Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Journal of
Materials Chemistry B
View Article Online

PAPER

View Journal | View Issue

Cite this: J. Mater. Chem. B, 2013, 1,

698

Thioglycerol-capped Mn-doped ZnS quantum dot

bioconjugates as ecient two-photon uorescent
nano-probes for bioimaging
Malgorzata Geszke-Moritz,ab Hanna Piotrowska,c Marek Murias,c Lavinia Balan,d
l Schneider*a
Michal Moritz,b Janina Lulekb and Raphae
Water-dispersible 1-thioglycerol (TG)-capped Mn-doped ZnS quantum dots were prepared in aqueous
solution using the nucleation-doping strategy. Using 4% Mn relative to Zn and a Zn(OAc)2/Na2S ratio of
0.9, Mn:ZnS nanocrystals with an average diameter of 3.9
0.5 nm, with pure Mn2+-related
photoluminescence (PL) at 585 nm, and with a PL quantum yield of 13.2% were obtained. Transmission
electron microscopy, X-ray powder diraction, electron spin resonance, X-ray photoelectron

Received 12th October 2012

Accepted 14th November 2012

spectroscopy, UV-visible spectroscopy and spectrouorometry have been used to characterize the crystal
structure, the doping status, and the optical properties of the doped-dots. Folic acid (FA) was linked to
TG-capped Mn:ZnS nanocrystals to produce Mn:ZnS@TG-FA nanobioconjugates that were used for

DOI: 10.1039/c2tb00247g

targeted in vitro delivery to a human cancer cell line. Folate receptor mediated cellular uptake of FA-

www.rsc.org/MaterialsB

functionalized dots is proven via confocal and two-photon imaging.

Introduction

Semiconductor nanocrystals (quantum dots, QDs) are regarded

as excellent uorescent probes due to the size-dependent
emission wavelengths and large extinction coecient which are
unavailable from conventional organic uorophores.13 In
recent years, the synthesis of binary metal chalcogenide QDs,
especially cadmium chalcogenides CdSe and CdTe, has become
a rapidly growing area in materials chemistry aimed at a
fundamental understanding for practical applications such as
light-emitting devices, lasers, and as biological uorescence
labels.410 Despite the usefulness of Cd-based QDs in numerous
applications, these nanocrystals have an intrinsic limitation;
because the photooxidation of QDs can cause the release of
cadmium and that photoexcited QDs can produce reactive
oxygen species (ROS), a doubt has been shed on the future
applicability of Cd-containing QDs, particularly in view of
environmental regulations.1114

Universite de Lorraine and CNRS, Laboratoire Reactions et Genie des Procedes (UPR
3349), 1 rue Grandville, BP 20451, 54001 Nancy, France. E-mail: raphael.schneider@
univ-lorraine.fr; Tel: +33 3 83 17 50 53
b

Department of Pharmaceutical Technology, Poznan University of Medical Sciences,

Grunwaldzka 6, 60-780 Poznan, Poland

c
Department of Toxicology, Poznan University of Medical Sciences, Dojazd 30 Street,
60-780 Poznan, Poland
d
Institut de Science des Materiaux de Mulhouse LRC 7228, 15 rue Jean Starcky, 68093
Mulhouse, France

Electronic supplementary
10.1039/c2tb00247g

information

698 | J. Mater. Chem. B, 2013, 1, 698706

(ESI)

available.

See

DOI:

In recent developments, doped QDs (d-dots) without heavy

metal ions have been explored as alternative emissive materials
in view of their ecient and stable uorescence from the doped
transition metal ions.1517 Additionally, d-dots exhibit several
advantages over QDs, such as longer luminescence excited state
lifetime, large Stoke shis leading to avoidance of self-absorption, and high thermal and photochemical stabilities. Among
d-dots, Mn-doped nanocrystals, especially Mn-doped ZnS dots,
have been regarded as a promising new class of nanophosphors
and stimulated many eorts of synthesis to investigate
the photoluminescence mechanism of the Mn2+ 4T1 / 6A1
transition.1517
With the development of a new doping strategy by decoupling the doping from nucleation and/or growth through
nucleation-doping or growth-doping approaches,18 many
improvements made in the preparation of high-quality Mndoped ZnS dots have come from organometallic routes.1924
Hydrophobic d-dots thus produced could be made waterdispersible by several methods, most of which rely on the
surface-exchange of hydrophobic ligands for hydrophilic ones.
Direct synthesis of Mn:ZnS d-dots in water is a promising
alternative route to organometallic reactions and facilitates the
use of the dots in biological systems. The aqueous route to
Mn:ZnS dots is however not so advanced that the organometallic one and only a few papers describe the preparation of
good quality Mn:ZnS dots with pure dopant emission, generally
using 3-mercaptopropionic acid as a stabilizer.2528
Herein, we report a simple aqueous synthetic procedure for
Mn:ZnS d-dots using 1-thioglycerol (TG) as a capping ligand. We
found that the use of TG ensured the reproducible access to

This journal is The Royal Society of Chemistry 2013

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Paper
Mn:ZnS nanocrystals with good photoluminescence quantum
yield (up to 13.2%), with pure dopant emission and without
shell introduction. To the best of our knowledge, there is no
report of such good quality Mn:ZnS d-dot preparation in
aqueous solution without shell deposition on the doped core.
Furthermore, we have chosen to decorate the surface of the dots
with a tumor-specic ligand, folic acid (FA), by taking advantage
of the functional polyhydroxyl shell of Mn:ZnS@TG nanocrystals. Folic acid was used as the targeting ligand for this
study because the a-folate receptor (FR) is observed to be upregulated in various types of human cancers, while it is only
minimally distributed in normal tissues.29,30 FA, a high anity
ligand to FR, is internalized into the cell through the receptor
mediated endocytosis even when conjugated with a wide variety
of molecules or nanoparticles.28,31,32 The uptake of FA-conjugated Mn:ZnS d-dots into T47D breast cancer cells was
conrmed by confocal imaging under biphotonic excitation. In
addition, the toxicity of the dots to human model cells was
assessed.

Materials and methods

2.1

Materials

Zinc acetate (Zn(OAc)2$2H2O, 99.999%), zinc nitrate

(Zn(NO3)2$6H2O, 98%), zinc sulfate (ZnSO4$7H2O, >99%),
manganese acetate tetrahydrate (Mn(OAc)2$4H2O, 99%),
Na2S$9H2O (98+%), 1-thioglycerol (TG, >97%), folic acid (FA,
>97%), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, >98%), N-hydroxysuccinimide (NHS, 98%), and
ethanol (HPLC grade) were used as received without additional
purication. All solutions were prepared using Milli-Q water
(18.2 MU cm, Millipore) as the solvent. Sodium borate buer
(0.1 M) was prepared from Na3BO3 and the pH adjusted to 8.8
using 0.1 M H3BO3.
2.2

Synthesis of Mn:ZnS@TG d-dots

The preparation of Mn:ZnS@TG dots was adapted from that

presented by Kim et al.33 Briey, solutions of 1 M
Zn(OAc)2$2H2O (5 mL), 0.1 M Mn(OAc)2$4H2O (1.5 mL) for a
Mn2+ doping of 4 at.%, and 1 M TG (20 mL) were mixed, titrated
to pH 10.3 with 2 M NaOH and saturated with N2 for 30 min. An
aqueous solution of Na2S$9H2O (1 M, 4.5 mL) was then quickly
injected in the reaction ask and the mixture reuxed for 20 h.
Aer cooling to room temperature, the d-dots were separated
from the aqueous solution by the addition of ethanol and by
centrifugation. Aer washing with ethanol and drying in a
vacuum at room temperature, the nanocrystals could be redispersed in water.
2.3

Conjugation of Mn:ZnS@TG d-dots with folic acid

TG-capped Mn:ZnS dots (5 mg) were dispersed in 6 mL of borate

buer to which 160 mL solution of FA in borate buer
(1.025 mmol, 0.45 mg), 160 mL solution of EDC in borate buer
(2.05 mmol, 0.38 mg) and 160 mL of a NHS solution in borate
buer (2.05 mmol, 0.24 mg) were added. The resulting solution
was then allowed to stir for 3 h under an inert atmosphere and

This journal is The Royal Society of Chemistry 2013

Journal of Materials Chemistry B

in the dark. Folic acid functionalized d-dots were precipitated
by adding ethanol to the reaction mixture, washed twice with
ethanol and dried in a vacuum.
2.4

Characterization of d-dots

Transmission electron microscopy (TEM) images were taken by

placing a drop of the particles in water onto a carbon lmsupported copper grid. Samples were studied using a Philips
CM20 instrument operating at 200 kV equipped with Energy
Dispersive X-ray Spectrometer (EDS). The X-ray powder diraction data were collected from an X'Pert MPD diractometer
(Panalytical AXS) with a goniometer radius 240 mm, xed
divergence slit module (1/2 divergence slit, 0.04 rd Sollers slits)
and an X'Celerator as a detector. The powder samples were
placed on zerobackground quartz sample holders and the XRD
patterns were recorded at room temperature using Cu Ka radiation (l 0.15418 nm). The average particle size was calculated
from line broadening using the Topas application (Bruker XAS).
We used the Fundamental Parameters (FP) approach34 and the
instrumental broadening was determined using standard LaB6
powder. A VARIAN 720-ES Inductively Coupled Plasma-Optical
Emission Spectrometer (ICP-OES) was used for multielemental
analyses. XPS measurements were performed at a residual
pressure of 109 mbar, using a KRATOS Axis Ultra electron
energy analyser operating with an Al Ka monochromatic source.
Absorption spectra were recorded on a Perkin-Elmer
(Lambda 2) UV-Visible spectrophotometer. Fluorescence
spectra were recorded on a uorolog-3 spectrouorimeter F222
(Jobin Yvon) using a 450 W xenon lamp. The quantum yield (QY)
values were determined from the following equation:
!





Fsample
Aref
nsample 2
QYsample
QYref
(1)
Fref
Asample
nref 2
where F, A and n are the measured uorescence (area under the
emission peak), absorbance at the excitation wavelength and
refractive index of the solvent respectively. PL spectra were
spectrally corrected and quantum yields were determined relative to Rhodamine 6G in water (QY 94%).35 Electron spin
resonance (ESR) experiments were carried out using a X-Band
EMX-plus spectrometer (Bruker Biospin). The samples were
investigated at room temperature. 2,2,6,6-Tetramethylpiperidine-1-oxyl (TEMPO) was used as standard for calibration (g
2.0061).
2.5

Cell cultures

T47D breast cancer cells were purchased from the European

Collection of Cell Cultures (ECACC, Porton Down Salisbury UK).
Cells were grown in phenol red-free DMEM (Dulbecco's modied Eagle's medium) tissue culture medium, supplemented
with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin under standard conditions at 37  C and humidied
atmosphere containing 5% CO2 and 95% air. For experiment,
logarithmically growing cells (2  104 cells) were subcultured in
96-well microtiter plates in a volume of 200 mL. Cells were
allowed to attach overnight and were then exposed to various

J. Mater. Chem. B, 2013, 1, 698706 | 699

View Article Online

Journal of Materials Chemistry B

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

concentrations of dots in dierent tests. All the materials used

in cell culture experiments were from Sigma-Aldrich, St Louis,
MO unless otherwise stated.
2.6

Confocal uorescent microscopy studies

Subconuent stock cultures of T47D cells were trypsinized and

seeded in 8-well chamber slides at a density of 4  104 cells per
well in growth medium. Cells were allowed to attach overnight
before treating with Mn:ZnS d-dots. Then, the supernatant was
discarded, followed by replacement of media supplemented
with 10% FBS containing Mn:ZnS d-dots (50 mM). Aer 24 h of
incubation at 37  C, the incubation medium was removed, and
mixed solution of Mitochondria Staining Kit and Hoechst 33342
was added. Hoechst 33342 is used as DNA stain while Mitochondria Staining Mix is used for detection of changes in the
mitochondrial inner-membrane electrochemical potential. The
Mitochondrial Staining Kit contains 5,50 ,6,60 -tetrachloro1,10 ,3,30 -tetraethylbenzimidazolocarbocyanine iodide (JC-1),
DMSO, JC-1 Staining Buer and Valinomycin Ready Made
Solution and was prepared according to the procedure provided
by the producer. The mixed solution of two dyes was a combination of 7.5 mL of Hoechst 33342 stock solution (1.5 mg of
Hoechst 33342 in 1.5 mL of ultra-pure water) and 1.5 mL of
Mitochondria Staining Mix. Aer 10 min of incubation, dye
solution was discarded, the cells were washed with culture
medium and xed using 4% formaldehyde (500 mL per well).
Then the cells were washed twice with PBS and mounted with
the mounting medium (Dako Faramount Aqueous Mounting
Medium). Mn:ZnS d-dots and Hoechst-labeled cells were
imaged on a Leica TCS SP5 confocal microscope equipped with
a Ti:Sapphire MaiTai biphotonic laser (Spectra Physics) and a
HCX PL APO CS 63 1.40 OIL objective. An argon laser was used
for JC-1-labeled cells. Fluorescence of d-dots and Hoechst 33342
was detected using excitation 800 nm (eective excitation
400 nm) and emission lters 500650 nm and 400500 nm,
respectively. Fluorescence of JC-1 was detected using excitation
488 nm and emission lters 500550 nm.
2.7

In vitro cytotoxicity

Dose dependent cytotoxicity eects of Mn:ZnS d-dots were

evaluated by using standard MTT and XTT assays.
MTT assay: The MTT assay is a colorimetric assay based on
the ability of viable cells to reduce a soluble yellow tetrazolium
salt,
(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide) (MTT), to blue formazan crystals. MTT assay was
employed to assess the cells viability aer treatment with ddots. Following the incubation of cells with dierent types and
concentrations of dots, the supernatant was removed and 200
mL of MTT solution (0.5 mg mL1 in serum-free DMEM) was
added. Aer 4 h, the medium was aspirated and the precipitated
formazan was dissolved in 200 mL of dimethylsulfoxide (DMSO).
Cell viability was determined by measuring the absorbance at
570 nm using a Biotek microplate reader (Biotek Instruments
USA). In this study, the nanocrystal concentration required to
inhibit cell growth by 50% (IC50) was determined from a plot of
percent cell viability of control untreated cells versus logarithm

700 | J. Mater. Chem. B, 2013, 1, 698706

Paper
of concentration. The experimental plate included wells without
cells (in order to exclude direct reaction between MTT and ddots), wells with cells treated with d-dots and wells of untreated
cells. Each treatment was performed in triplicate.
XTT assay: an XTT cell proliferation assay kit was purchased
from Cayman Chemical and used to study cell proliferation
according to the manufacturer's protocol. The assay is based on
the extracellular reduction of 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium hydroxide
(XTT) by a reduced form of nicotinamide adenine dinucleotide
(NADH) produced in the mitochondria via trans-plasma
membrane electron transport and an electron mediator.
Reduction of XTT produces a water-soluble formazan which
dissolves directly into the culture medium, eliminating the
need for an additional solubilization step. Immediately before
use the Electron Mediator Solution was used to reconstitute the
entire vial of XTT Reagent and mixed well.
The experimental plate included wells without cells (in order
to exclude direct reaction between XTT and d-dots), wells with
cells treated with d-dots and wells of untreated cells. Each
treatment was performed in triplicate.
T47D human breast cancer cells were seeded in 96-well
microtiter plates at a density of 2  104 cells per well and led to
growth for 24 h in a CO2 atmosphere at 37  C. Aer this time
dierent concentrations (0.085 mM) of Mn:ZnS@TG and
Mn:ZnS@TG-FA d-dots in culture medium were added. Aer
72 h, 10 mL of the reconstituted XTT mixture to each well were
added. The microtiter plates were gently shaken and then
incubated for two hours at 37  C in a CO2 atmosphere. The
plates were gently shaken for 1 minute and absorption of each
well was measured using a microplate reader at a wavelength of
450 nm.

Results and discussion

3.1

PL properties of Mn:ZnS d-dots

We rst studied the synthesis and the optical properties of Mndoped TG-capped ZnS nanocrystals. The synthetic conditions
were found to play a critical role in determining the PL properties of the resulting Mn:ZnS@TG d-dots. First, the reaction
temperature for the synthesis inuences the PL QY of the d-dots
markedly. The experimental results showed that to obtain
nanocrystals with good optical properties, the optimal reaction
temperature was 100  C. If the temperature was too low (60 or 80

C), the PL intensity (normalized to the same absorption at the
excitation wavelength) of the Mn2+ emission at ca. 585 nm of the
resulting d-dots was signicantly lower than that obtained at
100  C. Too high temperature (130 or 150  C), obtained by
heating the reaction mixture in a sealed Teon tube, led to the
formation of poorly water-dispersible and large-sized nanocrystals exhibiting only a surface defect emission located at ca.
450 nm. In our case, this situation probably originates from
partial decomposition of TG at high temperature leading to
dangling bond states on the d-dots surface due to incomplete
surface passivation by the ligand.36,37
Initial experiments performed with 4% Mn2+ relative to Zn2+
and a ca. 0.19 M Zn2+ solution showed that good quality d-dots

This journal is The Royal Society of Chemistry 2013

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Paper
were obtained when using Na2S in short supply (by ca. 0.9 fold)
relative to Zn2+. A decrease or an increase of the Zn2+/S2 ratio
yielded Mn-doped nanocrystals with poor PL eciency
(increase of the blue emission located at ca. 450 nm at the
expense of the orange emission originating from the 4T1 / 6A1
transition within the 3d shell of the Mn2+ ion). Aer injection of
Na2S, a strong orange emission evolved over a matter of hours,
reached its maximum intensity aer ca. 20 h of heating at
100  C, and remained stable if the heating time was
increased (Fig. 1a).
As shown in Fig. 1b, the Zn2+ precursor also has an inuence
on the optical properties of the Mn:ZnS@TG d-dots. The highest
PL QY (13.2%) was obtained with Zn(OAc)2. The use of ZnSO4 or
Zn(NO3)2 yielded d-dots with lower PL QYs, 11 and 2.5%,
respectively. It should also be pointed out that the purity and
the crystallinity of the d-dots prepared from Zn(OAc)2 were
higher than those prepared from ZnSO4 or Zn(NO3)2 as shown
by X-ray diraction (XRD) studies (data not shown). Finally, it
can be observed from Fig. 1b that when varying Zn2+ precursors
from Zn(OAc)2 to ZnSO4 and Zn(NO3)2, 4T1 to 6A1 luminescence
of Mn2+ ions shis from 585 to 590 and 593 nm, respectively.
Due to the limited degree of doping in Mn:ZnS@TG nanocrystals, MnMn interactions should be weak and therefore PL
emission shis should not be dependent on the Mn2+ concentration.38,39 It is more likely that the variations of the PL emission wavelengths observed in Fig. 1b are related to the
distribution of Mn2+ ions in the dots. Indeed, for the nucleation
or growth doping strategy, lattice diusion on Mn2+ dopants
plays an important role in PL intensity and PL emission wavelengths.40 This diusion into the host layer generates isolated
dopant centers within the ZnS host lattice, which could yield
highly emissive d-dots and seems to increase under our
synthetic conditions using Zn(OAc)2 compared to ZnSO4 and
Zn(NO3)2.
An investigation of the inuence of the Mn2+ concentration
on the PL eciency of Mn:ZnS@TG dots was also carried out.
Indeed, when the concentration of Mn2+ dopant ions exceeds a
certain threshold, the non-radiative energy transfers between
neighboring Mn2+ dopant ions can reduce and even annihilate
the PL.4143 A blank sample prepared without Mn2+ showed only
the ZnS-related trap emission at ca. 450 nm. As soon as Mn2+ is
incorporated in the ZnS nanocrystals, the intensity of the blue
emission decreased and Mn2+ emission at 585 nm comes up,
thus indicating that the energy transfers between the ZnS host
and the Mn2+ dopant is eective. With increasing the Mn/Zn
ratio from 2 to 4%, the PL QY gradually increased from 5.2 to
13.2% (Fig. 1c). Interestingly, a slight blue shi of the Mn2+
emission from 595 to 590 nm was observed during these variations of the Mn/Zn ratio. This blue shi may be ascribed to the
decrease of the symmetry of the crystal eld around the Mn2+
center in the ZnS host.18,40 Finally, above a Mn/Zn ratio of 4%, a
decrease of the PL intensity was observed probably due to the
formation of pairs of Mn2+ dopant ions.
Fig. 1d shows absorption, PL excitation, and PL emission of
Mn:ZnS@TG d-dots. A strong 585 nm ion PL band with a fullwidth at half-maximum of ca. 60 nm and a large Stokes shi of
ca. 270 nm arising from 4T1 / 6A1 transition from Mn2+ in the
This journal is The Royal Society of Chemistry 2013

Journal of Materials Chemistry B

Fig. 1 (a) Evolution of PL emission spectra during the growth of Mn:ZnS

nanocrystals, (b) inuence of the Zn2+ precursor on PL intensity, (c) inuence of
Mn2+ percentage on PL intensity, and (d) absorption (black), PL excitation (red)
and PL emission (blue) spectra of Mn:ZnS@TG d-dots.

ZnS lattice can be observed. The PLE spectrum of the orange

emission in the Mn:ZnS@TG d-dots is consistent with the
absorption onset. Finally, the dots showed nearly 100% pure

J. Mater. Chem. B, 2013, 1, 698706 | 701

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Journal of Materials Chemistry B

Fig. 2

Paper

A schematic illustration of the synthesis of folic acid-conjugated Mn:ZnS d-dots.

Fig. 3 Absorption (black), PL excitation (red), and PL emission spectra of FAconjuagted Mn:ZnS d-dots.

acidic pH environments. When the dots are dispersed in

ultrapure water, the pH of the resulting solution is about 7. The
highest PL intensity was observed at pH 11. From pH 11 to 7,
the PL intensity varies by less than 15%, and also remains stable
with time (over 7 days). A strong decrease of PL intensity was
observed below pH 6, the d-dots became unstable in the acidic
aqueous environment, they precipitated and the PL disappeared. A similar phenomenon was observed above pH 13.
The addition of EDC and NHS to FA results in the formation
of a highly reactive intermediate (NHS-folate). This activated
ester then reacted with hydroxyl groups present at the surface of
Mn:ZnS@TG QDs to give nanoconjugates of FA with the d-dots
as illustrated in Fig. 2.
Surface functionalization with FA did not aect UV-vis and
PL properties of Mn:ZnS@TG dots, the PL emission maximum
being located at ca. 585 nm (Fig. 3). According to our previous
reports,28,45 we observed a quenching of d-dots uorescence
when too high amounts of FA were used during the coupling
(ESI, Fig. S2). This probably originates from partial absorption
of the excitation light by FA (FA absorbs between 230 and
410 nm) and/or energy transfer between the d-dots and FA.45
Trials at dierent concentrations were conducted and we found
that using dilute solutions of FA (1.025 mM) and a Mn:ZnS dot/
FA ratio of 50/1 did not markedly alter the PL QY. The amount of
FA coupled to the dots using this ratio was sucient to induce
specic cell recognition (vide infra).
3.2

Fig. 4

ESR spectrum of Mn-doped ZnS nanocrystals (doping level of 4%).

dopant emission, indicating ecient energy transfer from the

ZnS host to the Mn2+ ions, owing to the strong coupling between
3d5 electrons of Mn2+ ions and s-p electrons of the ZnS host.42,44
The optical properties of Mn:ZnS@TG dots under various
biologically relevant conditions of pH were also examined.
Fig. S1 in the ESI shows the variation of PL intensity in basic-to-

702 | J. Mater. Chem. B, 2013, 1, 698706

Size, shape, and structure of the d-dots

To further elucidate the doping status of Mn2+ in Mn:ZnS@TG

d-dots, electron spin resonance (ESR) and X-ray photoelectron
spectroscopy (XPS) were performed. First, the ESR measurement was carried out to reveal the local environment of Mn2+ in
the TG-capped ZnS nanocrystals. A broad curve and six lines of
sharp splitting can be observed for Mn:ZnS@TG dots
(Fig. 4).46,47 The broad curve originates from the electron spin
spin interactions (+1/2 / 1/2) of the isolated Mn2+ ions in the
doped nanocrystals, which conrms that Mn2+ ions have
entered the ZnS host lattices and are homogeneously distributed near the core of the dots. The six lines show electron spin
nuclear interactions (I 5/2). The hyperne coupling constant
A 69.0 G from Mn:ZnS@TG d-dots agrees well with the
hyperne coupling constant from Mn at cubic ZnS lattice sites

This journal is The Royal Society of Chemistry 2013

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Paper

Journal of Materials Chemistry B

Fig. 5 TEM images of (a) Mn:ZnS@TG and (b) Mn:ZnS@TG-FA d-dots; (c) and (d) are the corresponding size distributions, and (e) powder X-ray diraction patterns of
the nanocrystals.

in bulk ZnS (A 69.6 G).48 ESR results explicitly evidence the

doping of Mn2+ in ZnS@TG d-dots.
XPS data are shown in the ESI (Fig. S3S6). The Zn 2p3/2, S
2p, O 1s, C 1s, and Na 1s photoelectron peaks were detected at
1019.5, 162.0, 531.8, 284.6, and 1069.6 eV, respectively, from the
Mn:ZnS@TG core. Since XPS is a surface-sensitive method of
analysis, the XPS spectra are dominated by the shell. The Mn2+
peak was not detected at the surface of the dots presumably
because Mn2+ atoms are buried by the ZnS host material. This
result shows also that Mn2+ ions are located near the core and
not at the periphery of Mn:ZnS nanocrystals. Aer surface
modication, the XPS spectrum also showed the signal of N 1s
at 399.1 eV, which indicates that FA was successfully anchored
at the periphery of the dots. The atomic percentage of Mn
relative to Zn in the Mn:ZnS@TG was determined by Inductively
Coupled Plasma-Optical Emission Spectrometer (ICP-OES). The
Mn/Zn atomic ratio was found to be 3.17, while the theoretical

value was 4.0. Since Mn:ZnS@TG has an average diameter of ca.

3.9 nm (vide infra), which represents ca. 198 ZnS structural units
in each nanocrystal (using a theoretical lattice parameter a of
0.5386 nm, JCPDS no. 05-0566), the number of Mn2+ ions per
nanocrystal was calculated to be 25.
The successful conjugation of FA to Mn:ZnS@TG d-dots was
further conrmed by FT-IR (see ESI, Fig. S7). The FT-IR spectrum of Mn:ZnS@TG-FA nanocrystals exhibits numerous bands
between 1695 and 1450 cm1 originating from amide C]O
stretching vibration, C]C bond stretching of the pteridine and
of p-aminobenzoic acid moieties, and from NH bending
vibrations of FA, while all these signals were absent in the
starting Mn:ZnS d-dots.
The morphology of the as-synthesized nanocrystals was
characterized by transmission electron microscopy (TEM). TEM
images and the corresponding size distributions indicate that
the average diameters of Mn:ZnS@TG and Mn:ZnS@TG-FA

Fig. 6 Confocal uorescence of T47D cells treated with (a) JC1, (b) Hoechst 33342, and (c) Mn:ZnS@TG-FA nanocrystals, (d) is the overlay of uorescence images. Scale
bars 25 mm. Confocal microscopy images (b) and (c) were obtained with laser excitation at 800 nm, while image (a) was recorded after excitation at 488 nm.

This journal is The Royal Society of Chemistry 2013

J. Mater. Chem. B, 2013, 1, 698706 | 703

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Journal of Materials Chemistry B

Paper
Mn:ZnS@TG d-dots aer biphotonic excitation. Fig. 6a and b
show the Hoechst and JC1 uorescence in the cells. The same
eld visualized showed internalized orange emission from FAconjugated d-dots (Fig. 6c). When the uorescence pattern of
the FA-conjugated dots was merged with the staining pattern of
Hoechst and JC1, it was evident that Mn:ZnS@TG-FA d-dots
appear to accumulate in the cytoplasm within the cells, thus
suggesting endocytotic uptake (Fig. 6d). To prove that
Mn:ZnS@TG-FA dots are specically targeted through the FA
FRa interaction, two control experiments were conducted. First,
cells were uncubated with unconjugated dots (see ESI, Fig. S9).
The second control experiment involved saturating the cells
with free FA for 2 hours in order to block the available FR on the
cell surface, followed by treatment with Mn:ZnS@TG-FA dots
(see ESI, Fig. S10). In both cases, minimal signal from the cells
was observed, thus conrming that the uptake of the dots
occurred predominantly via the FAFR interaction.
3.4

Fig. 7 Eect of d-dots concentration on the viability of T47D cells evaluated via
MTT assay. (a) Mn:ZnS@TG nanocrystals and (b) Mn:ZnS@TG-FA nanocrystals.

d-dots were 3.9
0.5 and 4.2
0.7 nm, respectively (Fig. 5ad).
The corresponding selected area electron diraction patterns
(insets of Fig. 5a and b) can be indexed to a zinc blende cubic
phase, this is in accordance with the results of powder X-ray
diraction (XRD) patterns. XRD spectra recorded on
Mn:ZnS@TG and on Mn:ZnS@TG-FA powders recorded aer
centrifugation and drying are shown in Fig. 5e. The diraction
peaks of both samples perfectly match the (111), (220), and
(311) crystalline planes of the cubic ZnS phase with very
broadened peaks indicating the formation of very small nanocrystals (JCPDS no. 05-0566). No characteristic peaks of any
foreign phase or manganese impurities were detected. In
addition, we can see that doping Mn2+ into the host ZnS
nanocrystals does not bring about a phase transformation on
the crystal structure.
3.3

Evaluation of cytotoxicity

Dose dependent cytotoxicity eects of the dots prepared were

rst evaluated by using standard 3-(4,5-dimethylthiazol-2-yl)2,5-diphenyl tetrazolium bromide (MTT) assays. The cell
viability (%) related to controls containing cell culture medium
without d-dots was calculated by test/control  100. Fig. 7
shows the cytotoxicity eects of Mn:ZnS@TG and of
Mn:ZnS@TG-FA dots on T47D cells. Increasing the concentration of Mn:ZnS@TG nanocrystals up to 2.5 mM caused no
signicant increase of toxicity, and the cell viability was ca. 80
90%. As demonstrated in the previous paragraph, folic acid

Fluorescence cell imaging

For in vitro cell-labelling studies with Mn:ZnS@TG-FA QDs, we

used T47D human breast cancer cells in culture medium. The
cells were incubated with FA-modied d-dots at a nal
concentration of 50 mM for two hours at 37  C. Aer two hours,
the cells were washed twice with PBS and subsequently stained
with JC1 and Hoechst 33342 to visualize mitochondria and
DNA, respectively. The cells were next imaged using a laser
scanning confocal microscope under biphotonic excitation
(excitation wavelength of 800 nm). This imaging technique
provides the possibility of long-term imaging of cellular
processes with reduced photodamage compared to UV-excited
imaging. Fig. S8 shows the PL emission spectrum of

704 | J. Mater. Chem. B, 2013, 1, 698706

Fig. 8 Eect of d-dots concentration on the viability of T47D cells evaluated via
XTT assay. (a) Mn:ZnS@TG nanocrystals and (b) Mn:ZnS@TG-FA nanocrystals.

This journal is The Royal Society of Chemistry 2013

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Paper
modication of Mn:ZnS dots was found to increase the uptake
of these nanocrystals by cells that overexpressed the a-folate
receptor. As seen in Fig. 7b, FA-capped dots are more toxic than
the uncapped dots at all dosages probably due to higher intracellular concentrations of these nanocrystals (by ca. 45 fold).
IC50 value determined for Mn:ZnS@TG dots was higher than
5000 mM, while that of Mn:ZnS@TG-FA d-dots was lower.
XTT
(2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxanilide) assays were also conducted to measure
the mitochondria activity and quantify the cell death aer
exposure of T47D cells to Mn:ZnS dots (Fig. 8). As previously
observed in the MTT assays, the toxicity was increased for the
FA-modied dots (Fig. 8b), which correlated with the aforementioned enhanced cellular uptake.
Finally, MTT and XTT assays of cell viability studies show
that Mn:ZnS d-dots demonstrate low cytotoxicity and can be
used at a high concentration for imaging or other biomedical
applications.

Conclusion

In summary, we report herein the rst aqueous route to good

quality TG-capped Mn-doped ZnS nanocrystals using the
nucleation-doping strategy. XRD and TEM analyses indicate
that Mn:ZnS@TG dots prepared by this method are quasiisotropic in shape, almost uniform in size with an average
diameter 3.9
0.5 nm, and have the cubic zinc blende structure. By rationally tailoring the experimental parameters,
Mn:ZnS d-dots with a PL QY of 13.2% and with pure dopant
emission located at ca. 585 nm could be obtained. TG-capped
Mn:ZnS nanocrystals can be readily conjugated with folic acid
for specic targeted bioimaging. The folate-receptor mediated
uptake of Mn:ZnS@TF-FA d-dots into T47D cancer cells was
demonstrated by presaturation of the cells with free folic acid.
Finally, the in vitro cytotoxicity results indicate that TG-capped
Mn:ZnS dots are a very low cytotoxic material that should oer
promising opportunities for various biological applications like
bimodal imaging and targeted drug delivery.

Notes and references

1 Y. Yin and A. P. Alivisatos, Nature, 2005, 437, 664.
2 A. M. Smith and S. M. Nie, Acc. Chem. Res., 2010, 43, 190.
3 B. N. G. Giepmans, S. R. Adams, M. H. Ellisman and
R. Y. Tsien, Science, 2006, 312, 217.
4 R. C. Somers, M. G. Bawendi and D. G. Nocera, Chem. Soc.
Rev., 2007, 36, 579.
5 I. L. Medintz, H. T. Uyeda, E. R. Goldman and H. Mattoussi,
Nat. Mater., 2005, 4, 435.
6 C. Burda, X. Chen, R. Narayanan and M. A. El-Sayed, Chem.
Soc. Rev., 2005, 105, 1025.
7 M. D. Regulacio and M. Y. Han, Acc. Chem. Res., 2010, 43,
621.
8 V. I. Klimov, A. A. Mikhailovsky, S. Xu, A. Malko,
J. A. Hollingworth, C. A. Leatherdale, H. J. Eisler and
M. G. Bawendi, Science, 2000, 290, 314.

This journal is The Royal Society of Chemistry 2013

Journal of Materials Chemistry B

9 R.-O. Moussodia, L. Balan, C. Merlin, C. Mustin and
R. Schneider, J. Mater. Chem., 2010, 20, 1147.
10 F. Aldeek, C. Mustin, L. Balan, T. Roques-Carmes,
M.-P. Fontaine-Aupart and R. Schneider, Biomaterials,
2011, 32, 5459.
11 R. Schneider, C. Wolpert, H. Guilloteau, L. Balan, J. Lambert
and C. Merlin, Nanotechnology, 2009, 20, 225101.
12 E. Dumas, C. Gao, D. Suern, S. E. Bradforth,
N. M. Dimitrijevic and J. L. Nadeau, Environ. Sci. Technol.,
2010, 44, 1464.
13 A. Aboulaich, C.-M. Tilmaciu, C. Merlin, C. Mercier,
H. Guilloteau, G. Medjahdi and R. Schneider,
Nanotechnology, 2012, 23, 335101.
14 D. R. Cooper, N. M. Dimitrijevic and J. L. Nadeau, Nanoscale,
2010, 2, 114.
15 S. C. Erwin, L. Zu, M. I. Hael, A. L. Efros, T. A. Kennedy and
D. J. Norris, Nature, 2005, 436, 91.
16 Y. Yan, O. Chen, A. Angerhofer and Y. C. Cao, J. Am. Chem.
Soc., 2006, 128, 12428.
17 D. J. Norris, A. L. Efros and S. C. Erwin, Science, 2008, 319,
1776.
18 N. Pradhan, D. Goorsky, J. Thessing and X. G. Peng, J. Am.
Chem. Soc., 2005, 127, 17586.
19 Z. Quan, Z. Wang, P. Yang, J. Lin and I. Fang, Inorg. Chem.,
2007, 46, 1354.
20 B. B. Srivastava, S. Jana, N. S. Karan, S. Paria, N. R. Jana,
D. D. Sarma and N. Pradham, J. Phys. Chem. Lett., 2010, 1,
1454.
21 O. Ehlert, W. B
ucking, J. Riegler, A. Markulov and T. Nann,
Microchim. Acta, 2008, 160, 351.
22 J. Zheng, W. Ji, X. Wang, M. Ikazawa, P. Jing, X. Liu, H. Li,
J. Zhao and Y. Matsumoto, J. Phys. Chem. C, 2010, 114, 15331.
23 W. Zhang, Y. Li, H. Zhang, X. Zhou and Y. Zhong, Inorg.
Chem., 2011, 50, 10432.
24 Z. Deng, L. Tong, M. Flores, S. Liu, J.-X. Cheng, H. Yan and
Y. Liu, J. Am. Chem. Soc., 2011, 133, 5389.
25 I. Q. Zhuang, X. D. Zhang, G. Wang, D. M. Li, W. S. Yang and
T. J. Li, J. Mater. Chem., 2003, 13, 1853.
26 W. P. Jian, J. Q. Zhuang, W. S. Yang and Y. B. Bai, J. Lumin.,
2007, 126, 735.
27 H.-B. Ren, B.-Y. Wu, J.-T. Chen and X.-P. Yan, Anal. Chem.,
2011, 83, 8239.
28 M. Geszke, M. Murias, L. Balan, G. Medjahdi, J. Korczynski,
M. Moritz, J. Lulek and R. Schneider, Acta Biomater., 2011, 7,
1327.
29 J. Sudimack and R. J. Lee, Adv. Drug Delivery Rev., 2000, 41,
147.
30 Y. J. Lu and P. S. Low, Adv. Drug Delivery Rev., 2002, 54, 675.
31 Y. J. Lu, E. Sega, C. P. Leamon and P. S. Low, Adv. Drug
Delivery Rev., 2004, 56, 1161.
32 J. Gravier, R. Schneider, C. Frochot, T. Bastogne, F. Schmitt,
J. Didelon, F. Guillemin and M. Barberi-Heyob, J. Med.
Chem., 2008, 51, 3867.
33 E. J. Kim, C.-S. Hwang and S. Yoon, Bull. Korean Chem. Soc.,
2008, 29, 1247.
34 R. W. Cheary, A. A. Coelho and J. P. Cline, J. Res. Natl. Stand.
Technol., 2004, 109, 1.

J. Mater. Chem. B, 2013, 1, 698706 | 705

View Article Online

Published on 15 November 2012. Downloaded by UNIVERSID FEDERAL DE UBERLANDIA on 30/05/2014 15:01:28.

Journal of Materials Chemistry B

35 M. Grabolle, M. Spiles, V. Lesnyak, N. Gaponik,
A. Eychm
uller and U. Resch-Genger, Anal. Chem., 2009, 81,
6285.
36 B. H. Dong, L. X. Cao, G. Su, W. Liu, H. Qu and H. Zhai,
J. Alloys Compd., 2010, 492, 363.
37 B. Dong, L. Cao, G. Su and W. Liu, J. Colloid Interface Sci.,
2012, 367, 178.
38 H. Yang, P. H. Holloway and B. B. Ratua, J. Appl. Phys., 2003,
93, 586.
39 W. Chen, R. Sammynaiken, R. Wallenberg and J. Bovin,
J. Appl. Phys., 2001, 89, 1120.
40 N. Pradhan and X. G. Peng, J. Am. Chem. Soc., 2007, 129,
3339.
41 A. A. Khosravi, M. Kundu, B. A. Kuruvilla, G. S. Shekhawat,
R. P. Gupta, A. K. Sharma, P. D. Vyas and S. K. Kulkarni,
Appl. Phys. Lett., 1995, 67, 2506.

706 | J. Mater. Chem. B, 2013, 1, 698706

Paper
42 K. Sooklal, B. S. Cullum, S. M. Angel and C. J. Murphy, J.
Phys. Chem., 1996, 100, 4551.
43 A. A. Bol and A. Meijerink, J. Phys. Chem. B, 2001, 105, 10197.
44 J. J. Zheng, X. Yuan, M. Ikezawa, P. T. Jing, X. Y. Liu,
Z. H. Zheng, X. G. Kong, J. L. Zhao and Y. Masumoto,
J. Phys. Chem. C, 2009, 113, 16969.
45 M. Geszke-Moritz, G. Clavier, J. Lulek and R. Schneider,
J. Lumin., 2012, 132, 987.
46 P. H. Borse, D. Srinivas, R. F. Shinde, S. K. Date, W. Vogel
and S. K. Kulkarni, Phys. Rev. B: Condens. Matter Mater.
Phys., 1999, 60, 8659.
47 F. V. Mikulec, M. Kuno, M. Bennati, D. A. Hall, R. G. Grin
and M. G. Bawendi, J. Am. Chem. Soc., 2000, 122, 2532.
48 J. J. Li, Y. A. Wang, W. Z. Guo, J. C. Keay, T. D. Mishima,
M. B. Johnson and X. G. Peng, J. Am. Chem. Soc., 2003, 125,
12567.

This journal is The Royal Society of Chemistry 2013