NIR-and FRET-based sensing of Cu2+ and S2- in physiological conditions and in live cells

Article abstract of DOI:10.1021/ic301872q

We have synthesized a new indole functionalized rhodamine derivative L ₁ which specifically binds to Cu²⁺ in the presence of large excess of other competing ions with visually observable changes in their electronic and fluorescence spectral behavior. These spectral changes are significant enough in the NIR and visible region of the spectrum and thus enable naked eye detection. The receptor, L₁, could be employed as a resonance energy transfer (RET) based sensor for detection of Cu²⁺ based on the process involving the donor indole and the acceptor Cu²⁺ bound xanthene fragment. Studies reveal that L₁-Cu complex is selectively and fully reversible in presence of sulfide anions. Further, fluorescence microscopic studies confirmed that the reagent L₁ could also be used as an imaging probe for detection of uptake of these ions in HeLa cells.

Full text of DOI:10.1021/ic301872q

Article
pubs.acs.org/IC
NIR- and FRET-Based Sensing of Cu2+ and S2‑ in Physiological
Conditions and in Live Cells
Chirantan Kar,† Manab Deb Adhikari,‡ Aiyagari Ramesh,*,‡ and Gopal Das*,†
†Department of Chemistry, Indian Institute of Technology Guwahati, Assam, 781 039, India
‡Department of Biotechnology, Indian Institute of Technology Guwahati, Assam, 781 039, India
S
* Supporting Information
ABSTRACT: We have synthesized a new indole functionalized
rhodamine derivative L1 which specifically binds to Cu2+ in the presence
of large excess of other competing ions with visually observable changes
in their electronic and fluorescence spectral behavior. These spectral
changes are significant enough in the NIR and visible region of the
spectrum and thus enable naked eye detection. The receptor, L1,
could be employed as a resonance energy transfer (RET) based sensor
for detection of Cu2+ based on the process involving the donor indole
and the acceptor Cu2+ bound xanthene fragment. Studies reveal that
L1−Cu complex is selectively and fully reversible in presence of sulfide
anions. Further, fluorescence microscopic studies confirmed that the
reagent L1 could also be used as an imaging probe for detection of
uptake of these ions in HeLa cells.
largely used in industrial processes, for instances, conversion
into sulfur, preparation of sulfuric acid and dyes, cosmetic
manufacturing, production of wood pulp, etc.9 Apart from
industrial processes sulfide anions can also be generated due to
microbial reduction of sulfate by anaerobic bacteria or formed
from the sulfur-containing amino acids in meat proteins.9 Con-
sequently, there are enough risks for the sulfide ions to be
exposed to drinking water. Sulfide can damage mucous mem-
branes and can cause unconsciousness and respiratory problems.9,10
The protonated forms HS− or H2S are even more toxic than the
sulfide itself. At a low concentration, H2S can produce dizziness,
while at a higher concentration it can result in loss of consciousness,
permanent damage of brain tissues, or even death through
suffocation.11 Therefore, development of a quick and sensitive
method for immediate sulfide detection in aqueous media and
in biological systems is of high interest.
In the recent literature, a large number of colorimetric and
fluorescent chemosensors have been reported for selective
sensing of copper and sulfide ions with absorbance and emis-
sion in the visible region.12−22 Although chemosensors with
spectroscopic responses (absorbance/emission) in the visible
region have important roles in various research areas, recently
molecular probes with near-infrared (NIR, 700−1000 nm)
optical responses are particularly gaining special interest.23 As
for NIR radiation the possibility of scattering is minimized, so
unlike UV−vis radiation, NIR radiation provides high sample
penetration. Additionally, the autofluorescence generated from
the chromophores and macromolecules present in the analytic
INTRODUCTION
■
The general basis of designing a molecular sensor for selective
recognition of different species depends upon host−guest
interaction promoted by hydrogen bonding, electrostatic force,
metal−ligand coordination, and hydrophobic and van der
Waals interaction.1 In recent years, the development of novel
colorimetric and fluorescent sensors of biologically active metal
ions has been extensively investigated because of their potential
applications in life sciences, medicine, chemistry, and bio-
technology.2 Copper is one of the most abundant essential trace
elements found in the human body and has a fundamental role
to sustain important physiological processes.3 Copper-containing
enzymes play a significant role in different catalytic processes
starting from providing energy for biochemical reactions to
assisting the formation of cross-links in collagen and elastin, and
thereby maintaining and repairing connective tissues related to
heart and arteries.4 On the basis of research findings, it has been
suggested that copper deficiency can increase the risk of develop-
ing coronary heart disease.5 However, copper in excessive
amounts could lead to detrimental effects by causing oxidative
stress and disorders associated with neurodegenerative diseases
including Alzheimer’s, Parkinson’s, Menkes, Wilson’s, and prion
diseases.6,7 Owing to the significant physiological relevance and
associated biomedical implications, there is considerable interest
in developing selective and sensitive copper sensors. However,
the challenge is in the development of sensors which are bio-
compatible and functional in the complex biological milieu.
Development of selective and efficient signaling units for
detection of various chemically and biologically important
anions has also attained significant interest.8 Being one of the
biologically and environmentally important anions, sulfide is
Received: August 28, 2012
Published: January 9, 2013
© 2013 American Chemical Society
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samples are less likely to interfere. This phenomenon enables
the assessment of molecular and physiological events in several
layers deep inside the analyte samples and tissues.24 There have
been few reports on UV−vis−NIR sensing of cation and
anions.25−29
live HeLa cells via a characteristic fluorescence switch ON/OFF
mechanism. We have reasonably chosen rhodamine and indole
derivatives as the two fluorophores for designing the receptor
L1; the binding of Cu2+ with L1 triggers the opening of the
spyrolactum ring of the rhodamine derivative, whose absorption
spectra shows a significant spectral overlap with the emis-
sion spectra of the indole moiety and offers the chance of a
RET process. In addition to the FRET signal, an increased
absorbance in the NIR region of the UV−vis spectra is also
noticed. The “switch ON” behavior of the receptor L1 is ob-
served selectively in the presence of Cu2+ ions. The formation
of the L1−Cu complex is reversible, but the reversibility of the
L1−Cu system to regenerate L1 is noticeable only in the
presence of S2‑ anions. Details about the synthesis of the
receptor L1 and the control compound L2 are discussed below,
and their characterization data are presented in the Supporting
Information.
Together with visible and NIR absorbance signals, a fluo-
rescence response at a visible region would be an added advan-
tage to the sensing of cations and anions over the methods
involving just one kind of optical response. Moreover, the fluo-
rescence chemosensors are known for their efficiency with
regard to sensitivity, specificity, and fast response real-time
monitoring.30 Commonly, one of the three different photoinduced
processes are involved for the signaling event of luminescence
based chemosensors, namely, PET (photoinduced electron
transfer),31 PCT (photoinduced charge transfer),32 and RET
(resonance energy transfer).33 RET is a nonradiative energy
transfer process in which the excitation energy of the donor is
transferred to the nearby acceptor via long-range dipole−dipole
interaction and/or short-range multipolar interaction. RET-
based probe molecules are particularly favorable for biological
applications rather than single dye-based probes, as the RET-
based process is not dependent on the concentration of a single
emissive probe, and one can quantitively measure the analyte
concentration by using the ratio of intensities of the well
separated fluorescence peaks with rational intensities at two
different wavelengths for free probe and analyte bound probe.34
Encouraged by the biological importance of Cu2+ and S2‑
ions, we envisioned combining the advantage of the character-
istics of a NIR optical response with the sensitivity of a RET
based fluorophoric response for the construction of a
chemosensor probe (Scheme 1), that may be potentially useful
EXPERIMENTAL SECTION
■
General Information and Materials. All of the materials for
synthesis were purchased from commercial suppliers and used without
further purification. The absorption spectra were recorded on a
Perkin-Elmer Lamda-25 UV−vis spectrophotometer using 10 mm
path length quartz cuvettes in the range 300−800 nm wavelengths,
while the fluorescence measurements were carried on a Horiba
Fluoromax-4 spectrofluorometer using 10 mm path length quartz
cuvettes with a slit width of 5 nm at 298 K. The mass spectra of L1 and
the L1−Cu complex were obtained using Waters Q-ToF Premier mass
spectrometer, and the mass spectrum of L2 was obtained using Agilent
Technologies 6520 Accurate mass spectrometer. NMR spectra were
recorded on a Varian FT-400 MHz instrument. The chemical shifts
were recorded in parts per million (ppm) on the scale. The following
abbreviations are used to describe spin multiplicities in 1H NMR
spectra: s = singlet; d = doublet; t = triplet; m = multiplet. Elemental
analyses were performed with a Perkin-Elmer 2400 elemental analyzer.
Synthesis of L1. Rhodamine B hydrazide was prepared following a
literature method.36 Rhodamine B hydrazide (456 mg, 1 mmol) and
indole-3-carboxaldehyde (145 mg, 1 mmol) were dissolved in 20 mL
of methanol. To this was added approximately 2 drops of acetic acid,
and the resulting solution was refluxed for 10 h. An off-white precip-
itate was found. The reaction mixture was allowed to attain room
temperature, and then the precipitate was collected through filtration.
The residue was washed thoroughly with methanol to isolate L1 in
pure form with 83% yield (the yield was calculated based on the
starting reagents). 1H NMR [400 MHz, CDCl3, SiMe4, J (Hz), δ
(ppm)]: 9.26 (1H, s), 8.51 (1H, s), 7.96 (1H, d, J = 8.4), 7.90 (1H, d,
J = 7.6), 7.49 (2H, t, J = 3.6), 7.05−7.20 (7H, m), 6.54 (1H, d, J = 8.8),
6.46 (1H, d, J = 2.4), 6.24 (1H, d, J = 2.4), 6.22 (1H, d, J = 2.4), 3.28
(8H, q, J = 7.2), 1.11 (12H, t, J = 7.2). 13C NMR [100 MHz, CDCl3,
SiMe4, δ (ppm)]: 164.20, 153.85, 151.09, 148.90, 148.01, 136.78, 132.87,
131.39, 128.79, 128.50, 124.85, 124.16, 123.11, 122.91, 122.75, 120.88,
113.62, 111.31, 108.04, 106.94, 102.02, 98.01, 66.80, 44.47, 12.77. ESI-MS
(positive mode, m/z) Calcd for C37H37N5O2: 583.29. Found: 584.30
(M + H+). Anal. Calcd. for C37H37N5O2 (583.29): C 76.13, H 6.39, N
12.00, O 5.48. Found: C 76.06, H 6.34, N 12.08, O 5.45;
Scheme 1. Synthetic Scheme for the Ligands L1 and L2
Synthesis of L2. 1-Methyl indole-3-carboxaldehyde was prepared
by the reported procedure.37 Rhodamine B hydrazide (456 mg,
1 mmol) and 1-methyl indole-3-carboxaldehyde (159 mg, 1 mmol)
were dissolved in 20 mL of methanol. To this was added approx-
imately 2 drops of acetic acid, and the resulting solution was refluxed
for 10 h. A white precipitate was found. The reaction mixture was
allowed to attain room temperature, and then the precipitate was
collected through filtration. The residue was washed thoroughly with
methanol to isolate L2 in pure form with 70% yield (the yield was cal-
for the detection of Cu2+ as well as S2‑ ions at physiological pH.
In our continuous effort to design a sensor for various anal-
ytes,35 here we discuss the metal and anion sensing capabilities
of an indole functionalized rhodamine based fluorophoric
ligand L1 and the absorption and fluorescence behavior of L1
upon metal complexation. We have also demonstrated by
fluorescence microscopic studies that ligand L1 could readily
detect the presence of intracellular Cu2+ as well as S2‑ ions in
1
culated based on the starting reagents). H NMR [400 MHz, CDCl3,
SiMe4, J (Hz), δ (ppm)]: 10.00 (1H, s), 8.12 (1H, d, J = 7.2), 7.92
(1H, d, J = 2.8), 7.44 (2H, t, J = 2.8), 7.08−7.21 (7H, m), 6.47 (1H, d,
J = 9.2), 6.40 (1H, d, J = 2.0), 6.25 (2H, q, J = 2.4, J = 4.0), 4.76 (3H, s),
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3.32 (8H, q, J = 7.2), 1.16 (12H, t, J = 7.2). 13C NMR [100 MHz,
CDCl3, SiMe4, δ (ppm)]: 185.44, 168.99, 168.68, 157.34, 154.03,
153.51, 149.01, 137.30, 132.64, 131.17, 129.84, 129.98, 128.75, 128.21,
125.54, 124.74, 123.99, 122.95, 122.84, 121.74, 121.40, 121.11, 119.20,
114.96, 112.28, 111.82, 108.38, 108.31, 105.59, 98.08, 97.94, 65.46,
44.48, 41.87, 12.76. ESI-MS (positive mode, m/z) Calcd for
C38H39N5O2: 597.31. Found: 598.3188 (M + H+). Anal. Calcd for
C38H39N5O2 (597.31): C 76.35, H 6.58, N 11.72, O 5.35. Found: C
76.38, H 6.51, N 11.76, O 5.29;
UV−Vis and Fluorescence Spectral Studies. Stock solutions of
various ions (1 × 10−3 mol L−1) were prepared in deionized water. A stock
solution of L1 (1 × 10−3 mol L−1) was prepared in DMSO. The solution of
L1 was then diluted to 1 × 10−5 mol L−1 with CH3CN/aqueous HEPES
buffer (1 mM, pH 7.3; 1:4 v/v). In titration experiments, each time a 1 ×
10−3 L solution of L1 (1 × 10−5 mol L−1) was filled in a quartz optical cell
of 1 cm optical path length, and the ion stock solutions were added into the
quartz optical cell gradually by using a micropipet. Spectral data were
recorded at 1 min after the addition of the ions. In selectivity experiments,
the test samples were prepared by placing appropriate amounts of the
anions/cations stock into 2 mL of solution of L1 (2 × 10−5 mol L−1). For
fluorescence measurements, excitation was provided at 340 and 495 nm,
and emission was collected from 390/500 to 650/700 nm.
Evaluation of the Apparent Binding Constant for the
Formation of L1·Cu2+. Receptor L1 with an effective concentration
of 10.0 × 10−6 M in an acetonitrile/aqueous HEPES buffer (1 mM;
1:4, v/v; pH 7.3) was used for the emission titration studies with a
Cu2+ solution. A stock solution of Cu(NO3)2, having a concentration
of 0.2 × 10−3 M, in an acetonitrile/aqueous HEPES buffer (1:4, v/v;
pH 7.3) solution was used. The effective Cu2+ concentration was
varied between 0 and 10 × 10−5 M for this titration. The solution pH
was adjusted to 7.3 using an aqueous HEPES buffer solution having an
effective concentration of 1 mM.
Calculations for the Apparent Binding Constants Using
Spectrophotometric Titration Data. The apparent binding
constant for the formation of the respective complexes were evaluated
using the Benesi−Hildebrand (B−H) plot (eq 1).35h,38
where f D(λ) is the normalized emission of the donor and ε(λ) is the
molar absorption coefficient (M−1 cm−1) of the donor. Energy transfer
efficiency (ΦET) was evaluated using the expression shown in eq 5
ΦET = 1 − (F′D/FD)
(5)
where F′D and FD denote the donor fluorescence intensity with and
without an acceptor, respectively.
Energy transfer rate constant (KET) was calculated using eq 6
ΦET = KET/(1/τD + KET
)
(6)
where τD denotes the fluorescence lifetime of the donor fragment in
the absence of acceptor.
Cytotoxic Effect on HeLa Cells. The cytotoxic effect of com-
pound L1 and L1−Cu complex was determined by an MTT assay
following the manufacturer instruction (Sigma-Aldrich, MO). HeLa
cells were initially propagated in a 25 cm2 tissue culture flask in
Dulbecco’s Modified Eagle Medium (DMEM) supplemented with
10% (v/v) fetal bovine serum (FBS), penicillin (100 μg/mL), and
streptomycin (100 μg/mL) in a CO2 incubator. For cytotoxicity assay,
cells were seeded into 96-well plates (approximately 104 cells per well),
and various concentrations of compound L1 and L1−Cu complex
(15, 25, 50, 75, and 100 μM) made in DMEM were added to the cells
and incubated for 24 h. Solvent control samples (cells treated with
DMSO alone) and cells treated with Cu(ClO4)2 alone were also
included in parallel sets. Following incubation, the growth media was
removed, and fresh DMEM containing MTT solution was added. The
plate was incubated for 3−4 h at 37 °C. Subsequently, the supernatant
was removed, the insoluble colored formazan product was solubilized
in DMSO, and its absorbance was measured in a microtiter plate
reader (Infinite M200, TECAN, Switzerland) at 550 nm. The assay
was performed in six sets for each concentration of compound L1 and
L1−Cu complex. Data analysis and calculation of standard deviation
was performed with Microsoft Excel 2010 (Microsoft Corporation).
For statistical analysis, a one way analysis of variance (ANOVA) was
performed using Sigma plot.
Cell Culture and Imaging Studies. HeLa cells were procured
from National Center for Cell Sciences (NCCS), Pune, India. The cells
were propagated in Dulbecco’s Modified Eagle Medium (DMEM) sup-
plemented with 10% (v/v) fetal bovine serum, penicillin (100 μg/mL), and
streptomycin (100 μg/mL). Cells were maintained under a humidified
atmosphere of 5% CO2 and at 37 °C incubator as mentioned before. For
cell imaging studies, cells were seeded into a 6 well plate and incubated
at 37 °C in a CO2 incubator for 3 days. After 3 days cells were washed
three times with phosphate buffered saline (pH 7.4) and incubated
with 10 μM L1 in DMEM at 37 °C for 1 h in a CO2 incubator and ob-
served under epifluorescence microscope (Nikon eclipse Ti). The cells
were again washed thrice with PBS (pH 7.4) to remove the free L1,
and then incubated in phosphate buffered saline with 20 μM Cu(ClO4)2
for 1 h. Again, images were taken using epifluorescence microscope. The
cells were then treated with 30 μM of Na2S solution, after incubation
for 1 h; the cells were washed with PBS three times to remove free
compound and ions before analysis. Then, fluorescence microscopic
images were acquired.
1/(A − A0) = 1/{K(Amax − A0)C} + 1/(Amax − A0)
(1)
A0 is the absorbance of L1 at absorbance maximum (λ = 702 nm), A
is the observed absorbance at that particular wavelength in the pres-
ence of a certain concentration of the metal ion (C), Amax is the maximum
absorbance value that was obtained at λ = 702 nm during titration with
varying metal ion concentration, K is the apparent binding constant
(M−1) and was determined from the slope of the linear plot, and C is the
concentration of the Cu2+ ion added during titration studies.
Finding the Detection Limit. The detection limit was calculated
on the basis of the UV−vis titration. The fluorescence emission
spectrum of L1 was measured 10 times, and the standard deviation of
blank measurement was achieved. To gain the slope, the ratio of the
UV−vis absorbance at 702 nm was plotted as a concentration of Cu2+.
So the detection limit was calculated with the following equation
(2)
detection limit = 3σ/k
where σ is the standard deviation of blank measurement, and k is the
slope between the ratio of UV−vis absorbance versus [Cu2+].
Evaluation of Different Parameters for FRET Process. The
RESULT AND DISCUSSION
■
UV−Vis Spectroscopic Studies of L1 in Presence of
Cu2+. UV−vis spectra recorded for L1 in CH3CN/aqueous
HEPES buffer (1 mM, pH 7.3; 1:4, v/v) indicated an absorp-
tion maximum at 324 nm, which may possibly be attributed to
intramolecular π−π* charge transfer (CT) transition. Accord-
ing to previous studies certain transition-metal ions bind selec-
tively with suitable derivatives of rhodamine,39 wherein metal−
ligand binding induces opening of the spirolactam ring and
generation of the xanthene form. This structural change is
manifested in the electronic and fluorescence spectral patterns.
Thus, the selectivity of L1 was checked with perchlorate or
nitrate salts of Na+, K+, Ca2+, Mg2+, Cr3+, Hg2+, Cu2+, Pb2+,
Zn2+, Fe2+, Co2+, Ni2+, Cd2+, and Ag+ in CH3CN/aqueous
Forster distance R was calculated using the expression shown in eq 3
̈
0
R0 = 0.211[(J)Q(n−4)(κ2)]
(3)
where n is the refractive index of the medium in between donor and
acceptor and was taken approximately to be equal to 1.4. κ2 is the
dipole orientation factor. Depending upon the relative orientation of
donor and acceptor, the value is in the range 0−4, and it is often
assumed to be 2/3. Q is the fluorescence quantum yield of the donor in
the absence of acceptor. J is the spectral overlap integral between the
emission spectrum of the donor and the absorption spectrum of the
acceptor and is shown in the following eq 4
J = f (λ)ε(λ)λ4 dλ
∫
D
(4)
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Figure 1. (A) UV−vis absorption spectra of receptor L1 (10 μM) observed upon addition of 10 equivalent metal ions (perchlorate or nitrate salts of
Na+, K+, Ca2+, Mg2+, Cr3+, Hg2+, Cu2+, Pb2+, Zn2+, Fe2+, Co2+, Ni2+, Cd2+, and Ag+) in a CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v).
(B) UV−vis titration spectra of L1 (10 μM) upon incremental addition of Cu(ClO4)2 in CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v).
Inset: Changes in the absorbance at 702 nm with incremental addition of Cu2+.
Scheme 2. Cu2+-Induced FRET OFF→ON of the Receptor L1
HEPES buffer (1 mM, pH 7.3; 1:4 v/v). A significant change in
UV−vis spectral pattern was observed only in presence of
Cu2+, among all the other metal ions used (Figure 1A). During
sequential titration (0 equiv to 3 equiv of Cu2+), a NIR absorp-
tion band appeared around 702 nm along with a hump in the
visible region near 557 nm (Figure 1B), and the solution turned
from colorless to blue (Figure S8). With further increase in the
concentration of Cu2+ the color of the solution changed from
blue to pink with the emergence of a sharp peak near 557 nm of
the electronic spectra. It is significant to mention that the
detection limit of L1 for Cu2+ ions was found to be 3.6 ppb,
which is much lower than the U.S. EPA maximum allowable
limit for Cu2+ ions (1.3 ppm) in drinking water.
In the recent literature, deprotonation induced NIR sensing by in-
dole moiety was reported by P. Ghosh and co-workers.38b
In the present study, visual color change of the solution from
colorless to blue is a consequence of the ratiometric
deprotonation of the NH proton of indole moiety in presence
of the metal cation. Such a huge shift of absorption spectra
could be attributed to the high conjugation and planarity of the
indole moiety of L1 with the binding site of the receptor
(Scheme 2) which favors maximum distribution of the negative
charge of the deprotonated receptor in the presence of the
guest metal ion. Regardless of the deprotonation of indole
moiety, the formation of the peak near 557 nm is mostly due to
the breakage of the spirolactum ring structure of L1 and sub-
sequent formation of the xanthene form. The spectral changes
due to formation of the L1−Cu complex and the deprotonation
of the indole unit occur simultaneously with addition of Cu2+.
However, in the present case the individual steps cannot be
analyzed in solution. Therefore, the apparent binding constant
for the formation of L1−Cu complex is calculated on the basis
of change in absorbance at 702 nm by considering a 1:1 binding
stoichiometry. The apparent binding constant (K) determined
by the B−H method was found to be 1.71 × 104 M−1.
To verify the significance of the deprotonation of indole −NH
in generating the NIR peak, a new control compound L2 was
synthesized where the indole −NH was replaced by −CH3. As
expected, compound L2 failed to show a NIR peak in presence
of Cu2+. The only change of the absorption spectra of L2 in
presence of Cu2+ ions was the formation of a sharp peak near
550 nm, predominantly due to the rupture of the spirolactum
ring of rhodamine into the xanthene form (Figure S9).
Fluorescence Spectroscopic Studies of L1 in Presence
of Cu2+. The selective binding of L1 with Cu2+ among all other
metal ions was also studied using the emission spectroscopy of
the solution of L1 (10.0 × 10−6 M) in the absence and presence
of an excess (10 equiv) of each of the metal ions in CH3CN/
aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v) (Figure 2A).
As the receptor L1 bears two different fluorophore units, we
consider it to be appropriate to study the metal binding event
of L1 at two different excitation wavelengths corresponding to
the excitation wavelength of the xanthene unit (495 nm) and
the indole unit (340 nm), respectively.
As evident from Figure 2B, excitation of the initial solution of
receptor L1 at 495 nm wavelength did not show any significant
emission over the range from 500 to 700 nm. This supports the
facts that in absence of metal ions the receptor remains in the
spirolactum form, and the nonexistence of the highly con-
jugated xanthene form results in the suppression of emission in
the above-mentioned region. Addition of Cu2+ to this receptor
solution induces a significant switch ON fluorescence response
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Figure 2. (A) Changes of the fluorescence emission of receptor L1 (10 μM) observed upon addition of metal ions (perchlorate or nitrate salts of
Na+, K+, Ca2+, Mg2+, Cr3+, Hg2+, Cu2+, Pb2+, Zn2+, Fe2+, Co2+, Ni2+, Cd2+, and Ag+) (10 equiv) in a CH3CN/aqueous HEPES buffer (1 mM, pH 7.3;
1:4 v/v). (B) Fluorescence titration spectra of L1 (10 μM) upon incremental addition of 5 equiv of Cu(ClO4)2 in CH3CN/aqueous HEPES buffer
(1 mM, pH 7.3; 1:4 v/v) λex = 495 nm. Inset: Changes in the fluorescence intensity at 557 nm with incremental addition of Cu2+.
Figure 3. Fluorescence spectra (λex = 340 nm) of (A) L1 (10 μM) with varying [Cu2+] (0−3 equiv), and (B) the overlap (shown with vertical
stripes) between emission and absorption spectra of the donor and acceptor, respectively. All studies were carried out in CH3CN/aqueous HEPES
buffer (1 mM, pH 7.3; 1:4 v/v) medium. Inset: (a) Visual change of the color of the L1 solution in presence of Cu2+ under UV light. (b) The changes
of fluorescent intensity ratios at 457 and 585 nm (I457/I585) with increasing concentration of Cu2+.
near 580 nm, with a visual display of reddish-pink fluorescence.
Switch ON responses for the absorption spectral band at
557 nm and the emission band at ∼580 nm on binding to Cu2+
suggest opening of the spirolactam ring in L1 on metal ion
coordination. It can also be observed from Figure 2A that the
metal−ligand binding induced ring-opening of L1 and the
generation of xanthene moiety is very much selective toward
Cu2+ ions and does not reveal any noticeable spectral change
for other tested metal ions (Na+, K+, Ca2+, Mg2+, Cr3+, Hg2+,
Pb2+, Zn2+, Fe2+, Co2+, Ni2+, Cd2+, and Ag+). To gain an insight
into the properties of L1 as a receptor for Cu2+, a titration of the
receptor was performed with increasing concentration of Cu2+.
As described in Figure 2B the fluorescence intensity of a 10 μM
solution of L1 was enhanced with incremental addition of Cu2+
ions, which also confirmed that receptor L1 exhibited a high
sensitivity toward Cu2+, with near about 600-fold increase of its
fluorescence intensity upon addition of only 5.0 equiv of Cu2+
ions.
initiates an intramolecular FRET process. In the free ligand L1 the
FRET pathway is totally suppressed, and only an emission
maximum near 480 nm is observed when excited at 340 nm.
Binding of the receptor to Cu2+ induces the FRET process to
produce an intense rhodamine-based red emission; i.e., energy
transfer from indole to xanthene is due to the ring-opening
resulting in increase of overlap integral between indole and
xanthene moiety.34g Thus, when titrated with increasing con-
centration of Cu2+ the emission band with a λmax near 457 nm
starts to decrease along with a concomitant generation of a new
fluorescence band at 585 nm. This change in fluorescence was
also observed visually, and the color changed to reddish-pink as
shown in the inset of Figure 3A.
The singlet−singlet excitation energy-transfer efficiency (ΦET)
between donor and acceptor was evaluated from steady state fluo-
rescence data. The value for ΦET was found to be 51.5% while the
Forster critical distance (R ) was calculated as 35.8 Å.
̈
0
The complex formed between L1 and Cu2+ is found to be 1:1
in stoichiometry, which is established with the help of Job’s plot
(Figure S10) from fluorescence spectrometry. Further con-
firmation of 1:1 stoichiometry was obtained from the ESI-MS
studies. The molecular-ion peak is observed at m/z 708.21 in
The binding of Cu2+ ion induces opening of the spirolactam
ring in L1 with an associated switch on UV−vis spectral response
in the range 500−580 nm, which has a significant spectral overlap
with the emission spectrum of the indole fragment, and this fact
unlocks a plausible route for nonradiative transfer of excitation
energy between donor indole to acceptor xanthene moiety and
the mass spectrum which is related to the mass of [L1 + Cu2+
+
NO3] (Figure S11).
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Figure 4. (A) Changes in the absorption spectra of L1−Cu complex in presence of different anions. (B) UV−vis titration spectra of L1 (10 μM) with
2 equiv of Cu2+ upon addition of sodium sulfide (30 μM) in CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v). Inset: Changes in the
absorbance at 702 nm with incremental addition of S2‑.
Figure 5. (A) Changes in the fluorescence spectra of L1−Cu complex in presence of different anions (λex = 495 nm). (B) Fluorescence titration
spectra (λex = 495 nm) of L1 (10 μM) with 2 equiv of Cu2+ upon addition of sodium sulfide (30 μM) in aqueous CH3CN/aqueous HEPES buffer
(1 mM, pH 7.3; 1:4 v/v). Inset: Changes in the fluorescence intensity at 557 nm with incremental addition of S2‑.
The role of the CO amide bond on binding Cu2+ cations
was examined using FTIR techniques. The FTIR spectra of L1
revealed that the peak at 1702 cm−1, the characteristic stretch-
ing frequency for the CO amide bond of the rhodamine unit,
shifted to 1630 cm−1 in presence of 2 equiv of the Cu2+ ion
(Figure S12). Such shift in the stretching frequency of CO
amide bond of the rhodamine unit on binding to a metal ion
has been reported earlier.40 Thus, FTIR studies suggest that the
shift in the stretching frequency of CO bond was due to its
participation in the binding event of the metal cation.
The UV−vis spectral pattern of the titration experiment (Figure 4B)
was similar but in reverse direction to the titration curve ob-
tained with Cu2+ (Figure 1B). This fact is evidence that ligand
L1 is regained from complex in presence of S2‑.
Fluorescence Spectroscopic Studies of L1−Cu Com-
plex in Presence of S2‑. Apart from the results obtained from
UV−vis studies, the fluorescence spectroscopy also shows that
the emission of the L1−Cu complex returns to its native L1
state, selectively in presence of sulfide anions (Figure 5A). To
further understand the fluorescence “ON−OFF’’ switching
property of the sensor, we have performed fluorescence titra-
tion experiment. The fluorescence intensity of the compound
L1 is enhanced to a moderate level in presence of 2 equiv of
Cu2+ ions; the resulting L1−Cu complex is then titrated by the
addition of various amounts of sulfide ions. Figure 5B shows
that the intensity of the fluorescence emission decreases with
increasing concentration of sulfide anion, and on addition of
near about 3 equiv of S2‑ anion both the intensity and overall
pattern of emission spectrum closely match those of compound
L1 (Figure 2B), so the fluorescence intensity along with the
maximum emission peak are totally regenerated. As the fluo-
rescence spectrum was recorded within 15 s after sulfide anion
addition, and the intensity does not change with time, so the
monitoring system is virtually real-time and stable. The results
of the spectroscopic studies indicated that the sensor L1 was
recycled during the detection of sulfide anions.
UV−Vis Spectroscopic Studies of L1−Cu Complex in
Presence of S2‑. Hence from the above-mentioned studies we
can conclude that L1 selectively binds with Cu2+ to form L1−Cu
complex with considerable change in its spectral properties.
We have further studied the influence of different anions on
the rupture of this metal−ligand complex and their effect on the
reversibility of this complex to regenerate L1. The optical
properties of the L1−Cu complex were studied in presence of
−
different anions such as F−, Cl−, Br−, I−, CN−, H2PO4 −, NO3 ,
ClO3 , ClO4 , SO42−, HSO3 , PO43−, and S2‑. It is worth
mentioning that the regeneration of compound L1 is observed
only by adding S2− to the solution containing L1−Cu, whereas
other anions failed to produce any discernible spectral change
(Figure 4A). For further understanding, a solution of L1 in
CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v) con-
taining 2 equiv of Cu2+ is titrated in presence of sulfide anions.
−
−
−
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From our Cu2+ binding studies it was evident that the
binding induced breakage of the spirolactum ring of L1 initiates
the FRET pathway for efficient transfer of energy from indole
moiety to the xanthene unit in the L1-Cu complex. It is obvious
that if the regeneration of L1 from L1-Cu complex is possible in
presence of sulfide anion then the removal of Cu2+ must affect
the FRET process. Thus, continuous addition of sulfide ions to
the L1-Cu complex generates a spectrum (Figure 6) which is
presence of other ions and sensed the respective analytes from
a competitive environment.
Biological Studies of L1 in Presence of Cu2+ and S2‑.
Owing to its favorable binding properties with copper(II) and
intense emission in visible region, it was conceived that com-
pound L1 could be exploited for fluorescence imaging of live
cells, particularly for sensitive detection of intracellular Cu2+.
However, to pursue this goal, it was pertinent to assess the
cytotoxic effect of compound L1 on live cells. Varying con-
centrations of compound L1 and L1−Cu complex were thus
chosen, and their cytotoxic effect on HeLa cells was ascertained
following an exposure period of 24 h. The well-established
MTT assay, which is based on mitochondrial dehydrogenase
activity of viable cells, was adopted. It is quite evident from
Figure 8 that compound L1 failed to exert any effect on the
Figure 6. Fluorescence titration spectra (λex = 340 nm) of L1 (10 μM)
with 2 equiv of Cu2+ upon continuous addition of sodium sulfide
(up to 30 μM) in CH3CN/aqueous HEPES buffer solution (1 mM,
pH 7.3; 1:4 v/v).
equivalent to the spectra of L1 (when excited at 340 nm). The
above-mentioned fact provides strong evidence of the
dissociation of L1-Cu complex in presence of S2‑ anions to
restore the native structure of L1.
Figure 8. MTT assay to determine the cytotoxic effect of compound
L1 and L1−Cu complex on HeLa cells. Statistically significant values
derived by ANOVA are indicated by asterisk marks. * indicates P value
<0.05, and ** indicates P value <0.001.
In order to verify the reason of the fluorescence “OFF−ON”
property, the mass spectrum of the L1−Cu system was also
studied in presence of S2−. The mass spectrum of the L1−Cu
system shows a molecular-ion peak at m/z 708.21, correspond-
ing to [L1 + Cu + NO3], while subsequent addition of S2‑ ions
to the above solution gives a molecular ion peak at m/z 584.30
(Figure S13) which confirmed the identity of free L1 and
substantiated the mechanism of the sensing of sulfide anions.
The sensing efficiency of L1 and L1−Cu complex was further
tested in presence of other cations and anions, which may
interfere in estimation of copper and sulfide (Figure 7). The
receptor L1 and the L1−Cu complex both performed well in
viability of HeLa cells, irrespective of the chosen concentrations
of the compound. However, exposure of HeLa cells to the
lowest concentration of L1−Cu complex (15 μM) resulted in a
decline in cell viability. In presence of higher concentrations of
L1−Cu complex, the effect was more prominent and indicated a
dose-dependent cytotoxic effect on HeLa cells. Previous litera-
ture reports also suggest cytotoxic and antiproliferative effects
Figure 7. (A) Normalized fluorescence responses of L1 (10 μM) to various cations in CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v). The
blue bars represent the emission intensities of L1 in the presence of cations of interest (50 μM). The red bars represent the change of the emission
that occurs upon the subsequent addition of Cu2+ to the above solution. (B) Normalized fluorescence responses of L1−Cu to various anions in
CH3CN/aqueous HEPES buffer (1 mM, pH 7.3; 1:4 v/v). The red bars represent the emission intensities of L1−Cu in the presence of anions of
interest (50 μM). The blue bars represent the change of the emission that occurs upon the subsequent addition of S2‑ to the above solution. The
intensities were recorded at 585 nm.
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Figure 9. Fluorescence microscopic images of HeLa cells (A) after treating with 10 μM L1 (under green light), (B) after adding 20 μM of Cu2+
(under green light) to the L1 treated cells, and (C) after adding 30 μM S2‑ (under green light) to the (L1 + Cu2+) treated cells. Scale bar for the
images is 50 μm.
of copper complex on cancer cells.41 The viability of HeLa cells
was not influenced by the solvent (DMSO) or the copper salt
(Figure 8), substantiating that the observed cytotoxic effect
could be attributed to L1−Cu complex.
Cu2+ and S2‑. It is significant to mention here that brightfield
images of treated cells did not reveal any gross morphological
perturbations, which suggested that HeLa cells were viable.
This finding is encouraging for future in vivo biomedical applica-
tions of the sensor.
The results obtained in the in vitro cytotoxic assay suggested
that, in order to pursue fluorescence imaging studies of L1−Cu
complex in live cells, it would be prudent to choose a working
concentration below 15 μM for compound L1. Hence, to assess
the effectiveness of compound L1 as a probe for intracellular
detection of Cu2+ by fluorescence microscopy, HeLa cells were
treated with 10 μM L1 solution for 1 h followed by incubation
with 20 μM Cu(ClO4)2 to promote formation of L1−Cu
complex. On the basis of the established 1:1 stoichiometry of
binding between L1 and Cu2+ (Figure S10 and S11), it can be
reasonably assumed that the concentration of L1−Cu complex
formed in HeLa cells would be much lower than the con-
centration (15 μM) at which a marginal cytotoxic effect of the
complex was observed (Figure 8).
Fluorescence microscopic studies revealed a lack of fluo-
rescence for HeLa cells treated with compound L1 alone (Figure 9,
panel A). Upon incubation with Cu(ClO4)2, a striking switch-
ON fluorescence was observed inside HeLa cells, which
indicated the formation of L1−Cu complex, as observed earlier
in solution studies. Further, an intense red fluorescence was
conspicuous in the perinuclear region of HeLa cells (Figure 9,
panel B). Interestingly, sulfide sensing inside HeLa cells by
L1−Cu complex could also be pursued as evident from the
remarkable switch-OFF of the red fluorescence emission inside
cells following incubation with Na2S solution (Figure 9, panel C).
Essentially, the fluorescence microscopic analysis strongly
suggested that compound L1 could readily cross the membrane
barrier, permeate into HeLa cells, and rapidly sense intracellular
CONCLUSION
■
In summary, we have developed an NIR sensitive receptor
L1 which selectively binds with Cu2+ ions and propels a switch
ON response in optical and fluorescence spectra in the visible
region. Apart from the NIR and visible changes the FRET-
based fluorescence response makes it a dual probe for naked
eye detection through change in color and fluorescence. The
detection limit for Cu2+ was found to be much lower than the
permissible Cu2+ concentration in drinking water as per
standard norms. The complex formed between L1 and Cu2+
is dissociable only in presence of sulfide anion, which makes the
L1−Cu complex an efficient sensor for sulfide anions. From the
extensive spectroscopic studies it is clear that the receptor L1
and L1−Cu complex could be used as a ratiometric sensor for
the detection of Cu2+ and S2‑, respectively, based on NIR
response and RET process involving the donor indole and the
acceptor Cu2+ bound xanthene moiety of L1. The receptor L1
shows intense change in its fluorescence emission when bound
to Cu2+ in physiological conditions. Hence, the effectiveness
of compound L1 as a probe for intracellular detection of Cu2+
by fluorescence microscopy was also studied. Moreover, the
fluorescence microscopic analysis strongly suggested that
compound L1 could readily cross the membrane barrier,
permeate into HeLa cells, and rapidly sense intracellular
Cu2+ and S2‑.
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ASSOCIATED CONTENT
* Supporting Information
■
S
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516. (b) Martinez-Manez, R.; Sancenon, F. Chem. Rev. 2003, 103,
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1H NMR, 13C NMR, mass spectra, and IR spectra as
characterization data of L1 and L1−Cu complex. Benesi−
Hildebrand plot, Job’s plot, UV−vis spectra, and photograph of
the L1 solution in presence and absence of Cu2+ ions. This
material is available free of charge via the Internet at http://
pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*E-mail: gdas@iitg.ernet.in; aramesh@iitg.ernet.in. Fax: +91-
361-258-2349. Phone: +91-361-258-2313.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
G.D. gratefully acknowledges Department of Science and
Technology (DST), New Delhi, India, for financial support.
C.K. and M.D.A. acknowledge the Department of Chemistry,
Central Instrumental Facility, and IIT Guwahati for instru-
mental support and fellowship. We thank the Department of
Biotechnology, Government of India, for a research grant (BT/
01/NE/PS/08). Special thanks are due to Debraj Biswas for his
kind cooperation.
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