Turn-On Ratiometric Fluorescent Probe for Selective Discrimination of Cr3+ from Fe3+ in Aqueous Media for Living Cell Imaging

Article abstract of DOI:10.1002/chem.201501892

Pyrene-based turn-on ratiometric fluorescent probe 1 demonstrates high sensitivity and exceptional selectivity toward Cr³⁺ in the presence of other metals, including Fe³⁺ in aqueous media. Interaction of Cr³⁺ with probe 1

Full text of DOI:10.1002/chem.201501892

DOI: 10.1002/chem.201501892
Communication
&
Host-Guest Systems
Turn-On Ratiometric Fluorescent Probe for Selective
Discrimination of Cr3+ from Fe3+ in Aqueous Media for Living Cell
Imaging
Lubna Rasheed,[a] Muhammad Yousuf,[a, b] Il Seung Youn,[a, b] Taeseung Yoon,[a] Kwang-
Youn Kim,[c] Young-Kyo Seo,[c] Genggongwo Shi,[a, b] Muhammad Saleh,[a, b] Jin-Hoe Hur,[d]
and Kwang S. Kim*[a]
vitro and in vivo levels of biologically relevant species and un-
derstanding their functions.[3]
Abstract: Pyrene-based turn-on ratiometric fluorescent
probe 1 demonstrates high sensitivity and exceptional se-
Chromium ion (Cr3+), as an essential micronutrient in
lectivity toward Cr3+ in the presence of other metals, in-
humans, has great impacts on the metabolism of carbohy-
cluding Fe3+ in aqueous media. Interaction of Cr3+ with
drates, fats, proteins, and nucleic acids by activating certain en-
zymes and stabilizing proteins and nucleic acids.[4] Chromium
probe 1 brings pyrene moieties close enough to have
better aligned p–p stacking, thus enhancing the excimer
deficiency leads to an increase in risk factors associated with
diabetes and cardiovascular disease, including elevated levels
peak many fold. On the other hand, the interaction of
Fe3+ with probe 1 brings forth a negligible difference in
of circulating insulin, glucose, triglycerides, and total cholester-
ol.[5] Excessive intake of Cr3+ negatively affects cellular struc-
stacking, resulting in an insignificant change in fluores-
tures and functions, although its toxicity observed in vivo is
cence intensity. Exceptional selectivity of probe 1 with
much lower compared to that of Cr4+ [6]
.
Cr3+ over Fe3+ and other metals has been confirmed by
theoretical studies in addition to experimental results.
Imaging of HeLa cells observed by confocal fluorescence
microscopy reveals that probe 1 can be used to monitor
Cr3+ in live cells to map its subcellular distribution.
The development of suitable turn-on fluorescence sensors
for monitoring intercellular Cr3+ is limited due to fluorescent
quenching of paramagnetic Cr3+ and lack of Cr3+ selective
multichelating ligand.[7] Hitherto, few methods have been de-
veloped to monitor Cr3+ transformation in the intracellular
compartment or its distribution in living systems.[8] Real-time
monitoring of Cr3+ in living organisms requires a suitable che-
mosensor, which selectively distinguishes Cr3+ from Fe3+ with
high selectivity in aqueous media. Liu and co-workers reported
rhodamine-based chemosensors which can distinguish Cr3+
from Fe3+ with moderate selectivity.[8a] Most of the probes re-
ported for Cr3+ are usually based on a single emission intensity
change,[7,8e] which tends to be affected by a variety of factors
such as probe molecule concentration, instrumental efficiency,
and microenvironment. Ratiometric probes can circumvent
these limitations, which are advantageous for applications in
biological systems.[8c] To the best of our knowledge, none of
the reported ratiometric sensors are capable in selective dis-
crimination of Cr3+ from Fe3+ in aqueous media.
Most of first two rows of transition-metal ions are ubiquitous
in nature and involve several biologically important functions.[1]
Excess accumulation of these ions in biosystems can lead to
diseases and ultimately to fatal consequences.[2] Distribution of
these metal ions inside and outside the cells is, therefore, of
paramount importance to understand physiological condi-
tions.[1–2] On account of simplicity and high sensitivity, fluores-
cent sensors have become powerful tools for monitoring in
[a] Dr. L. Rasheed,+ M. Yousuf,+ I. S. Youn, T. Yoon, G. Shi, M. Saleh,
Prof. Dr. K. S. Kim
Center for Superfunctional Materials, Department of Chemistry
Ulsan National Institute of Science and Technology (UNIST)
Ulsan 689-798 (Republic of Korea)
In an attempt to overcome the limitations associated with
the previously developed methods, we have synthesized
pyrene-based probe 1 (Figure 1) that selectively differentiates
Cr3+ from Fe3+ ratiometrically in aqueous media by complexa-
tion-enhanced excimer formation between two pyrene moiet-
ies. Moreover, probe 1 has been successfully used to image
Cr3+ in living cells. To the best of our knowledge, probe 1 is
the first sensor that selectively discriminates Cr3+ from Fe3+ ra-
tiometrically in aqueous media.
E-mail: kimks@unist.ac.kr
[b] M. Yousuf,+ I. S. Youn, G. Shi, M. Saleh
Department of Chemistry, Pohang University of Science and Technology
Pohang, 790-784 (Republic of Korea)
[c] Dr. K.-Y. Kim, Dr. Y.-K. Seo
School of Life Sciences, Ulsan National Institute of Science and Technology
Ulsan 689-798 (Republic of Korea)
[d] J.-H. Hur
UNIST Central Research Facilities (UCRF)
Ulsan National Institute of Science and Technology
Ulsan 689-798 (Republic of Korea)
The synthesis of probe 1 is described in the Supporting In-
formation. First, probe 1 was applied for cation recognition
studies. Visual features clearly showed fluorescence enhance-
ment upon addition of Cr3+ (Figure 2a). Probe 1 displayed mo-
[+] Authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/chem.201501892.
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Figure 1. Probe 1.
Figure 3. a) Fluorescent titration of 1 (10 mm) upon the addition of the ni-
trate salt of Cr3+ at pH 7.4 (0.01m HEPES buffer), (slit width=3 nm;
lexc =350 nm, 1% DMSO). Inset: change in peak intensity at 384 nm. b) Cor-
responding binding isotherm. Inset: Change in fluorescence intensity vs.
equivalent of Cr3+
.
Figure 2. a) Visual fluorescence features of 1 (10 mm) upon the addition of
Cr(NO3)3 at pH 7.4 (0.01m HEPES buffer), 99% H2O and 1% DMSO. b) Fluo-
rescent emission changes of 1 (10 mm) upon addition of nitrate salts of Ca2+
,
Cr3+, Co2+, Cu2+, Cd2+, Al3+, Fe3+, Fe2+, Pb2+, Li1+, Mn2+, Mg2+, Ni2+, K1+
,
and Zn2+ (slit width=3 nm; lexc =350 nm, 1% DMSO).
ure S9, Supporting Information). Probe 1 showed 1:1 binding
stoichiometry with Cr3+ as evident from the job’s plot (Fig-
ure S10, Supporting Information). The binding constant of
probe 1 upon complexation with Cr3+ was calculated to be
7.22”105 mÀ1 (Figure 3 and Figure S7, Supporting Informa-
tion)[11] and the detection limit was calculated to be 1.98”
10À5 m (Figure S8, Supporting Information).[12] The absorption
nomer (385 and 402 nm) and excimer (475 nm) peaks arising
from intramolecular p–p stacking between pyrene rings (quan-
tum yield=0.07).[9] Probe 1 showed a minor change in fluores-
cence upon addition of cations except Cr3+ (Figure 2b). Probe
1 displayed the most significant fluorescence enhancement for
excimer (475 nm) and quenching for monomer (385 nm) (Fig-
ure 2b and 3) upon addition of Cr3+ (quantum yield=0.85).
The complexation of probe 1 with Cr3+ brings pyrene moieties
close enough to experience enhanced p–p stacking which
might be the main cause of fluorescence enhancement for ex-
cimer and quenching for monomer.[10] This fluorescence en-
hancement and quenching can be exploited as an effective
fluorescence discrimination of Cr3+ from other cations ratio-
metrically (Figure 3). Moreover, the ratio of the fluorescence in-
tensities of Probe 1 at 475 and 385 nm linearly correlates with
the concentration of Cr3+ with a correlation coefficient of R=
0.9978 (Figure S6, Supporting Information).
spectra of probe
1 (Figure S10, Supporting Information)
showed the characteristic absorption maximum at 366 nm due
to the pyrene moiety. Absorption spectra of probe 1 with Cr3+
(11 equiv) showed a slight decrease in absorption with distin-
guished redshift by 4 nm at 324 nm and 2 nm at 366 nm (Fig-
ure S11, Supporting Information).
To monitor the physical interaction of probe 1 with Cr3+
1
,
a H NMR titration experiment was conducted (Figure S5, Sup-
porting Information). Quenching with downfield shift (d<
1.2 ppm) of iminic proton (Ha) was observed upon interaction
of probe 1 with Cr3+, which suggests strong interaction of
imine N with Cr3+ and increased p–p stacking between the
pyrene moieties. This is supported by quenching and broaden-
ing of pyrene protons (Hb, Hc, and Hd), which caused the de-
shielding effect at the iminic functionality. Quenching and
broadening associated with an upfield shift (d<0.2 ppm) of
The competitive binding experiment of probe 1 for Cr3+
was conducted in the presence of competing cations, and fluo-
rescence response was observed to be almost unaffected (Fig-
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benzene and pyridine ring protons (He–i) were observed which
might be due to the strong interaction of O and N with Cr3+
.
In the computational results, partial p–p interaction be-
tween two pyrene moieties is found, which is responsible for
the excimer peak (Figure 4, and Figures S18 and S19, Support-
ing Information). There are two possibilities for cations to be
Figure 5. Confocal fluorescence images in HeLa cells. a) Cells with probe 1.
b) Cells incubated with probe 1 for 30 min, washed, and then further incu-
bated with 50 mm Cr3+ for 30 min (lex =405 nm). c) Brightfield image of
cells. d) overlay image of (b) and (c).
Figure 4. Computed structures of 1-Cr3+ and 1-Fe3+ at the B97D3/aVDZ
level (white: H; grey: C; red: O; blue: N; pale blue: Cr; purple: Fe; red-
dotted line: coordination bond).
the peri-nuclear area of the cytosol, indicating a subcellular dis-
tribution of Cr3+ and good cell-membrane permeability of
probe 1 (Figure 5c and Figure S16, Supporting Information). A
high-resolution image was obtained from differential interfer-
ence contrast (DIC) by excitation of probe 1 loaded with HeLa
cells in the presence and absence of Cr3+ at 405, 488, and
559 nm, while emission was collected at 450, 510, and 610 nm
for blue, green, and red channels, respectively (Figures S12–14,
Supporting Information). The relative fluorescence intensity
(450 nm) in each picture was measured and the results
showed that a submicromolar concentration of probe 1 is suffi-
cient for practical imaging purposes (Figure S15, Supporting In-
formation).
added; they can either be intercalated into two pyrene moie-
ties or interact with N and O atoms through coordination
bonding. The calculation results suggest that the latter is pre-
ferred by both Cr3+ and Fe3+. However, the addition of Cr3+
causes better aligned p–p stacking of pyrene moieties. The ad-
dition of Fe3+ results in no substantial difference in stacking,
while one of the pyrene rings strayed out followed by
a weaker p–p interaction (Figure 4). Therefore, the perfect
stacking after adding Cr3+ between the pyrene rings is proba-
bly the main reason of fluorescence enhancement.
Probe 1 can effectively be used for bioimaging applications
owing to the significant difference in quantum yield between
1 and 1+Cr3+. Prior to the bioimaging studies, a good level of
cell viability in the presence of probe 1 was confirmed by MTT
assay (Figure S17, Supporting Information).[13] A practical bio-
imaging application of probe 1 for Cr3+ in biological samples
was conducted by confocal fluorescence microscopy using
LSM 780 NLO (Carl Zeiss) and a 63” oil-immersion objective
lens. Human cervical cancer cell lines HeLa were used for bio-
imaging purposes. Under selective excitation of probe 1 at
405 nm, staining HeLa cells with a 1 mm solution of probe
1 for 30 min at 378C led to very weak intracellular fluorescence
(Figure 5a and Figure S15, Supporting Information). The cells
were then supplemented with 50 mm Cr3+ in the growth
medium for 30 min at 378C loaded with probe 1 under the
same conditions; a significant increase in the fluorescence
from the intracellular area was observed (Figure 5b). Bright-
field measurements confirmed that the cells, after treatment of
probe 1 and Cr3+, were visible throughout the imaging experi-
ments (Figure 5d). Overlay of fluorescence and brightfield
images revealed that the fluorescence signals were localized in
In summary, we have developed a new pyrene-based fluo-
rescent probe 1 which exhibits high sensitivity and excellent
selectivity toward Cr3+ ratiometrically in the presence of other
metals including Fe3+ in aqueous media. Experiments and cal-
culations reported here provide new insights into the selectivi-
ties of novel fluorescent probe 1 which senses Cr3+. Addition
of Cr3+ causes better aligned p–p stacking of pyrene moieties
of probe 1 which results in many fold enhancement of excimer
formation. The addition of Fe3+ marks no substantial difference
in stacking compared to probe 1 alone, while one of the
pyrene rings strayed out, followed by weaker p–p interaction.
Confocal fluorescence microscopy experiment reveals that
probe 1 can be used to monitor Cr3+ in living cells and to map
its subcellular distribution. We anticipate that this probe will
be of great benefit to biomedical researchers for studying the
bioactivity of Cr3+ in biological systems. The experimental re-
sults of this study will provide the basis for a new strategy for
the design and development of fluorescent chemosensors
showing high selectivity towards Cr3+
.
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NÀ); 13C NMR (100 MHz, (CD3)2SO, 258C): d=71.070, 114.212,
120.358, 120.890, 121.724, 123.120, 123.264, 124.023, 124.941,
124.987, 125.950, 126.239, 126.535, 126.709, 127.286, 127.362,
128.864, 128.963, 129.759, 130.055, 130.731, 132.764, 141.998,
150.929, 156.695, 160.307, 162.667 ppm; elemental analysis calcd
(%) for C53H35N3O2: C 85.35, H 4.73, N 5.63; found: C 85.05, H 4.77,
N 5.61.
Experimental Section
General
The synthesized compounds were fully characterized with standard
spectroscopic techniques. Microanalyses were performed on
a Carlo 1102 elemental analysis instrument. Electronic absorption
(UV/Vis) spectra were recorded using a Shanghi 756 MC UV/Vis
spectrometer.1H and 13C NMR spectra were performed on an Agi-
lent (400 MHz) spectrometer at 298 K. HRMS were obtained on
a Micromass Platform II mass spectrometer. Fluorescence studies
were carried out on Shimadzu RF-5301 PC spectrofluorophotome-
ter at 298 K. Confocal fluorescence imaging was performed by
using LSM 780 NLO (Carl Zeiss) and a 63” oil-immersion objective
lens. Human cervical cancer cell lines HeLa were obtained from the
American Type Culture Collection (ATCC, Manassas, VA, USA). Dul-
becco’s modified Eagle’s medium, fetal bovine serum, penicillin,
and streptomycin were obtained from BioWhiteker, Walkersville,
MD. Pyrene aldehyde, 2-amino phenol, 2,6-bis(bromomethyl)pyri-
dine, potassium carbonate, and metabolize 3-(4,5-dimethyldiazol-2-
yl)-2,5- diphenyltetrazolium bromide (MTT) were purchased from
Aldrich and were used without further purification. Barium nitrate,
calcium nitrate, chromium(III) nitrate, cobalt nitrate, copper(II) ni-
trate, cadmium nitrate, iron(III) nitrate, iron(II) nitrate, lead nitrate,
lithium nitrate, manganese(II) nitrate, magnesium nitrate, nickel ni-
trate, potassium nitrate and zinc nitrate were also purchased from
Aldrich and used without further purification.
Acknowledgements
This work was supported by the NRF (National honor scientist
program) (2010-0020414) and KISTI (KSC-2014-C3-019).
Keywords: chromium
·
density functional theory
·
fluorescence · pyrene · ratiometric
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(400 MHz, DMSO): d=6.962 (dt, J=7.2, 1.2 Hz 1H), 7.013 (dd, J=
7.2, 1.2 Hz 1H), 7.183 (dt, J=5.2, 2 Hz 1H) 7.535 (dd, J=6.4 Hz, J=
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Synthesis of probe 1
A
solution of 2-[(E)-(1-pyrenylmethylene)amino]phenol (a)
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82 mg) dissolved in anhydrous of CH3CN (10 mL) was added to the
reaction mixture. The reaction mixture was then refluxed for 24 h.
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1 in 80% yield. H NMR (400 MHz, (CD3)2SO, 258C): d=5.312 (s, 8H,
ÀCH2À), 7.073 (m, 2H), 7.191 (dd, 4H, J=3.8, 0.8 Hz), 7.35 (d, 2H,
J=7.6 Hz), 7.614 (d, 2H, J=8 Hz), 7.815 (t, 1H, J=8 Hz), 8.081 (t,
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Received: May 14, 2015
Published online on September 29, 2015
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