A phenanthrene based highly selective fluorogenic and visual sensor for Cu2 + ion with nanomolar detection limit and its application in live cell imaging

Article abstract of DOI:10.1016/j.inoche.2012.08.012

A new phenanthrene based chemosensor has been synthesized and investigated to act as highly selective fluorescence and visual sensor for Cu^{2 +} ion with very low detection limit of 1.58 nM; this has also been used to image Cu^{2 +} in human cervical HeLa cancer cells.

Full text of DOI:10.1016/j.inoche.2012.08.012

Inorganic Chemistry Communications 25 (2012) 26–29
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Inorganic Chemistry Communications
journal homepage: www.elsevier.com/locate/inoche
A phenanthrene based highly selective fluorogenic and visual sensor for Cu2+ ion
with nanomolar detection limit and its application in live cell imaging
Sellamuthu Anbu a, Sankarasekaran Shanmugaraju a, Rajendran Ravishankaran b,
Anjali A. Karande b, Partha Sarathi Mukherjee a,
⁎
a
Department of Inorganic and Physical Chemistry, Indian Institute of Science, Bangalore, 560 012, India
Department of Biochemistry, Indian Institute of Science, Bangalore-560 012, India
b
a r t i c l e i n f o
a b s t r a c t
Article history:
A new phenanthrene based chemosensor has been synthesized and investigated to act as highly selective
fluorescence and visual sensor for Cu2+ ion with very low detection limit of 1.58 nM; this has also been
used to image Cu2+ in human cervical HeLa cancer cells.
Received 11 July 2012
Accepted 14 August 2012
Available online 21 August 2012
© 2012 Elsevier B.V. All rights reserved.
Keywords:
Fluorogenic Cu2+ sensors
Visual sensors
Live cell imaging
Intracellular Cu2+ detection
Research on highly selective and sensitive fluorescent probes for
3d-series metal ions has attracted great interest due to their vital
roles in many biological processes [1,2]. In this regard, substantial
efforts have been given and a large number of chemosensors have
been developed for selective sensing of particular metal cations
both in vitro and in vivo [3,4]. Fluorescent chemosensors have several
advantages over the other optical sensors due to their intrinsic high
sensitivity, easy handling and real-time monitoring with fast response
time [5,6]. In recent time, the development of selective and sensitive
imaging tools capable of rapid monitoring Cu2+ ions has attracted
great attention due to the environmental and bio-relevant nature of
Cu2+ [7–9]. Cu2+ is a vital (both useful and cytotoxic) trace ion in
various enzymatic processes. Copper deficiency may lead to several
neurological problems and excess copper in the body causes Alzheimer's,
Wilson's, and Menke's etc. diseases [10,11]. Hence, search for selective
fluorescent chemosensors for Cu2+ ions has become a great promising
area of research. Although, several fluorescent sensors for Cu2+ are
known, due to the slow response, low (mM or μM) sensitivity [12–15]
lack of high selectivity and cytotoxicities of these sensors limit them to
use for practical application [7–9]. In light of these issues, very recently
an ethynyl based fluorescent Cu2+ sensor has been reported with the
detection limit of ppb (0.1 μM) level [16]. Herein, we report highly
sensitive (nanomolar), selective phenanthrene-based fluorescent and
visual sensor (R) for Cu2+ ion with very low detection limit of ppt
level (picomolar) and its potential application in human cancer cell
bio-imaging. The fluorogenic receptor R (Scheme 1) was synthesized
in a single step by reacting 5-(4-hydroxyphenyl)salicylaldehyde with
9,10-phenanthrenequinone in 58% yield (Scheme 1). R was characterized
by FTIR, multinuclear (1H, 13C) NMR, HRMS and elemental analyses
(Figs. S1–S4).
The binding behavior of the receptor R towards different metal
cations as their chloride salts was monitored using UV–vis absorption
and fluorescence spectroscopy. All the titration studies were carried
out in H2O/CH3CN (8:2, v/v) solvent mixture. The electronic absorption
spectrum of R (5 μM) in H2O/CH3CN (8:2, v/v) exhibited three sharp
bands at 259, 351 and 366 nm (Fig. 1). Upon gradual addition of the
aqueous solution of Cu2+ ion in increasing concentration (0–100 μM),
the bands at 351 and 366 nm show slight enhancement in the initial
absorption intensity and a new absorption band centered at 396 nm
started to appear, which is attributed to the charge-transfer complex
R–Cu2+ (Fig. 1).
The appearance of a well defined isobestic point centered at λ=
282 nm is consistent to an equilibrium between R and copper complex
R–Cu2+ in solution. Furthermore, a perfect linear relationship was
obtained from the absorption titration profile for the plot (R=0.9983)
of measured [1/(A−A0)] at 396 nm as a function of 1/[Cu2+] using the
well known linear Benesi–Hildebrand expression, which indicates a
~1:1 stoichiometry complex formation between R and Cu2+ ion in
solution (Fig. 1). Calculated association constant is Ka=2.48×103 M−1
.
,
Notably, the addition of other metal cations (Na+, K+, Mg2+, Ca2+
Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Hg2+) did not alter the initial
absorption spectrum of the receptor R significantly. From these UV–vis
studies, it is clear that receptor R shows very high selective binding
affinity in the ground state only for Cu2+ ion even in the presence of
different other metal ions (Fig. 1).
⁎
Corresponding author. Tel.: +91 80 2293 2827; fax: +91 80 2360 1552.
E-mail address: psm@ipc.iisc.ernet.in (P.S. Mukherjee).
1387-7003/$ – see front matter © 2012 Elsevier B.V. All rights reserved.
http://dx.doi.org/10.1016/j.inoche.2012.08.012

S. Anbu et al. / Inorganic Chemistry Communications 25 (2012) 26–29
27
Scheme 1. Synthesis of phenanthrene-based receptor R and its Cu2+ complex.
Fig. 1. Change in the absorption spectrum (left) of R (5 μM) in the presence of increasing concentration (0–100 μM) of Cu2+ ions (inset: the Benesi–Hilder Brand plot) and change
in the absorption spectrum (right) of R upon mixing with different other metal cations.
The fluorescence spectrum of the receptor R (5 μM) exhibits a
strong emission at 435 nm in H2O/CH3CN (8:2, v/v) medium. Upon
gradual addition of increasing amounts of aqueous Cu2+ solution
(0–10 μM) to the solution of R, the fluorescence emission at 435 nm
is almost completely quenched after the addition of ~1.0 equivalent
of Cu2+ (Fig. 2). This dramatic quenching of initial fluorescence
intensity of R induced by Cu2+ ion is attributed to the reverse
photo-induced electron transfer from phenanthrene moiety to the
phenolic-OH and imidazole-N atoms due to the decrease in electron
density upon the metal ion complexation [17]. Furthermore, the
time resolved fluorescence study showed no changes in life time
(2.13 ns) of R upon the gradual titration with Cu2+, which support
that the observed fluorescence quenching follows static quenching
mechanism via the ground state complex (R–Cu2+) formations
(Fig. S5). The stoichiometry plot (Fig. 2) analysis of the fluorescence
titration profile of R (5 μM) revealed a 1:1 stoichiometry between R and
Cu2+ species and the calculated Stern–Volmer binding constant is
166.5×103 M−1. The formation of a 1:1 complex was also indicated
by ESI-MS, where obtained spectra show a peak at m/z of 518.53
corresponding to the expected [R+CuCl(H2O)] complex (Fig. S6).
In order to prove the selectivity of receptor R towards Cu2+, we
carried the fluorescence titration experiment of R with other alkali
Fig. 2. Reduction in the initial fluorescence intensity of R (5 μM) upon gradual addition of Cu2+ solution (0–10 μM) (inset: Stern–Volmer plot) (left) and its stoichiometry plot (right).

28
S. Anbu et al. / Inorganic Chemistry Communications 25 (2012) 26–29
spectroscopic conditions as used for Cu2+. The possible reason for
the high selectivity of R might be due to the paramagnetic and
unfilled d-shell of Cu2+ ion. That eventually makes the Cu2+ ion to
exhibit discernable quenching of the fluorescence intensity via
electron and/or energy transfer process [18,19]. Thus, receptor R
could be used as a highly selective fluorescence sensor for Cu2+ ion
over other metal species in aqueous medium.
To corroborate the practical applicability of receptor R as a selective
fluorescence probe for Cu2+ ion, we carried out a competitive
fluorescence titration study with other competing metal ions. As
shown in Fig. 4, the initial fluorescence intensity of R did not changed
significantly (red bar) upon mixing R with one equivalent of different
other metal cations (Na+, K+, Mg2+, Ca2+, Mn2+, Fe2+, Co2+, Ni2+
,
Zn2+, Cd2+ and Hg2+). But the subsequent addition of one equivalent
of Cu2+ solution elicited a prominent fluorescence quenching (green
bar), which further confirmed the excellent selectivity of the sensor R
for Cu2+ ion in aqueous medium even in the presence of other aforesaid
interfering metal cations.
Fig. 3. Change in the initial fluorescence intensity of receptor R (5 μM) in the presence
of 1.0 equiv of different metal cations in H2O/CH3CN (8:2, v/v) medium.
Moreover, search for visual sensors for the trace detection of desired
analytes has been a popular target in modern chemistry owing to their
ease of interpretation and more suitable tool to practice in field. The
ability of receptor R as a colorimetric probe for Cu2+ ion was imaged
using a hand-held camera in the presence of other competing metal
cations. As depicted in Fig. S8, R exhibited a distinct visual color
change from colorless to green (under room light) and blue emission
to almost dark (under UV light) after the addition of Cu2+ solution.
However, there were no observable color changes noticed upon the
mixing of R with other interfering metal cations (Na+, K+, Mg2+
,
Ca2+, Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Hg2+) solutions.
Thus, receptor R can be used as selective colorimetric sensor for Cu2+
ion over other competing metal ions in various environmental and
biological systems.
As far as real-time application is concerned, the sensing process of a
probe molecule must be a highly reversible one. To examine whether
sensing process of R is reversible, one equivalent of ethylenediamine
(en) solution was added into the solution of R which is pre-incubated
with one equivalent of Cu2+ solution. After the addition of en solution,
the initial emission intensity of R was almost recovered immediately
from non-fluorescent R–Cu2+ complex including a sharp visual color
change (Fig. 5). This result suggests the high reversibility of R towards
Cu2+ sensing and potential in application of real-time monitoring. In
addition, the fluorescence titration profile also demonstrates that R
has a detection limit of 1.58 nM (100.33 ppt) for Cu2+ (Fig. 5), which
is higher than that of other reported Cu2+ chemosensors [12–15] and
this level of detection limit is sufficient enough to sense Cu2+ ion
Fig. 4. Competitive selective binding affinity of R (5 μM) towards Cu2+ ions in the
presence of 1.0 equiv of different metal cations in H2O/CH3CN (8:2, v/v) medium.
(Na+, K+), alkaline-earth (Mg2+, Ca2+) and 3d-series (Mn2+, Fe2+
,
Co2+, Ni2+, Zn2+, Cd2+ and Hg2+) metal ions. As shown in Fig. 3,
only Cu2+ elicited a dramatic fluorescence quenching response,
while the other tested metal ions such as Na+, K+, Mg2+, Ca2+
,
Mn2+, Fe2+, Co2+, Ni2+, Zn2+, Cd2+ and Hg2+ exhibited almost
no fluorescence quenching response (Fig. S7) under the identical
Fig. 5. Changes in fluorescence intensity (left) of R (5 μM, orange line) in the presence of Cu2+ (red line) and Cu2++en (blue line) in H2O/CH3CN 8:2 (v/v) and detection limit plot
(right).

S. Anbu et al. / Inorganic Chemistry Communications 25 (2012) 26–29
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Fig. 6. Confocal fluorescence images of Cu2+ in HeLa cells (ApoTome [ZEISS] Fluorescence microscope). (A) Brightfield transmission images of HeLa cells. (B) Hoechst 33342 stained
fluorescence images of HeLa cells incubated with R (5 μM). (C) Fluorescence image of HeLa cells incubated with R (5 μM). (D) Merged images of (B and C). (E) Cells supplemented
with R (5 μM) in the growth media for 0.5 h at 37 °C and then incubated with CuCl2 (10 μM) for 1 h at 37 °C. (F) Reversibility of fluorescence was achieved by addition of EDTA
(20 μM). λex=435 nm; fluorescence images are recorded at single (465 20 nm) channel.
even in the biological systems. Because the average concentration of
Cu2+ in blood is 100–150 μg/L (15.7–23.6 μM) [20].
Appendix A. Supplementary material
Having studied the interesting photophysical properties of R such as
high sensitivity, selectivity and fast-response towards Cu2+ ion, we
further extended our study to evaluate its potential use in imaging
Cu2+ in living cells. The human cervical HeLa cancer cell lines incubated
for 0.5 h at 37 °C with different concentrations of R (1.0 and 5.0 μM)
showed bright fluorescence due to the accumulation of R within the
cells (Figs. 6 and S9). But in contrast, the staining of pre-incubated cell
with Cu2+ (5.0 and 10.0 μM) for 1 h at 37 °C exhibited almost no
fluorescence and subsequent addition of EDTA (5.0 and 10.0 μM)
regenerated the initial emission intensity of R. This result implies
that receptor R is reversible and highly cell membrane permeable
and thus R can be used as bio-sensor to probe the intracellular
Cu2+ concentration and investigate its bioactivity in living cells.
In conclusion, we have synthesized a new phenanthrene-based
visible and fluorescent sensor R, which shows highly selective, sensitive
and reversible fluorescence quenching response towards Cu2+ in
aqueous medium. In addition, we further demonstrated that receptor
R can be utilized in live cell imaging of Cu2+ ion. To the best of our
knowledge this represents a fluorescence and visual sensor with lowest
detection limit of nanomolar in solution. The excellent detection limit
[100.33 ppt] of this sensor would be useful in detection of trace quantity
of Cu2+ in biological and environmental samples.
Supplementary data to this article can be found online at http://
dx.doi.org/10.1016/j.inoche.2012.08.012.
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Acknowledgment
S. A. is grateful to UGC for D. S. Kothari fellowship, and S. S is thankful
to CSIR, India for research fellowship. Financial support from the DST,
New Delhi, India, is gratefully acknowledged.
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