An NBD fluorophore-based colorimetric and fluorescent chemosensor for hydrogen sulfide and its application for bioimaging

Article abstract of DOI:10.1016/j.tet.2012.10.106

Hydrogen sulfide (H₂S) is an important gaseous mediator in cellular physiology and pathology. For understanding the biological action of H₂S to both healthy and disease states, it is important to develop H₂S-selective sensor. Herein we report a colorimetric and fluorescent probe, which employs an NBD moiety as fluorophore, and is equipped with azide group as H₂S-active unit. The probe exhibits selective and sensitive response to H₂S in aqueous solution. By employing 1 mM sensor, the detection limit was evaluated to be 680 nM with a signal-to-noise ratio of 3. Confocal microscopy imaging experiments demonstrate the probe has potential as a powerful tool for the imaging of hydrogen sulfide in living cells. Crown Copyright

Full text of DOI:10.1016/j.tet.2012.10.106

Tetrahedron 69 (2013) 867e870
Contents lists available at SciVerse ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
An NBD fluorophore-based colorimetric and fluorescent chemosensor
for hydrogen sulfide and its application for bioimaging
*
Guodong Zhou, Huilin Wang, Yang Ma, Xiaoqiang Chen
State Key Laboratory of Materials-Oriented Chemical Engineering, College of Chemistry and Chemical Engineering, Nanjing University of Technology, Xinmofan Road 5#, Nanjing
210009, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Hydrogen sulfide (H2S) is an important gaseous mediator in cellular physiology and pathology. For un-
derstanding the biological action of H2S to both healthy and disease states, it is important to develop
H2S-selective sensor. Herein we report a colorimetric and fluorescent probe, which employs an NBD
moiety as fluorophore, and is equipped with azide group as H2S-active unit. The probe exhibits selective
Received 4 August 2012
Received in revised form 26 September
2012
Accepted 9 October 2012
Available online 15 November 2012
and sensitive response to H2S in aqueous solution. By employing 1 mM sensor, the detection limit was
evaluated to be 680 nM with a signal-to-noise ratio of 3. Confocal microscopy imaging experiments
demonstrate the probe has potential as a powerful tool for the imaging of hydrogen sulfide in living cells.
Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
Keywords:
Fluorescent sensor
Hydrogen sulfide
7-Nitrobenz-2-oxa-1,3-diazole (NBD)
Bioimaging
1. Introduction
and sludge, they are hard to be applied for intracellular detection
due to their limitations to in vivo studies. Fluorescent methods in
Hydrogen sulfide is considered as the third biologicallyactive gas
with the other two endogenous gasotransmitter: nitric oxide and
carbon monoxide. Most endogenous hydrogen sulfide results from
the metabolism of sulfhydryl-containing amino acids (e.g., cysteine)
by bacteria present in both the intestinal tract and the mouth.1 Hy-
drogen sulfide is also produced via enzymatic reaction in the brain
and several smooth muscles.2 Hydrogen sulfide play important roles
in various physiological processes, such as vasodilation,3 neuro-
transmission,2a apoptosis,4 inflammation,5 oxygen sensing,6 in-
hibition of insulin signaling.7 On the other hand, hydrogen sulfide
was known as a toxic gas, and inhalation of high concentrations can
lead to death in animals. Single, short-term and medium-term in-
halation exposures to hydrogen sulfide have also resulted in re-
spiratory, olfactory, cardiovascular, neurological, hepatic, and
developmental neurochemical effects and abnormal growth in de-
veloping cells.8 Therefore, it is highly important to develop an im-
aging probe for hydrogen sulfide with excellent selectivity and
sensitivity that is applicable to various biological systems.
conjunction with suitable probes are more desirable for the mea-
surement of biological species since they are simple, sensitive, and
can afford real information on the localization and quantity of the
targets of interest.13 In the past several years, a few fluorescent
probes for hydrogen sulfide based on different mechanisms have
been exploited, including hydrogen sulfide-induced reduction of
azide to amine,14 fluorescence recovery after copper dissociation
from complex-fluorophore ensemble,15 removal of 2,4-
dinitrobenzenesulfonyl group,16 cleavage of disulfide bond and
H2S-specific Michael additionecyclization.17
In this work, we introduced azide group to 7-nitrobenz-2-oxa-
1,3-diazole (NBD) fluorophore to construct a H2S-selective sensor.
NBD was chosen as the fluorophore because of long emission
wavelength and good cell permeability. The azide-functionated NBD
derivate was expected to react with H2S, producing a strongly
fluorescent amino-substituted NBD (Scheme 1). The NBD derivate 1
was synthesized as shown in Scheme 1. NBD-Cl was reacted with
NaN3 in EtOH to afford 1 in 18.5% yield. The experimental details and
characterization data for 1 are given in Supplementary data.
To date, several detection methods for hydrogen sulfide have
been developed, such as gas chromatography,9 electrochemical
analysis,10 colorimetric method,11 metal-induced sulfide pre-
cipitation.12 Although these methods are useful to monitor hydro-
gen sulfide in environmental samples, such as air, water, sediment,
2. Results and discussion
2.1. Absorbance and fluorescence studies in aqueous solution
To verify the selectivity of 1 to hydrogen sulfide, the absorbance
and fluorescence studies in aqueous solution were carried out. The
* Corresponding author. Tel./fax: þ86 25 8358 7856; e-mail address: chenxq@
njut.edu.cn (X. Chen).
0040-4020/$ e see front matter Crown Copyright Ó 2012 Published by Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.tet.2012.10.106

868
G. Zhou et al. / Tetrahedron 69 (2013) 867e870
Cl
N3
NH2
N
N
N
N
N
O
N
NaHS
NaN3
EtOH
O
O
PBS, pH 7.5
NO2
NO2
NO2
NBD-NH2
NBD-Cl
1
Scheme 1. Synthesis of sensors 1 and the reaction in the presence of H2S.
fluorescence and absorbance changes that 1 undergoes upon the
addition of various analytes were shown in Fig.1. Afteradding 100 mM
of anions, such as S2O42ꢀ, HSꢀ, S2O32ꢀ, S2O52ꢀ, HCO3ꢀ, HPO4ꢀ, Fꢀ,
Clꢀ, Brꢀ, Iꢀ, SO42ꢀ, HSO3ꢀ, NO3ꢀ, H2PO4ꢀ, OAcꢀ, andcitratetothePBS
buffer (20 mM, pH 7.5, 1% CH3CN) solution containing 10 mM of 1 for
5 min, only NaHS induced a remarkable fluorescence enhancement at
550 nm. The absorption spectrum also displays that the addition of
NaHS lead to a red-shift of absorption peak from 396 nm to 468 nm.
Accordingly, the addition of 1 produce a colorimetric change from
pale-yellow to deep-yellow, which can be detected by naked-eye
(Fig. S1). Interestingly, the mixing of Na2S2O4 and 1 creates a blue-
shift in maxium absorption wavelength from 396 to 329 nm
(Fig. S2), accompanied by the color change from pale-yellow to col-
orless. It was reasoned that Na2S2O4 as a strong reductant, destroyed
the conjugated structure of NBD fluorophore, resulting in the fade of
color and the blue-shift of absorption wavelength. Besides, other
analytes including Na2S2O3, Na2S2O5, NaHCO3, Na2HPO4, NaF, NaCl,
NaBr, NaI, Na2SO4, NaHSO3, NaNO3, NaH2PO4, NaOAc, and sodium
citrate show neglectable changes in fluorescence and absorbance
spectra under the same conditions.
Fig. 2. Fluorescence (top) and absorption spectra (bottom) of 1 (10
mM) in the presence
of various concentrations of NaHS (0, 5, 10, 20, 30, 40, 50 and 60
mM) in PBS buffer
(20 mM, pH 7.5, 1% CH3CN) (lex¼474 nm, slit: 5/5 nm). Each spectrum is recorded at
5 min after addition of NaHS to 1.
appeared, and an enhancement of the fluorescence intensity by up
to 16-fold was observed upon addition of 6 equiv of NaHS. Con-
comitantly, a gradual decrease of the absorption peak at 396 nm
and a progressive increase of a new absorption band at around
468 nm by addition of NaHS were observed (Fig. 2). A well-defined
isosbestic point was noted at 428 nm, which may indicate the
formation of a new product upon treatment of 1 with NaHS.
The time-dependent fluorescence responses were monitored at
550 nm (Fig. 3, top), and the results show that the reaction is
complete within 3 min when 6 equiv of NaHS was mixed with 1. To
investigate the detection limit of 1 for hydrogen sulfide, 1 (1 mM)
was treated with various concentrations of NaHS (0e700 nM). The
fluorescence intensity at 550 nm was plotted as a function of the
NaHS concentration. The fluorescence intensity of 1 is linearly
proportional to NaHS concentrations of 0e700 nM, and as low as
680 nM concentration of HSꢀ was detected by using 1 with a signal-
to-noise ratio of 3 (Fig. 3, bottom).
The fluorescence intensity changes of 1 induced by HSꢀ were
measured at various pHs for investigating the effect of pH on the
fluorescence response. As shown in Fig. 4 (top), in the absence of
NaHS, the fluorescence of 1 was weak in buffers at various pH
values from 4.5 to 10. When NaHS was added to the solution con-
taining 1, strong fluorescence enhancement was detected at pH 6.5
to 9, which indicated that 1 enable to detect NaHS within a wide pH
range.
Fig. 1. Top: fluorescence spectra of 1 (10
(100
Bottom: UV/vis absorption spectra of 1 (10
mM) upon incubation with various analytes
m
M) for 5 min in PBS buffer (20 mM, pH 7.5, 1% CH3CN) (lex¼474 nm, slit: 5/5 nm).
m
M) in PBS buffer (20 mM, pH 7.5, 1%
2ꢀ
CH3CN) in the presence of 100
m
M analytes including S2O42ꢀ, HSꢀ, S2O32ꢀ, S2O5
,
HCO3ꢀ, HPO4ꢀ, Fꢀ, Clꢀ, Brꢀ, Iꢀ, SO42ꢀ, HSO3ꢀ, NO3ꢀ, H2PO4ꢀ, OAcꢀ, citrate.
The changes in the fluorescence spectra of probe 1 (10
mM) in
the absence or presence of NaHS (0e60 mM) in PBS buffer are dis-
2.2. Sensing mechanism
played in Fig. 2. The probe 1 is weak-fluorescent in the absence of
NaHS; however, the addition of NaHS caused a dramatic change in
the fluorescence spectra. A strong new emission peak at 550 nm
On the detection mechanism, the fluorescence enhancement
and UVevis spectral change should be attributed to the reduction

G. Zhou et al. / Tetrahedron 69 (2013) 867e870
869
Fig. 3. Top: time-dependent fluorescence intensity of 1 at 10
60
m
M in the presence of
m
M hydrogen sulfide. Bottom: the change in the fluorescence intensity of 1 at 1 mM
against varied concentrations of hydrogen sulphide in CH3CNePBS buffer (20 mM, pH
7.5) (1:99, v/v).
Fig. 4. Top: the fluorescence intensity of 1 (10
mM) at 550 nm in the presence and
absence of hydrogen sulfide in CH3CNeH2O system with different pHs. (NaH2PO4
buffer was used for pH 4.5 and 5.5, MOPS buffer for pH 6.5, HEPES buffer for pH 7.4,
TriseHCl buffer was used for pH 8.5, CHES buffer was used for pH 9.0 and CAPS buffer
was used for pH 10.0.) Bottom: Job’s plot of the reaction between 1 and hydrogen
sulfide in CH3CNePBS (20 mM, pH 7.5, 1:99, v/v) solutions at pH 7.5. Total concen-
reaction between hydrosulfide anion and azide group in the probe,
which afford the amino substituted NBD fluorescein (Scheme 1). To
examine this plausible mechanism, the stoichiometry of a reaction
event between NaHS and 1 was initially determined. The results
obtained from Job’s plot show the 1:1 stoichiometry for the re-
action between 1 and NaHS (Fig. 4, bottom). NMR analysis of
a product NBDeNH2 obtained from the reaction of 1 with NaHS also
supports the formation of amino substituted NBD (Fig. S3 and 4).
The peak at 181.0364 corresponding to the resulting product
[NBDꢀNH2þHþ]þ was clearly observed in the mass spectra
(Fig. S5).
tration of 1 and hydrogen sulfide was kept constant at 10 mM.
2.3. Living cell imaging
In order to further demonstrate that the permeability and the
monitoring of hydrogen sulfide in living cells, confocal microscopy
experiments were carried out. When MCF-7 cells were incubated
with 1 (20
cells (Fig. 5, top). However, in a control experiment, cells were pre-
treated with 1 (20 M), followed by treatment with an excess
(150 M) of NaHS, a resource of hydrogen sulfide. The confocal
microscopic images show a significant fluorescence signal (Fig. 5,
bottom). This confirms that 1 can be used as the imaging agent for
hydrogen sulfide in living cells.
mM), only weak fluorescence was exhibited inside the
Fig. 5. Phase contrast and fluorescence images of MCF-7 cells. Top: PC-12 cells were
treated with 20 M of 1 for 30 min; Bottom: PC-12 cells were pre-incubated with
20 M of 1 for 30 min and then treated with 150 M of NaHS for 30 min (a and d, phase
m
m
m
m
m
contrast images; b and e, fluorescence images; c and f, merged images).
suggested that the present probe has potential as a powerful tool
for the imaging of hydrogen sulfide in living cells.
3. Conclusions
4. Experimental
In summary, we have developed a new NBD fluorophore-based
colorimetric and fluorescent chemosensor for the detection of hy-
drogen sulfide with high selectivity and sensitivity. The fluores-
cence enhancement and UVevis spectral changes are attributed to
reduction of azido group to amine. Confocal microscopy imaging
4.1. Materials and equipments
Unless otherwise noted, materials were obtained from com-
mercial suppliers and were used without further purification.

870
G. Zhou et al. / Tetrahedron 69 (2013) 867e870
Chromatography was carried out on silica gel 60 (230e400 mesh
ASTM). 1H NMR and 13C NMR spectra were recorded using Bruker
300 or Bruker 500. Mass spectra were obtained using a Waters
Micromass Q-Tof micro mass spectrometer. Fluorescence emission
spectra were obtained using RF-5301/PC Spectrofluorophotometer.
microscope (LSCM) culture dishes with a density of 5ꢂ105 cells/
well. The cell lines were cultured in RPMI-1640 medium supple-
mented with 10% (v/v) calf serum, penicillin (100 U mLꢀ1) and
streptomycin (100 mg mLꢀ1). Cells were maintained at 37 ꢁC in
a humidified atmosphere containing 5% CO2. When the whole cells
took up 70e80% space of culture dishes, the cells were first in-
UV absorption spectra were obtained on
a-1860A UVevis Spec-
trometer. All pH measurements were made with Sartorius PB-10
meter. The IR spectra were obtained using Nicolet 8700 FT-IR
Spectrometer.
cubated with 20
m
M of 1 in culture media for 30 min at 37 ꢁC. Then
M hydrogen
the cells were further treated without or with 150
m
sulfide in culture media for 30 min at 37 ꢁC. After washing with
phosphate buffered saline (PBS) to remove the remaining hydrogen
sulfide, the cells were imaged by confocal laser scanning micros-
copy (Olympus FV-1000).
4.2. Synthesis
Synthesis of sensor 1. A suspension of NBDeCl (185.6 mg,
0.93 mmol) in 15 mL of EtOH was added dropwise into a stirred
solution of sodium azide (130 mg, 2 mmol) in 7 mL of a mixed
solvent (H2O/EtOH, 1:1). Then the reaction mixture was stirred at
room temperature for 3 h. The organic solvent was evaporated in
vacuum, and the aqueous solution was extracted by CH2Cl2. The
combined organic layers was washed with brine and then dried
over MgSO4 (3 h). Solvent evaporation gave the crude product,
which was purified by column chromatography to give 1 (35.5 mg,
18.5%) as a dark yellow solid. Mp 99e100 ꢁC; 1H NMR (CDCl3,
Acknowledgements
This work was supported by grants from NSFY of China
(21002049) and the Natural Science Foundation of the Jiangsu
Higher Education Institutions of China (Grant No. 10KJB150005).
The work was also supported by the Scientific Research Foundation
for the Returned Overseas Chinese Scholars, State Education
Ministry.
300 MHz)
d
(ppm): 8.54 (d, 1H, J¼9.0 Hz, ArH), 7.09 (d, 1H, J¼9.0 Hz,
ArH); 13C NMR (CDCl3, 75 MHz)
d
(ppm): 145.87, 143.53, 138.06,
Supplementary data
132.09, 114.87; elemental analysis calcd (%) for C6H2N6O3: C 34.96,
H 0.98, N 40.77; found: C 34.83, H 1.19, N 40.66; IR nmax (KBr) 3101.1,
2143.8, 2119.4, 1632.6, 1537.2, 1449.8, 1375.4, 1340.9, 1324.1, 1278.5,
These data include characterization of 1 and 2, mass spectra of 2,
absorption spectra of 1 (10 mM) in the presence of Na2S2O4. Sup-
plementary data related to this article can be found online at http://
dx.doi.org/10.1016/j.tet.2012.10.106.
1209.0, 1120.1, 1071.8, 1056.0, 1017.2, 993.6, 890.6 cmꢀ1
.
The confirmation of the product NBDeNH2 from the reaction be-
tween 1 and H2S. A solution of sodium hydrosulfide (67.2 mg,
1.2 mmol) in 2 mL H2O was added into a stirred solution of 1
(180 mg, 1 mmol) in 40 mL acetonitrile. The reaction mixture was
stirred 3 h at room temperature. Then the organic solvent was
evaporated in vacuum, and the aqueous solution was extracted by
CH2Cl2. The combined organic layers gave the crude product, which
was purified by column chromatography to give NBD-NH2 (30 mg,
References and notes
1. (a) Tonzetich, J.; Carpenter, P. A. W. Arch. Oral Biol. 1971, 16, 599; (b) Beauchamp,
R. O., Jr.; Bus, J. S.; Popp, J. A.; Boreiko, C. J.; Andjelkovich, D. A. Crit. Rev. Toxicol.
1984, 13, 25.
2. (a) Abe, K.; Kimura, H. J. Neurosci. 1996, 16, 1066; (b) Hosoki, R.; Matsuki, N.;
Kimura, H. Biochem. Biophys. Res. Commun. 1997, 237, 527.
3. Yang, G.; Wu, L.; Jiang, B.; Yang, B.; Qi, J.; Cao, K.; Meng, Q.; Mustafa, A. K.; Mu,
W.; Zhang, S.; Snyder, H.; Wang, R. Science 2008, 322, 587.
4. Yang, G.; Wu, L.; Wang, R. FASEB J. 2006, 20, 553.
5. Li, L.; Bhatia, M.; Zhu, Y. Z.; Zhu, Y. C.; Ramnath, R. D.; Wang, Z. J.; Anuar, F. B.;
Whiteman, M.; Salto-Tellez, M. P. K. FASEB J. 2005, 19, 1196.
6. Peng, Y. J.; Nanduri, J.; Raghuraman, G.; Souvannakitti, D.; Gadalla, M. M.; Ku-
mar, G. K. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 10719.
7. (a) Kaneko, Y.; Kimura, Y.; Kimura, H.; Niki, I. Diabetes 2006, 55, 1391; (b) Yang,
W.; Yang, G. D.; Jia, X. M.; Wu, L. Y.; Wang, R. J. Physiol. (London) 2005, 569, 519.
8. International Program on Chemical Safety (IPCS), World Health Organization.
Hydrogen Sulfide: Human Health Aspects; 2003.
16.7%). Mp >200 ꢁC; 1H NMR (DMSO, 300 MHz)
d (ppm): 8.85 (s,
2H, NH2), 8.49 (d, 1H, J¼9.0 Hz, ArH), 6.39 (d,1H, J¼9.0 Hz, ArH); 13
C
NMR (DMSO, 125 MHz)
d (ppm): 147.24, 144.24, 144.03, 137.86,
120.49, 102.59; ESI-MS m/z¼180.0364 [MþH]þ, calcd for
C6H5N4O3¼181.0362; IR nmax (KBr) 3433.7, 3342.5, 3235.7, 2360.3,
2341.9, 1650.0, 1619.7, 1557.5, 1531.3, 1505.3, 1447.3, 1422.5, 1386.5,
1318.0, 1290.7, 1125.2, 1042.1, 994.2 cmꢀ1
.
4.3. Absorbance and fluorescence studies in aqueous solution
9. (a) Radford-Knoery, J.; Cutter, G. A. Anal. Chem. 1993, 65, 976; (b) Brub, P. R.;
Parkinson, P. D.; Hall, E. R. J. Chromatogr., A 1999, 830, 485.
10. (a) Lawrence, N. S.; Davis, J.; Jiang, L.; Jones, T. G. J.; Davies, S. N.; Compton, R. G.
Electroanalysis 2000, 18, 1453; (b) Spilker, B.; Randhahn, J.; Grabow, H.; Beikirch,
H.; Jeroschewski, P. J. Electroanal. Chem. 2008, 612, 121.
11. (a) Fisher, E. Chem. Ber. 1883, 16, 2234; (b) Choi, M. M. F. Analyst 1998, 123, 1631;
(c) Choi, M. G.; Cha, S.; Lee, H.; Jeon, H. L.; Chang, S. K. Chem. Commun. 2009,
7390; (d) Edison, T. K., Jr.; Cristiana, M. P. Y.; Arlete, B. R.; Itamar, S. M.; Telma, T.
F. Polym. Int. 2011, 60, 951.
Stock solutions (10 mM) of analytes including Na2S2O4, NaHS,
Na2S2O3, Na2S2O5, NaHCO3, Na2HPO4, NaF, NaCl, NaBr, NaI, Na2SO4,
NaHSO3, NaNO3, NaH2PO4, NaOAc, sodium citrate in distilled water
were prepared. Stock solution of probe 1 (1 mM) was also prepared
in acetonitrile. In a typical experiment, test solution was prepared
by placing 30 mL of the probe stock solution into a test tube, diluting
12. Ishigami, M.; Hiraki, K.; Umemura, K.; Ogasawara, Y.; Ishii, K.; Kimura, H. An-
the solution to 3 mL with PBS buffer (20 mM, pH 7.5), and adding an
appropriate volume of each analyte stock. Normally, excitation was
at 474 nm, and both the excitation and emission slit widths were
5 nm. Fluorescence spectra were measured after the addition of
analyte for 5 min. For low concentration titration of hydrogen
sulfide, fluorescence spectra were measured after addition of hy-
drogen sulfide for 5 min, and both the excitation and emission slit
widths were 5 nm.
tioxid. Redox Signalling 2009, 11, 205.
13. (a) Chen, X.; Zhou, Y.; Peng, X.; Yoon, J. Chem. Soc. Rev. 2010, 39, 2120; (b) Zhou,
Y.; Yoon, J. Chem. Soc. Rev. 2011, 41, 52; (c) Chen, X.; Pradhan, T.; Wang, F.; Kim,
J. S.; Yoon, J. Chem. Rev. 2012, 112, 1910; (d) Du, J.; Hu, M.; Fan, J.; Peng, X. Chem.
Soc. Rev. 2012, 41, 4511; (e) Wang, H.; Zhou, G.; Gai, H.; Chen, X. Chem. Commun.
2012, 8341; (f) Wang, H.; Zhou, G.; Mao, C.; Chen, X. Dyes Pigm. 2013, 96, 232.
14. (a) Peng, H.; Cheng, Y.; Dai, C.; King, A. L.; Predmore, B. L.; Lefer, D. J.; Wang, B.
Angew. Chem., Int. Ed. 2011, 50, 9672; (b) Yu, F. B.; Li, P.; Song, P.; Wang, B.; Zhao,
J. Z.; Han, K. Chem. Commun. 2012, 2852; (c) Lippert, A. R.; New, E. J.; Chang, C. J.
J. Am. Chem. Soc. 2011, 133, 10078.
15. (a) Sasakura, K.; Hanaoka, K.; Shibuya, N.; Mikami, Y.; Kimura, Y.; Komatsu, T.;
Ueno, T.; Terai, T.; Kimura, H.; Nagano, T. J. Am. Chem. Soc. 2011, 133, 18003; (b)
Zhang, Q.; Jin, W. S. Spectrochim. Acta, Part A 2012, 90, 35.
4.4. Cell culture and fluorescence imaging
16. Yang, X. F.; Wang, L. P.; Xu, H. M.; Zhao, M. L. Anal. Chim. Acta 2009, 631, 91.
17. Liu, C.; Pan, J.; Li, S.; Zhao, Y.; Wu, L. Y.; Berkman, C. E.; Whorton, A. R.; Xian, M.
Angew. Chem., Int. Ed. 2011, 50, 10327.
MCF-7 cells were purchased from American Type Culture Col-
lection (ATCC, USA), and were seeded in Laser scanning confocal