Rationally designed fluorescence 'turn-on' sensor for Cu2+

Article abstract of DOI:10.1039/c0cc05421f

A rationally designed, coumarin-based fluorescent sensor imino-coumarin (IC) displays high selectivity for Cu²⁺ over a variety of competing metal ions in aqueous solution with a significant fluorescence increase. DFT/TDDFT calculations support that the fluorescence 'turn-on' of IC originates from blocking the electron transfer of the nitrogen lone pair upon complexation with Cu²⁺. IC was successfully applied to microscopic imaging for detection of Cu²⁺ in LLC-MK2 cells (in vitro) and several living organs (in vivo).

Full text of DOI:10.1039/c0cc05421f

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COMMUNICATION
Rationally designed fluorescence ‘turn-on’ sensor for Cu2+w
Kyoung Chul Ko,a Jia-Sheng Wu,b Hyun Jung Kim,b Pil Seung Kwon,c Jong Wan Kim,d
Richard A. Bartsch,e Jin Yong Lee*a and Jong Seung Kim*b
Received 7th December 2010, Accepted 6th January 2011
DOI: 10.1039/c0cc05421f
A rationally designed, coumarin-based fluorescent sensor imino-
coumarin (IC) displays high selectivity for Cu2+ over a variety of
competing metal ions in aqueous solution with a significant fluores-
cence increase. DFT/TDDFT calculations support that the fluores-
cence ‘turn-on’ of IC originates from blocking the electron transfer
of the nitrogen lone pair upon complexation with Cu2+. IC was
successfully applied to microscopic imaging for detection of Cu2+ in
LLC-MK2 cells (in vitro) and several living organs (in vivo).
charge transfer (MLCT),6 intramolecular charge transfer
(ICT),7 excimer/exciplex formation,8 and excited-state intra-/
intermolecular proton transfer (ESIPT).9 Recently, Wang et al.
raised a new signal mechanism, CQN isomerisation, for the
design of fluorescent sensors.10 We further developed this
mechanism as fluorescence enhancement by orbital control
(FEOC) for Cu2+ 11
To design a new fluorescent ‘‘turn-on’’
.
Cu2+ sensor, herein, we intend to adapt the recent strategy
FEOC.11 The basic architecture of the FEOC sensor is comprised
of a fluorophore, the sp2 hybridized nitrogen lone pair (–CQN–),
and a chelator site.10 Based on such a logical strategy,
we designed and synthesized the imino-coumarin IC (Fig. 1).
IC exhibits a distinct fluorescence increase in the presence of
Cu2+, while competing metal ions do not induce fluorescence
changes. The fluorescence behaviour was originated from the
blocking of the electron transfer from the nitrogen lone pair
to the fluorophore upon complexation with Cu2+ as revealed
by DFT/TDDFT calculations. IC was successfully applied to
microscopic imaging for detection of Cu2+ in LLC-MK2 cells
(in vitro) and several living organs (in vivo).
Copper is the third element in abundance among the essential
heavy metals (next to iron and zinc) in the human body and plays
an important role in various physiological processes.1 Abnormal
uptake of certain levels of Cu2+ by animals is known to cause
Wilson’s disease, gastrointestinal disease, hypoglycemia, dyslexia,
and infant liver damage.2 Thus, detecting trace amounts of Cu2+ is
important not only for environmental applications, but also for
toxicity determinations in living organs. One of the tools for
detecting Cu2+ is to utilize a fluorescent chemosensor. Cu2+
complexation is well known to induce intrinsic fluorescence
quenching,1,3 and most artificial fluorescent chemosensors give rise
to quenching of signals upon Cu2+ binding.1 Only a few examples
IC was synthesized by refluxing 7-diethylamino-coumarin-
3-aldehyde10 and 4-amino-5-phenyl-4H-1,2,4-triazole-3-thiol in
absolute ethanol to give an 85% yield of the chemosensor. The
were reported to be fluorescence turn-on sensors toward Cu2+ 4
.
Most of these studies have been conducted in organic media due to
poor solubility of the chemosensors in aqueous solution. For
practical applications, fluorescence sensors which ‘turn-on’ in the
presence of the analyte are superior to those which ‘turn-off’. Also,
sensing in organic media has intrinsic difficulties for applications in
environmental and biological systems.4a,b Therefore, a fluorescence
‘turn-on’ chemosensor with high selectivity towards Cu2+ that
functions in aqueous solution is highly desirable.
1
molecular structure of IC was verified by H- and 13C-NMR
spectroscopy (Fig. S12 and S13, ESIw, respectively), FAB-MS,
and X-ray crystal structure analysis (Fig. 1).z
IC shows a characteristic UV-Vis absorption band at 450 nm
in 80% aqueous MeCN (v/v). In the presence of Cu2+, the
absorption at 450 nm decreased and a small irregular band
appeared at longer wavelength (Fig. S1, ESIw). Fluorescence
titration spectra of Cu2+ for 3.0 mM IC in 80% aqueous MeCN
are shown in Fig. 2. IC is a weak fluorescent molecule with a
To date, a variety of signaling mechanisms have been proposed
and utilized for optical detection of different species, including
photo-induced electron/energy transfer (PET),5 metal–ligand
maximum emission at 515 nm. Upon gradual addition of Cu2+
,
fluorescence was remarkably enhanced with a red shift to
a Department of Chemistry, Sungkyunkwan University,
Suwon 440-746, Republic of Korea. E-mail: jinylee@skku.edu
b Department of Chemistry, Korea University, Seoul 136-701,
Republic of Korea. E-mail: jongskim@korea.ac.kr
525 nm. Job’s plot analysis of the fluorescence spectra
(Fig. S2, ESIw) showed a maximum at 0.5 mole fraction of Cu2+
,
c Department of Clinical Laboratory Science, Wonkwang Health
Science University, Iksan 570-750, Korea
d Department of Laboratory Medicine, Dankook University Hospital,
Cheonan 330-715, Republic of Korea
e Department of Chemistry and Biochemistry, Texas Tech University,
Lubbock, Texas 79401, USA
w Electronic supplementary information (ESI) available: Experimental
procedures, calculation details, cell incubation procedures, and additional
figures. CCDC 641149. For ESI and crystallographic data in CIF or
other electronic format see DOI: 10.1039/c0cc05421f
Fig. 1 Structure of a new chemosensor IC and crystal structure of IC
with displacement atomic ellipsoids drawn at the 30% probability level.
c
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more stable than CS by 4.41 kcal molꢁ1, however, in PCM
calculation considering the solvent effect, the energy difference
was only 0.88 kcal molꢁ1 (Fig. S5, ESIw).14 The gas-phase
structure is not always the same as the solid or liquid one, as
previously reported.15 In addition, the bare structure may be
changed when coordinated as observed in our recent study.11
Furthermore, for IC, CS can bind Cu2+ more tightly than BS
because it is better organized. Therefore, we adopted CS to
study the fluorescence behavior of IC and IC–Cu2+ complex.
The optimized structures of IC and IC–Cu2+ are shown in
Fig. 4. For IC–Cu2+, various geometries were tested, and
reported in ESI.w According to calculated results and our
recent study,16 in IC–Cu2+ complex, two water molecules are
believed to be involved as shown in Fig. 4. IC has a little
structural change from IC upon Cu2+ binding; the dihedral
Fig. 2 Fluorescence titration spectra of IC (3.0 mM) in 80% aqueous
MeCN(phosphatebuffer, pH=7.2)uponadditionofCuCl2 (0–20.0mM)
with excitation at 460 nm.
indicating the formation of a 1 : 1 adduct between IC and
Cu2+. A peak at m/z 482.1674 corresponding to the adduct
(IC–H + Cu)+ in the MALDI-TOF mass spectrum (Fig. S3,
ESIw) provides further evidence for a 1 : 1 complex between
IC and Cu2+. Based upon a 1 : 1 stoichiometry, the associa-
tion constant of IC with Cu2+ was calculated to be 3.34 ꢀ 104
Mꢁ1 from the fluorescence titration data.12
angle CQN–N–C is 44.31 in IC and 41.31 in IC–Cu2+
.
From the TDDFT calculations, lmax of IC and IC–Cu2+
were calculated to be 386 and 416 nm, respectively, which are in
good agreement with the experiment. In IC, the contributions of
HOMO-2 - LUMO, HOMO-1 - LUMO and HOMO -
LUMO transitions were 2.6, 94.0 and 3.4%, respectively. Due to
the importance of the lone pair electrons of the nitrogen atom,11
we carefully analyzed its contribution to the relevant orbitals. It
was found that the nitrogen lone pair electron belongs to
HOMO-2 in IC. Thus, HOMO-2 - LUMO transition (2.6%)
is responsible for the fluorescence quenching. On the other
hand, in IC–Cu2+, the nitrogen atom has strong interaction
with Cu2+ d-orbital to produce bonding type orbitals that block
the PET process from the nitrogen lone pair electron to the
coumarin, hence block the fluorescence quenching (Fig. S7–S10,
ESIw). The possibility of the restricted CQN isomerization
process of IC on coordination to Cu2+ could also contribute
to the fluorescence enhancement along with the PET process.10
Biosensor molecules that can selectively monitor guest species
in living systems have attracted a great attention.17 In this
context, we carried out an in vitro biological test utilizing IC.
To determine the cell permeability of IC, kidney cells of a
monkey (LLC-MK2) were incubated with IC (3.0 mM). As
shown in Fig. 5, IC displays a non-fluorescent image in the
absence of Cu2+, whereas a strong confocal image with green
fluorescence upon addition of Cu2+. Thus, IC has potential
To assess the pH dependence of the fluorescent response of IC
to Cu2+, fluorescence changes of IC (3.0 mM) in the absence and
presence of 25 equivalents of Cu2+ in 80% aqueous MeCN were
determined (Fig. S4, ESIw). In the absence of Cu2+, IC gave only
slight fluorescence variation as the pH of buffer solution varies
from 1 to 13. On the other hand, in the presence of Cu2+, the
fluorescence intensity at 515 nm exhibited marked increase in the
region of pH 4–11 witha goodplateau between pH5 and 8. Thus,
the optimal pH range for detection of Cu2+ is pH 5–8. Hereafter,
the discussion will be done for the spectral data performed in
phosphate buffer solution (pH = 7.2) in 80% MeCN.
Fig. 3 displays the fluorescence changes of IC in the presence
of various metal cations. The very weak fluorescence of IC was
only marginally increased upon addition of a mixture includ-
ing the four alkali metal cations, the four alkaline earth metal
cations, Cd2+, Co2+, Fe2+, Hg2+, Ni2+, Pb2+ and Zn2+
.
However, when Cu2+ was added, the fluorescence was
remarkably enhanced, indicating that IC is an excellent selective
fluorescent ‘‘turn-on’’ sensor for Cu2+
.
The crystal structure (bare structure, BS) seems to be
inadequate for metal ion coordination, thus, we calculated
another structure (coordination structure, CS) as well as the
crystal structure by DFT calculations with 6-31G* basis sets
using a suite of Gaussian 03 programs.13 In gas-phase, BS was
applicability for in vitro qualitative detection of Cu2+
.
In addition to the cell line in vitro study, the contrast
enhancing effect of IC with Cu2+ was further evaluated in
ICR (Institute for Cancer Research) mice, in vivo. Necropsy
was performed on a mouse after 3 days of oral feeding with
Cu2+. Lung, heart, liver, muscle, kidneys, and spleen were
extracted, washed with phosphate buffered saline, and
Fig. 3 Fluorescence spectra of IC (3.0 mM) (phosphate buffer,
pH = 7.2) in 80% aqueous MeCN in the presence of Cu2+ (20 mM)
and a metal ion mixture of the four alkali metal cations, the four
alkaline earth metal cations, Cd2+, Co2+, Fe2+, Hg2+, Ni2+, Pb2+
and Zn2+ (100 mM in each) with excitation at 460 nm.
Fig. 4 Calculated structures of (a) IC and (b) IC–Cu2+
.
c
3166 Chem. Commun., 2011, 47, 3165–3167
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a = 9.57100(10) A, b = 15.5340(8) A, c = 16.3360(6) A, V =
2057.69(13) A3, T = 293(2) K, Z = 4, D = 1.354 Mg mꢁ3, r =
0.187 mmꢁ1, F(000) = 880; 11 777 reflections measured, of which 4187
were unique (Rint = 0.0419). 273 refined parameters, final R1 = 0.0599 for
reflections with I > 2s(I), wR2 = 0.1448 (all data), GOF = 1.222. Final
largest diffraction peak and hole: 0.547 and ꢁ0.511 e Aꢁ3. CCDC 641149.
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Fig. 5 Confocal fluorescence images of IC with Cu2+ in LLC-MK2
cells (excitation at 458 nm, Zeiss LSM 510 META confocal micro-
scope, ꢀ20 objective lens) in D-MEM (Dulbeccos-Modified Eagles
Medium). Fluorescence image of (a) LLC-MK2 cells with IC (3.0 mM)
(phosphate buffer, pH = 7.2) in 80% aqueous MeCN and (b) after
addition of Cu2+. (c) Bright-field transmission image of LLC-MK2
cells (50.0 mM). (d) An overlay image of (b) and (c).
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Fig. 6 Fire scale images of mouse organs with IC (20.0 mM)
(phosphate buffer, pH = 7.2) in 80% aqueous MeCN and after
addition of Cu2+ (80.0 mM).
subjected to imaging. The images reveal that Cu2+ accumu-
lated only in the liver and kidney of the fed mouse as seen in
Fig. 6, which is consistent with a previous report that Cu2+
preferentially accumulates in these organs.8a Thus, IC has a
capability to sense Cu2+-accumulation in specific organs.
Since mouse urine is deeply related to fundamental meta-
bolic processes,18 using IC we also tried to detect Cu2+ in
urine collected from a Cu2+-fed mouse and a control mouse.
Fig. S11 (ESIw) shows the fluorescence spectra of IC exposed
to collected urine of the two mice, monitored at wavelengths of
460–700 nm upon excitation at 450 nm. The fluorescence
intensity was strong at 531 nm when IC was added to the
urine which was collected from a Cu2+-fed mouse. On the
other hand, urine from the control mouse gave only weak
fluorescence, as in the case of a Cu2+-fed mouse without IC.
These results suggest that IC could be developed as a signaling
system for Cu2+ in biological samples and encourages the
possible application for Cu2+ detection in human urine.
This work was supported by CRI project (2010-0000728)
from National Research Foundation of Korea. The work at
SKKU was supported by the NRF grants (No. 2010-0001630)
and (No. 2010-0000166) funded by MEST, Republic of Korea.
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Notes and References
z Single crystal data for IC: C22H21N5O2S, Mw = 419.50, plate orange
crystal, size: 0.30 ꢀ 0.15 ꢀ 0.12 mm, monoclinic, space group P21/c,
c
This journal is The Royal Society of Chemistry 2011
Chem. Commun., 2011, 47, 3165–3167 3167