Functionalized Fe3O4 nanoparticles for detecting zinc ions in living cells and their cytotoxicity

Article abstract of DOI:10.1002/chem.201200294

The zinc tank: A new fluoro-chromogenic chemosensor based on BODIPY-functionalized Fe₃O₄ nanoparticles (1) has been prepared. Chemoprobe 1 exhibits high selectivity for Zn²⁺ over other competing metal ions tested. Moreover, confocal microscopy experiments established that 1 can be used for detecting Zn²⁺ levels in living cells (see figure). Copyright

Full text of DOI:10.1002/chem.201200294

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DOI: 10.1002/chem.201200294
Functionalized Fe3O4 Nanoparticles for Detecting Zinc Ions in Living Cells
and Their Cytotoxicity
Gyusik Kang,[a] Hyunjong Son,[a] Jung Mi Lim,[b] Hee-Seek Kweon,[c] In Su Lee,[d]
Dongmin Kang,*[b] and Jong Hwa Jung*[a]
The design and synthesis of metal chemosensors with high
selectivity and sensitivity is an active field in supramolecular
chemistry.[1] Particular attention has been focused on heavy
and/or transition metal (HTM) ions and their detection in
the cell environment.[2] Zinc(II) ions are the second most
abundant transition metal ions essential for the human body
and play an indispensable role in various biological process-
es, such as gene transcription, regulation of metalloenzymes,
cell apoptosis, neural signal transmission, and insulin secre-
tion.[3] The total concentration of Zn2+ varies widely in dif-
ferent types of cells, ranging from the nanomolar level to
approximately 0.3 mm.[4] The detection and imaging of Zn2+
in biological samples are of significant interest due to this
cationꢀs unique role in physiological functions. Numerous
scientific endeavors have focused on the development of flu-
tion, and protein purification, because of their biocompati-
bility and stability against degradation.[7] Receptor-immobi-
lized magnetic nanoparticles have some important advantag-
es as solid chemosensors and adsorbents in heterogeneous
solid–liquid phases.[8] First, such nanoparticles are readily
synthesized by hydrolysis reactions—a versatile technique
that allows introduction of chemical functionalities. Second,
immobilized receptors on inorganic nanoparticles can
remove guest molecules (toxic metal ions and anions) from
the pollutant solution. Third, magnetic nanoparticles can be
easily isolated and controlled from pollutants by a small
magnet and can be repeatedly utilized with suitable regener-
ative treatment. Magnetic nanoparticles can also provide ef-
ficient binding to guest molecules because of their high sur-
face-to-volume ratio, which simply offers more contact area.
With these concepts in mind, we undertook the construc-
tion of a new magnetic nanoparticle-based OFF–ON fluo-
rescent Zn2+ probe (1) that operates by a photoinduced
electron transfer (PET) mechanism. Probe 1 was shown to
be highly selective for Zn2+ in aqueous media. The applica-
tion of this new magnetic nanoparticle-based probe for the
detection of zinc ions in living cells is also described. To
obtain a highly selective OFF–ON probe for Zn2+ in aque-
ous solution for practical applications, we introduced
a BODIPY moiety as the fluorophore because of its charac-
teristically strong fluorescence behavior (including high fluo-
rescence quantum yields) and its high chemical stability.
Fe3O4 nanoparticles were employed as the inorganic support
because of their noncytotoxicity and the ease of their mag-
netic separation.
orescent chemosensors for the in vitro and in vivo detection
[5]
of Zn2+
.
For example, Yoon et al. have recently reported
a highly selective, cell permeable, and ratiometric fluores-
[6]
cent probe for Zn2+
.
However, some of the reported Zn2+
probes have poor water solubility and are vulnerable to in-
terference by the presence of other metal ions. Furthermore,
the majority of existing probes might not be able to perform
within the cell environment. Therefore, the development of
fluorescent probes with greater versatility and improved per-
formance is still in great demand.
Magnetic nanoparticles are of great interest for biomedi-
cal and environmental research applications, such as biose-
paration, drug targeting, cell isolation, enzyme immobiliza-
[a] G. Kang, H. Son, Prof. Dr. J. H. Jung
Department of Chemistry and Research Institute of
Natural Sciences Gyeongsang National University
Jinju 660-701 (Korea)
The overall synthesis procedure for nanomaterials 1 is de-
picted in Scheme 1. First, we prepared the supporting nano-
materials from superparamagnetic Fe3O4 nanoparticles of
about 20 nm in diameter stabilized by oleic acid through
E-mail: jonghwa@gnu.ac.kr
[b] J. M. Lim, Prof. Dr. D. Kang
Division of Life and Pharmaceutical Sciences
Ewha Women’s University, Seoul 120-750 (Korea)
E-mail: dkang@ewha.ac.kr
[c] Dr. H.-S. Kweon
Division of Electron Microscopic Research
Korea Basic Science Institute, Daejeon 305-333 (Korea)
[d] Prof. Dr. I. S. Lee
Department of Chemistry
Pohang University of Science and Technology
Nam-Gu, Pohang 790-784 (Korea)
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/chem.201200294.
Scheme 1. Preparation of BODIPY-functionalized Fe3O4 nanoparticles
(1).
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a previously reported procedure.[8b,c,d] The BODIPY-based
derivative was selected as the fluorogenic group due to its
sharp fluorescence peaks with high quantum yields, and in-
sensitivity to the polarity and pH of the environment. Sec-
ondary, compound 3 was synthesized by following a similar
method to that described previously (Scheme S1 in the Sup-
porting Information).[9] Then, compound 3 was treated with
3,4-dihydroxy benzaldehyde in the presence of acetic acid
and piperidine to give the desired product 2 in toluene.
Third, the Fe3O4 nanoparticles were treated with 2 in tolu-
ene with vigorous stirring, overnight, to link them onto the
surface of the Fe3O4 nanoparticles by covalent bonding (see
the Experimental Section and Scheme 1). Finally, 1 was fully
characterized by transmission electron microscopy (TEM),
FTIR spectroscopy, time-of-flight second ion mass spectros-
copy (ToF-SIMS), and fluorophotometry.
atom in the benzoyl moiety. Upon addition of increasing
Zn2+ concentrations, 1 showed a large chelation-enhanced
fluorescence (CHEF) effect in the fluorescence emission
spectra; this results from the blocking of the PET process
and we observed an overall emission increase of approxi-
mately 32-fold (F=0.058, Figure 2) at the emission maxi-
mum (lem =594).
TEM imaging of 1 revealed a spherical structure with
a narrow size distribution (ca. 20 nm) that maintained its
nanocrystalline appearance (Figure 1). The IR and ToF-
Figure 1. a) TEM image of 2-immobilized Fe3O4 nanoparticles (1); scale
bar: 50 nm. b) Photograph of a magnet attracting 1 in aqueous solution.
SIMS results were in accord with bond formation; the IR
spectrum of 1 showed strong new bands at 3469, 3313, 3145,
3003, 2927, 2833, 2591, 2553, 2455, 1484, 1465, 1235, 1162,
and 1043 cmÀ1, which originated from receptor 2; this is in
accord with 2 residing on the Fe3O4 nanoparticles (Figure S1
in the Supporting Information). The ToF-SIMS spectrum of
1 displayed fragments attributable to 2 (m/z 414 and 441),
and thereby provides evidence that 2 was anchored onto the
surface of the Fe3O4 nanoparticles (Figure S2 in the Sup-
porting Information).
Figure 2. a) Fluorescence changes of 1 (10 mm) upon addition of Zn2+
ions in the presence of other metal ions (150 equiv) in aqueous solution.
b) Fluorescence responses of 1 to various metal ions. The bars labeled as
1+Mn+ represent the addition of selected metal ions (150 equiv) to a so-
lution of 1 (10 mm). The bars labeled as 1+Mn+ +Zn2+ represent subse-
quent addition of Zn2+ (150 equiv) to the solution. For all measurements,
the pH value was adjusted by using HEPES (20 mm) in pure aqueous so-
lution, pH 7.4. Excitation was achieved at 570 nm, and the emission was
monitored at 594 nm.
Spectroscopic measurements for 1 were performed in
With few exceptions,[11] most reported BODIPY-fluores-
cent probes are based on an intramolecular charge transfer
(ICT) mechanism.[10] However, in our case, the noticeable
fluorescence intensity enhancement of receptor 2 attached
to Fe3O4 nanoparticles can be due to the inhibition of the
PET process, which quenches fluorescence, during the bind-
ing of Zn2+. Preliminary computational simulations indicat-
ed that 2 is twisted at the BODIPY moiety; this presumably
HEPES
(4-(2-hydroxyethyl)-1-piperazineethanesulfonic
acid; 20 mm) buffer, pH 7.4. The UV/Vis absorption spec-
trum of free 1 showed one absorption band at 570 nm (e=
4.17ꢂ104 mÀ1 cmÀ1). As expected, nanoparticle 1 is virtually
nonfluorescent in its apo state (F<0.0018, lex =570 nm),
which is a consequence of the efficient PET[10] quenching of
the fluorophore by the lone-pair electrons of the nitrogen
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Detection of Zinc Ions in Living Cells
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results in a blocking of the ICT. In addition, the direct con-
jugation of the amine to the styryl group results in a very
significant red shift, and high fluorescence quantum yields
for this BODIPY-based receptor.[10a]
We also investigated the ability of 1 to serve as an ion-se-
lective fluorogenic probe by testing the binding of other
cle 1 were re-exposed to Zn2+, the fluorescence emission
was again present, with no reduction in response (Figure S6
in the Supporting Information). The fluorescence change
was reproducible over several cycles of detection/stripping.
The Job plot of the fluorescence changes indicated 1:1 bind-
ing for 1 with Zn2+ (Figure S7 in the Supporting Informa-
tion). With the use of the fluorescence titration data, the as-
sociation constant (Ka) for Zn2+ coordination to nanoparti-
cle 1 was calculated to be (2.31ꢂ103)mÀ1.[13,14]
Spectral changes upon addition of the previously men-
tioned biologically and environmentally relevant metal ions
were also screened by fluorophotometry. The emission pro-
files of apo or Zn2+-bound 1 were unchanged in the pres-
metal ions, including Ca2+, Hg2+, Cd2+, Li+, Ag+, Cu2+
,
Fe2+, Mg2+, Pb2+, K+, Na+, Mn2+, Co2+, Fe3+, Al3+, and
Ni2+. However, no significant spectral changes were ob-
served upon addition of any of these metal ions (Figure 2);
this indicates that nanoparticle 1 is a highly selective che-
moprobe for the detection of Zn2+
.
For comparison, we performed spectroscopic measure-
ments using ligand 2 in acetonitrile solution (2 was insoluble
in water); under these conditions 2 gave a single absorption
band at 570 nm (e=5.0ꢂ104 mÀ1 cmÀ1). In the absence of
Zn2+, 2 also exhibited no fluorescence emission when excit-
ed at 570 nm. Upon the addition of Zn2+, the fluorescence
emission intensity of 2 increased by approximately 1.5-fold
(F=0.0045, Figure S3 in the Supporting Information) with
an emission maximum at 594 nm. Other metal ions, such as
Ni2+, Hg2+, and Al3+, also induced the fluorescence intensi-
ty enhancement of 2. In particular, the fluorescence en-
hancement of 1 upon the addition of Al3+ can occur because
the two hydroxyl groups of BODIPY dye act as binding
sites for Al3+. Al3+ can be classified as a hard acid by
HSAB theory.[12] The lower selectivity of 2 for Zn2+ com-
pared to 1 can be attributed to its high flexibility and its acy-
clic structural character prior to immobilization on the sur-
face of the nanoparticles.
ence of 10 mm Ca2+, Hg2+, Cd2+, Li+, Ag+, Cu2+, Fe2+
,
Mg2+, Pb2+, K+, Na+, Mn2+, Co2+, Fe3+, Al3+, or Ni2+ (Fig-
ure 2b, and Figure S8 in the Supporting Information); this
indicates that nanoparticle 1 shows great promise as a useful
selective chemoprobe for detection of Zn2+, in vivo.
Ideally, for biological applications, sensing should be prac-
tical over a range of pH values, and thus we investigated the
effect of pH on the spectrophotometric behavior of nano-
particle 1 in both the absence and presence of Zn2+ (Fig-
ure S9 in the Supporting Information). Over the pH range
from 3 to 11, nanoparticle 1 showed no fluorescence emis-
sion in the absence of Zn2+, whereas upon the addition of
Zn2+, the fluorescence intensity of nanoparticle 1 was
strongly dependent to pH values. The fluorescence intensity
of nanoparticle 1 in the presence of Zn2+ was highest at
pH 7.4. On the other hand, under acidic conditions, the fluo-
rescence intensity of nanoparticle 1 slightly increased after
addition of Zn2+; this can be attributed to protonation of
the nitrogen atoms of ligand 2 immobilized on Fe3O4 nano-
particles.
To further demonstrate the practical application of the
nanoparticle-based probe 1, we established its ability to
track Zn2+ levels in living cells using a model for respiratory
zinc exposure. Live cell confocal microscope imaging experi-
ments were carried out that utilized 1 to enhance membrane
permeability (Figure 3a–c). HeLa cells (human cancer cells)
were incubated with nanoparticle 1 (5.0 mm) for 30 min at
378C and then washed with phosphate buffered saline
(PBS) to remove excess nanoparticle 1, which would other-
wise contribute to weak intracellular fluorescence (Fig-
The highly selective Zn2+ recognition of nanoparticle-
based fluorescence chemoprobe 1 demonstrates that the ap-
proach employed in the present study cooperatively enhan-
ces and controls the selectivity towards this metal ion. More
importantly, quantitative measurements of the emission
maximum of Zn2+-bound 1 indicated that the fluorescence
change correlated linearly with the [Zn2+] over the 0–30 ppb
range investigated. As shown in Figure S4 in the Supporting
Information, the detection limit for Zn2+ is approximately
0.2 nm. Considering that the Zn2+ concentration in living
cells can vary from nanomolar levels to about 0.3 mm, the
observed detection limit for nanoparticle 1 in aqueous
media could be highly advantageous under certain circum-
stances. Evaluation of the time course for the fluorescence
intensity of nanoparticle 1 at 594 nm (Figure S5 in the Sup-
porting Information) indicated that immediately after the
addition of Zn2+, its fluorescence intensity started to in-
crease, and that by 60 s the fluorescence intensity was
almost saturated. Thus, the response time of this system is
within 1 min; this makes it a rapid and convenient method
for the quantification of Zn2+ in aqueous solutions (Fig-
ure S5 in the Supporting Information).
ure 3B). On treating the cells with ZnACHTNURTGNEUNG(ClO4)2 (5.0 mm) for
30 min at 378C and then staining with nanoparticle 1 under
the same loading conditions resulted in an increase in the
observed intracellular fluorescence intensity of 1 (Fig-
ure 3C). This procedure allowed us to successfully perform
selective Zn2+ imaging in living cells, despite the presence
of many potential interfering substances, such as proteins
and amino acids. Thus, nanoparticle 1 is potentially useful
for studying the toxicity and/or bioactivity of Zn2+ in living
cells. We further evaluated the internalization of 1 in HeLa
cells by TEM (Figure S10 in the Supporting Information).
Intact nanoparticles 1 within the cells are evident, as con-
firmed by measurement of their diameters. Conversely, few
intact nanoparticles 1 are aggregated. This result provides
After exposure to Zn2+, nanoparticle 1 was successfully
regenerated. When the Zn2+-bound nanoparticle 1 was
treated with an aqueous EDTA solution (10 mm), the fluo-
rescence intensity of Zn2+-bound nanoparticle
1
was
quenched. However, when the washed, stripped nanoparti-
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J. H. Jung, D. Kang et al.
Figure 3. a) Bright-field confocal image of HeLa cells. b) Fluorescence
image of HeLa cells incubated with 1 (5.0 mm) for 30 min at 378C.
c) Fluorescence image of 1-loaded HeLa cells incubated with Zn
(5.0 mm) after 30 min at 378C.
ACHTUGNRTEN(NUGN ClO4)2
visual evidence for cellular uptake of fluorescence receptor-
immobilized Fe3O4 nanoparticles 1.
Furthermore, we evaluated the reversibility of the above
Zn2+ detection procedure in living cells (Figure S11 in the
Supporting Information). The fluorescence emission of Zn2+
-bound 1 was observed to decrease to the initial level upon
addition of EDTA (10 mm) to the HeLa cells. The fluores-
cence change was reproducible over several cycles of detec-
tion/stripping. This result strongly indicates that Zn2+ ions
are dissociated from Zn2+-bound 1 by EDTA. Clearly, che-
moprobe 1 is not only very useful for detection and removal
of toxic Zn2+ ions in living cells, but 1 is also readily regen-
erated by using the above procedure. This is a rare example
of the reversible sensing of a target molecule or ions in
living cells.
Figure 4. MTT cytotoxicity assay showing the effect of varying concentra-
tions of 1 on growth inhibition of: a) HeLa, and b) Cos7 cell lines.
10 mm. These findings show considerable promise for the de-
velopment of a new category of tailor-made biocompatible
sensing systems built by immobilization of appropriate fluo-
rogenic receptors on the surface of other novel nanomateri-
als for fluorescence microscopic imaging. They also show
considerable promise for the detailed study of the biological
action of heavy metal toxins in living systems.
The cytotoxicity of 1 was evaluated against two different
cell models: HeLa and Cos7 cells (monkey kidney fibro-
blasts; Figure 4). The two cell lines were chosen as represen-
tative models of the various cellular environments that 1 is
likely to encounter, in vivo. In both cell lines, MTT (3-(4,5-
Experimental Section
Preparation of Fe3O4 nanoparticles: Fe3O4 nanocrystals with 20 nm aver-
age core size were prepared by the previously reported procedure.[15]
Fe3O4 nanocrystals (80 mg) were dispersed in cyclohexane. Then, the re-
sulting nanocrystals of Fe3O4 were collected by magnetic decantation.
The collected nanocrystals of Fe3O4 were redispersed in EtOH and re-
covered by using a magnet. For purification, the dispersion of Fe3O4 into
an EtOH suspension, followed by magnetic separation, was repeated
three times.
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide)
assays showed that nanoparticle 1 did not exhibit any toxici-
ty at levels of up to 10 mm under same the conditions in
which staurosporin (1.0 mm) induced 70–90% apoptotic cell
death. These findings strongly suggest that the nanoparticles
1 are quite suitable as chemoprobes, in vivo, and they con-
trast markedly with the cationic polymers, amino- or polyly-
sine-functionalized silica nanoparticles, which exhibit high
toxicity in cell lines.
In summary, we have readily prepared BODIPY-function-
alized Fe3O4 nanoparticles (1) by a straightforward proce-
dure. These nanoparticles act as a new type of synthetic flu-
orogenic chemoprobe for imaging Zn2+ ions in living cells.
Magnetic nanoparticle 1 exhibits a high affinity and selectiv-
ity for Zn2+ over other competing metal ions tested and
they successfully detected Zn2+ in cultured cells. Further-
more, nanoparticle 1 did not exhibit any toxicity up to
Preparation of 2-immobilized Fe3O4 (1): Compound
2
(50 mg,
0.054 mmol) was dissolved in anhydrous toluene (5 mL) to which Fe3O4
nanoparticles (100 mg) were added. The mixture was stirred under reflux
in N2 for 24 h. The collected solid was washed several times with di-
chloromethane and acetone to rinse away any excess 2 and then dried
under vacuum.
Cell incubation: HeLa cells were cultured in Dulbeccoꢀs modified
Eagleꢀs medium (DMEM, Gibco BRL, USA) supplemented with fetal
bovine serum (FBS, 10%), penicillin (50 mgmLÀ1), and streptomycin
(50 mgmLÀ1). The cells were grown on a microscopy culture dish (diame-
ter, 35 mm) with poly-l-lysine coating. To determine the cell permeability
of 1, the cells were incubated with 1 (5.0 mm) for 30 min at 378C, and
washed with PBS to remove excess 1. To observe fluorescence changes,
ZnACHTNURTGNEG(UN ClO4)2 (5.0 mm) was added to the 1-loaded cells and then further incu-
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Detection of Zinc Ions in Living Cells
COMMUNICATION
bated for 30 min at 378C. Fluorescence changes was observed by confo-
cal microscope. In addition, the reversibility of 1 loaded in HeLa cell was
observed by treatment with EDTA solution (10 mm).
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cultured on poly-l-lysine-coated glass-bottom dishes (diameter, 35 mm)
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taraldehyde (2.5%) in phosphate buffer (0.1m), postfixed with OsO4
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Acknowledgements
This work was supported by a grant from Environmental-Fusion Project
(191-091-004), and World Class Project (WCU) supported by Ministry of
Education Science and Technology, Korea (R32-2008-000-20003-0 and
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Keywords: biosensors
nanoparticles · zinc
· chromophores · fluorophores ·
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Received: January 27, 2012
Published online: April 19, 2012
Chem. Eur. J. 2012, 18, 5843 – 5847
ꢁ 2012 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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5847