter and with uniform size distributions are identified(Fig. 2A–C). For MWCNT-QD hybrids, we have found that both pro-posed strategies can realize efficient functionalization of QDs ontoMWCNTs (Fig. 2D–I) and the diameter of MWCNT-QD hybrids isabout 35 nm. The tethered QDs are well-distributed on the singlenanotube surface, avoiding the QD aggregation that results fromthe influence of cross linker [15]. However, it appears that moreamount of QDs have been coupled to MWCNT-PAMAM (Fig. 2G–I)using covalent linkage followed by mercapto-mediated assem-

bly (Fig. 1C) as compared with that using mercapto-mediatedassembly only (Fig. 1B). As described in experimental sections,QDs were tethered onto MWCNT-PAMAM twice (steps (iii) and(iv), Fig. 1C) when preparing MWCNT-QD-2, whereas only once

350 Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 346– 352

resce

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in water have been evaluated and the results (Fig. S4, Support-ing information) show that the homogeneity of MWCNT-QD hybridsuspensions remains within 1 h (Fig. S4A) at room temperature

Fig. 5. Optical bright-field (A) and fluo

or MWCNT-QD-1. Moreover, Photoluminescence spectrum anal-sis results (Supporting information, Fig. S2) revealed that nobvious fluorescence emission was observed in the supernatantfter step (iii), while the supernatant after step (ii) showed sig-ificant fluorescence emission, suggesting the complete reactionetween MWCNT-PAMAM and activated TGA-modified QDs in stepiii). Based on these results, we consider the strategy demon-trated in Fig. 1C has higher QD-tethering efficiency. On thether hand, we found that during the synthesis of MWCNT-QD-1ybrids, some of the intermediate product (MWCNT-PAMAM-TGA)dhered to the inside wall of centrifuge tube, resulting in lowield of MWCNT-QD-1. This phenomenon was not observed dur-ng preparing MWCNT-QD-2, and the possible reason need to beurther studied. It should be noted that TGA-modification is verymportant for the uniform functionalization of QDs on the singleanotube. It was attempted to couple the received carboxylatedDs to MWCNT-PAMAM (without TGA modification) by amidationetween carboxyl group and amine group terminated PAMAM den-rimers using EDC and NHS as cross linker. The HRTEM resultshow that the QDs are not well tethered onto the single nan-tube but form coacervation with MWCNT-PAMAM (Supportingnformation, Fig. S3), which may be due to too small amount of car-oxyl groups on the surface of received QDs and the influence ofross linker during fabrication process. In this paper, if the prepara-ion conditions of MWCNT-QD hybrids are not specially described,he hybrids were synthesized by the covalent linkage followed by

ercapta-mediated assembly demonstrated in Fig. 1C and here-fter the MWCNT-QD hybrids are referred to MWCNT-QD-2.

Fig. 3 shows the typical UV–vis curves of QDs and MWCNT-QDybrids. The UV–vis absorption behavior for MWCNT-QD hybridsred line) revealed significant absorption at 246 nm, which is accor-ant with the characteristic absorption of the CdSe/ZnS QDs (black

ine); whereas the absorption peaks of the hybrids at 490 and40 nm (insert plot of Fig. 3) exhibit about a 80 nm blue shifthen compared with those of the corresponding band gap of bulkdSe/ZnS core–shell QDs (570 and 607 nm), indicating the quan-um confinement effect of the CdSe/ZnS nanocrystal that tetherednto MWCNT-PAMAM surface [39]. These results also confirm thathe CdSe/ZnS core–shell QDs have been functionalized on MWCNT-AMAM surface through the proposed strategy.

The optical properties of MWCNT-QD hybrids were charac-erized by fluorescence spectroscopy and optical fluorescence

icroscopy. As shown in Fig. 4, upon light excitation at 311 nm,here is no observable luminescence peak in the case of MWC-Ts after treatment with PAMAM dendrimers (Fig. 4, black line);

hereas the MWCNT-QD hybrids (Fig. 4, red line) exhibit photo-

uminescence with a maximum emission at 620 nm that similaro the characteristic luminescence peak of CdSe/ZnS core–shellDs (Fig. 4, blue line), which suggests the assembly of QDs onto

nt (B) images of MWCNT-QD hybrids.

MWCNT-PAMAM does not change the fluorescence emission wave-length of QDs and is also further indicative of the quantum sizeeffect [39]. Robel et al. [40] have reported that the luminescenceof CdS quantum dots is totally quenched when they are bound tothe SWCNTs due to the charge or electron transfer to the SWCNTs.In contrast, the MWCNT-QD hybrids show luminescence but a sig-nificantly decreased emission density as compared with that of thefree QDs. A possible pathway for this phenomenon is that the elec-trons of the excitons can be partly transferred to MWCNTs by anelectron-injection mechanism while the reminder of electrons ren-der a reduced emission by an electron–hole recombination process[39–41]. On the other hand, from the view of potential utilizationsof the MWCNT-QD hybrids in CNT cellular tracking or imaging,their fluorescence properties are usually characterized by opticalfluorescence microscopy. The optical fluorescent image (Fig. 5B)indicates that the MWCNT-QD hybrids possess intense red flu-orescence signal under fluorescence microscopy. Moreover, afterstored at 4 ◦C under heliophobe environment for two months, theMWCNT-QD hybrids keep good fluorescence stability in aqueoussuspensions (results not shown).

The dispersive property and stability of the MWCNT-QD hybrids

Fig. 6. Viability of HeLa cells after incubated with MWCNT-QD hybrids at differentconcentrations for 24 h in RPMI-1640 medium at 37 ◦C in a humidified atmospherecontaining 5% CO2. The cellular viability is calculated as a percentage from the via-bility of the control (untreated) cells. The viability of the control cells is considered100%. The results are means ± SD (n = 3).

Y. Zhang et al. / Colloids and Surfaces B: Biointerfaces 87 (2011) 346– 352 351

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ig. 7. Fluorescent images of HeLa cells after incubated with MWCNT-QD hybrids fof HeLa cells after incubated with MWCNT-QD hybrids for 6 h and subsequently all

nd part of the hybrids aggregate after stored 3 h (Fig. S4B). How-ver, it seems no further accumulation in the prolonged time (7.5 h,ig. S4C). Based on these results, it is possible to deduce that theres part aggregation in the MWCNT-QD hybrids while the remain-er has relative good dispersive property in water within 7.5 h. Inuture experiments, the dispersibility can be improved through are-centrifugation of MWCNT-QD suspensions to remove the accu-ulated hybrids [42].

.2. Cytotoxicity of MWCNT-QD hybrids to HeLa cells

CNTs [43–46] and QDs [47] have shown signs of cytotoxicityependent on several factors such as dose, dimension, chemicalunctionalization and physical aspects. Therefore, before develop-ng any biological applications, the cytotoxic effect of MWCNT-QDybrids is an important factor that should be considered. Theytotoxicity of MWCNT-QD hybrids were evaluated by both MTTssay (Fig. 6) and morphological observation of HeLa cells withright-field optical microscopy (Supporting information, Fig. S5).o study the reproducibility of the MTT viability assay, three inde-endent experiments were performed under identical conditions.rror bar in Fig. 6 shows the standard deviation (SD) of the threexperiments. After incubated with MWCNT-QD hybrids over 24 h,he metabolic activity of HeLa cells decrease with the increase ofybrid concentration, exhibiting a dose-dependent cytotoxicity.owever, the cellular viability retains about 84% after incubatedith 100 �g mL−1 MWCNT-QD hybrids, indicating that the hybrids

how a little effect on cellular viability in the studied concen-ration and time range. Additionally, the morphological studiesave confirmed that the number and morphology of HeLa cellsave no obvious change when the hybrid concentration is lower

) and then allowed to grow over 72 h (B). (C) The average integrated optical densityto grow over different time. The results of (C) are means ± SD (n = 3).

than 50 �g mL−1 (Supporting information, Fig. S5B–D), but thecell number slightly decreases at higher concentrations (50 and100 �g mL−1, Supporting information, Fig. S5E and F). These resultssuggest that MWCNT-QD hybrids have good biocompatibility.

3.3. Fluorescence property of MWCNT-QD hybrids in living cellsystem

Besides cytotoxicity, the fluorescence property and stability ofMWCNT-QD hybrids in living cell system are also critical for poten-tial CNT cellular tracking or imaging applications. We probed theseproperties through incubating HeLa cells with the hybrids andthen characterizing by optical fluorescence microscopy (Fig. 7A andB). After incubated with MWCNT-QD hybrids for 6 h, the mediumwas renewed (without the hybrids) and the cells were allowed togrow 0, 16, 24, 48, and 72 h, respectively. One can observe thatlots of MWCNT-QD hybrids bind to the cell surface (Fig. S6, Sup-porting information), showing the hybrids have good affinity withHeLa cells. Moreover, significant red fluorescence are observed forMWCNT-QD hybrids interacted with cells (Fig. 7A), suggesting thehybrids have good fluorescence property in living cell system. Addi-tionally, the fluorescence of MWCNT-QD hybrids seems to have noobvious change when the growing time prolonged to 72 h (Fig. 7B).In order to quantitatively analyze the fluorescence stability ofMWCNT-QD hybrids in living cell system, the obtained fluorescentimages were analyzed by digital image analysis technique [48,49].Fig. 7C shows the dependence of average integrated optical density,

which is related to the fluorescence density of MWCNT-QD hybrids,on the growing time. Three independent experiments were car-ried out under identical conditions to evaluate the reproducibilityof the digital image analysis. Error bars in Fig. 7C show the SD of

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he three experiments. The average integrate optical density at 72 hetains about 80% of that at 0 h, indicating good fluorescence stabil-ty of MWCNT-QD hybrids over a long-time exposure in cell culture

edium. The decrease of MWCNT-QD hybrid fluorescence may bettributed to the effect of cell culture medium and the interaction ofhe hybrids with HeLa cells (adhering to cell surface or penetratingnto cell). It has been reported that the CNT internalization dependsn various factors including the CNT size, surface functional groups,nd dispersive property and so on [50–52]. Therefore, it needs fur-her experiments, for example, optimizing the physical/chemicalharacteristics of the hybrids and recording the confocal images ofreated cells, to investigate the interaction of the hybrids with cellsnd its consequent effect on the fluorescence property.

. Conclusions

We have fabricated highly biocompatible MWCNT-QD hybridsy covalent linkage followed by mercapto-mediated assembly. TheDs were effectively tethered onto MWCNT-PAMAM surface inigh density and well-distributed on the single nanotube sur-

ace. The MWCNT-QD hybrids show a little effect on HeLa celliability over 24-h incubation even at very high concentrations100 �g mL−1). They possess intense red fluorescence signal underuorescence microscopy and good fluorescence stability over 72-

exposure in living cell system. These features suggest that theWCNT-QD hybrids will have potential for CNT cellular tracking

r imaging.

cknowledgments

This work was supported by the National Natural Scienceoundation of China under Grants (20705008, 20905025 and0975037), the Hunan Provincial Natural Science Foundation ofhina (09JJ6026), Aid Program for Science and Technology Inno-ative Research Team in Higher Educational Institutions of Hunanrovince and the Program for Excellent Talents in Hunan Normalniversity (ET20804).

ppendix A. Supplementary data

Supplementary data associated with this article can be found, inhe online version, at doi:10.1016/j.colsurfb.2011.05.038.

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