Visible light excitable on fluorescence and naked eye detection of Cu 2+via hydrolysis of rhodamine-thiophene conjugate: Human breast cancer cell (MCF7) imaging studies

Article abstract of DOI:10.1039/c4dt00002a

Thiophene appended rhodaminehydrazone derivative, RDHDTCA allows selective colorimetric and fluorescence recognition of Cu²⁺ as low as 2.4 × 10^-8 M. RDHDTCA is capable to detect intracellular Cu ²⁺ in human breast cancer c

Full text of DOI:10.1039/c4dt00002a

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Visible light excitable ON fluorescence and
naked eye detection of Cu2+ via hydrolysis of
rhodamine–thiophene conjugate: human breast
cancer cell (MCF7) imaging studies†
Cite this: Dalton Trans., 2014, 43,
7747
Sangita Adhikari,a Abhijit Ghosh,a Sandip Mandal,a Archya Sengupta,b
Ansuman Chattopadhyay,b Jesús Sanmartín Matalobos,*c Sisir Lohar*a and
Debasis Das*a
Received 1st January 2014,
Accepted 28th January 2014
Thiophene appended rhodaminehydrazone derivative, RDHDTCA allows selective colorimetric and
fluorescence recognition of Cu2+ as low as 2.4 × 10−8 M. RDHDTCA is capable to detect intracellular
Cu2+ in human breast cancer cells, MCF7 under fluorescence microscope.
DOI: 10.1039/c4dt00002a
www.rsc.org/dalton
causes gastrointestinal disturbance, neurodegenerative dis-
eases, and liver/kidney damage. Thus, development of a Cu2+
Introduction
Rhodamine derivatives possess excellent photo-physical pro- selective, water soluble fluorescence probe functioning at phys-
perties1 and transform from spirocyclic form to open-ring iological pH is highly desirable. Although, a significant
form in the presence of analytes capable to activate the carbo- number of rhodamine-based fluorescent probes for different
nyl group.2 Consequently the fluorescence properties also metal ions viz. zinc,11 iron,12 chromium13 and mercury14 have
change significantly. Fluorescence sensors offer several advan- been developed, relatively few have been reported for copper.15
tages in terms of sensitivity, selectivity, simplicity, response Herein, we report a thiophene appended rhodaminehydrazone
time and spatial resolution. Several photosensing processes derivative, RDHDTCA for selective determination of Cu2+ at
viz. photo-induced electron transfer (PET),3 photo-induced physiological pH. Experimental data suggest Cu2+ assisted
charge transfer (PCT),4 fluorescence resonance energy transfer spirolactam ring opening of RDHDTCA and subsequent hydro-
(FRET),5 inter molecular charge transfer (ICT),6 chelation lysis are responsible for fluorescence enhancement.
enhanced fluorescence (CHEF),7 perturbation of optical tran-
sitions and polarizabilities, excimer/exciplex formation,8a,b
modification of redox potentials in ground or excited states,
and photo-regulation of binding properties are well known.
Results and discussion
Copper, the third most abundant soft transition metal ion in
the human body, is an essential element for various biochemi-
cal and physiological processes. Due to its redox sensitivities,
it acts as a vital cofactor for varieties of enzymes such as tyrosi-
nase, cytochrome oxidase, and Cu/Zn superoxide dismutase
etc.9 However, at higher concentration, Cu2+ is a significant
environmental pollutant and exerts toxicity to all living organ-
isms.10 Excess accumulation of copper in the human body
Single crystals of RDHDTCA are grown from its ethanol solu-
tion and characterized by X-ray crystallography (Fig. 1). The
most significant geometric data, which are in the expected
range for rhodaminehydrazone derivatives,16 are shown in
Tables S1 and S2 (see ESI†). The crystal structure clearly shows
the formation of a spirolactam-ring, where two planes of the
spiro compound are almost in mutually vertical position. The
dihedral angle between the spirolactam-ring and the xanthene-
ring planes is about 88.7°. Weak intermolecular C–H⋯N
(3.5 Å) and C–H⋯O (3.3 and 3.4 Å, bifurcated) hydrogen bonds
along with other weaker interactions are responsible for the
crystal packing. The absence of significant interactions invol-
ving the aldehyde residue explains the disorder found for the
corresponding branch.
aDepartment of Chemistry, The University of Burdwan, Burdwan, West Bengal, India.
E-mail: ddas100in@yahoo.com; Fax: +91-342-2530452; Tel: +91-342-2533913
bDepartment of Zoology, Centre for Advanced Studies, Visva Bharati University,
Santiniketan, West Bengal, India
cDepartamento de Química Inorgánica, Facultade de Química, Avda. Das Ciencias
s/n, 15782 Santiago de Compostela, Spain
The effect of pH on the emission characteristics of
RDHDTCA has been examined. Different sets of an equimolar
mixture of RDHDTCA and Cu2+ are adjusted to different pH
†Electronic supplementary information (ESI) available. CCDC 947579. For ESI
and crystallographic data in CIF or other electronic format see DOI: 10.1039/
c4dt00002a
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Fig. 3 Changes in the absorbance spectra of RDHDTCA (20 µM,
CH3CN–water = 9 : 1, v/v) upon gradual addition of Cu2+ (0, 0.1, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90 ,
100 µM). Inset: color changes of RDHDTCA in the presence of Cu2+. All
spectra are recorded after 40 minutes of mixing of Cu2+ and RDHDTCA.
Fig. 1 View of the molecular structure of RDHDTCA showing only one
of the three sets of positions found for the disordered aldehyde residue.
Displacement atomic ellipsoids are drawn at 50% probability level.
Fig. 2 Effect of pH on the emission intensity of free RDHDTCA (20 μM)
and [Cu2+–RDHDTCA] system (10 : 1, mole ratio, λex
λem = 581 nm).
= 500 nm,
Fig. 4 Changes in the fluorescence spectra of RDHDTCA (20 µM,
CH3CN–water = 9 : 1, v/v) upon gradual addition of Cu2+ (0, 0.1, 0.5, 1,
2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90,
100 µM) (λex = 500 nm). Inset: color changes of RDHDTCA in the
presence of Cu2+. All spectra are recorded after 40 minutes of mixing of
Cu2+ and RDHDTCA.
(pH 2.0–11.0) and their emission intensities are measured.
Fig. 2 indicates that over pH 7.4, the emission intensity of the
[Cu2+–RDHDTCA] system becomes maximum allowing deter-
mination of Cu2+ at physiological pH.
ment. This is because, Cu2+ released to the medium after
hydrolysis, may be available for chelation to other RDHDTCA
molecules, so a lower amount of external Cu2+ is necessary
for the same extent of fluorescence enhancement (Fig. S1,
ESI†).
The Job’s plot (Fig. S2, ESI†) indicates a binding stoichio-
metry of RDHDTCA to Cu2+ as 1 : 1 (mole ratio). The associ-
ation constant (Ka) of RDHDTCA for Cu2+ is determined by
fluorescence (Fig. S3, ESI†) and UV-Vis (Fig. S4, ESI†) tech-
niques following the Benesi–Hildebrand method.17,18 The
values are 4.297 × 104 M−1 and 3.3 × 104 M−1 respectively.
Quantum yield of free RDHDTCA (1.19 × 10−2) increases 8.54
fold in the presence of Cu2+ (details in ESI†). In the presence
of water, the [RDHDTCA–Cu2+] system further undergoes
rapid hydrolysis leading to open ring rhodamine unit,
evidenced by the mass spectra of the final stable product
(Fig. S5, ESI†).
Fig. 3 shows the changes in the absorbance spectra of
RDHDTCA upon gradual addition of Cu2+. The absorbance of
free RDHDTCA gradually enhances with increasing concen-
tration of Cu2+ while the colorless RDHDTCA solution turns to
red-pink indicating the ring-opening of rhodamine unit of
RDHDTCA.
Changes in the emission spectra of RDHDTCA upon
gradual addition of Cu2+ are presented in Fig. 4. Addition of
Cu2+ to RDHDTCA results a 581 nm emission band along with
generation of pink-red color, attributed to Cu2+ induced spiro-
lactam ring opening of RDHDTCA. The sigmoidal nature of
fluorescence enhancement may be rationalized in the light of
Cu2+ assisted hydrolysis of RDHDTCA (vide infra). With
increasing Cu2+ concentration, more and more RDHDTCA
being chelated followed by their hydrolysis, so the emission
intensity increases. Moreover, at higher [Cu2+] region, a small
addition of Cu2+ causes much higher fluorescence enhance-
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Fig. 7 Fluorescence microscope images of MCF7 cells: (A) and (B) are
images of cells after 2 h incubation with Cu2+ (50 µM) and RDHDTCA
(20 µM) respectively, while (C) shows the cells after 2 h incubation with
50 µM Cu2+ followed by the addition of 20 µM RDHDTCA solution.
Fig. 5 Proposed Cu2+ sensing mechanism.
Conclusion
Thiophene appended rhodaminehydrazone derivative func-
tions as an excellent colorimetric and fluorescent probe for
trace level determination of Cu2+ at physiological pH with
LOD, 2.4 × 10−8 M. Initial chelation of RDHDTCA to Cu2+ fol-
lowed by hydrolysis is attributed to the fluorescence enhance-
ment. RDHDTCA is capable to detect intracellular Cu2+ in
human breast cancer cells, MCF7 under fluorescence
microscope.
Fig. 6 Color of RDHDTCA (20 µM) in the presence of different cations:
naked eye (top); under UV lamp (bottom) in CH3CN–water (9 : 1, v/v).
Experimental
General procedure
High-purity rhodamine B, thiophene-2-carboxaldehyde, hydra-
zine hydrate (Sigma Aldrich, India) and Cu(NO3)3·3H2O
(Merck, India) are used. Spectroscopic grade solvents are used.
Metal salts used are either in nitrate or chloride forms. Other
analytical reagent grade chemicals are used without further
purification except when specified. Mili-Q Milipore® 18.2 MΩ
cm−1 water is used in all the experiments. A Shimadzu Multi
Spec 1501 spectrophotometer is used for recording UV-Vis
spectra. FTIR spectra are recorded on a PerkinElmer FTIR
(model RX1) spectrophotometer. Mass spectra are carried out
using a QTOF Micro YA 263 mass spectrometer in ES positive
mode. 1H NMR spectra are recorded using Bruker Advance
DPX 300 (300 MHz) in CDCl3. The steady-state fluorescence
emission and excitation spectra are recorded with a Perkin
Elmer LS 55 precisely, spectrofluorimeter. Systronics digital
pH meter (model 335) is used to measure the solution pH. HCl
or NaOH (50 µM) are used for pH adjustment.
From the above discussions, the plausible binding inter-
action of RDHDTCA for Cu2+ is presented in Fig. 5. The limit
of detection (LOD) for Cu2+ is 2.4 × 10−8 M (Fig. S6, ESI†).
Selectivity and interferences
Fig. 6 shows the naked eye and UV illuminated color of
RDHDTCA in the presence of different cations. Common
cations viz. Na+, Mg2+, Hg2+, Co2+, Mn2+, Ni2+, Cr3+, Sn2+, Pb2+,
Cd2, Al3+ and Zn2+ do not show any color change of free
RDHDTCA. Only Fe3+/Fe2+ show slight coloration, however it
can be masked by F− ion without disturbing the [RDHDTCA–Cu2+]
system. Again, except for iron, other cations, present in excess
together with Cu2+ (50 equivalent) in a ternary mixture, do not
show any significant change to the emission intensities of the
[RDHDTCA–Cu2+] system. Insignificant effect of iron is nulli-
fied using F− ion as masking agent. Thus, RDHDTCA can
selectively sense Cu2+ through fluorescence enhancement
(Fig. S7 and S8, ESI†).
Synthesis of rhodaminehydrazone unit
N-(Rhodamine-B) lactam-hydrazine is prepared by refluxing
rhodamine B and hydrazine hydrate for 3 hour following the
Cell imaging
RDHDTCA is capable to detect intracellular Cu2+ in human literature method.19
breast cancer cells, MCF7 under fluorescence microscope.
Synthesis of RDHDTCA. N-(Rhodamine-B) lactam-hydrazine
Cells treated only with RDHDTCA (in the absence of Cu2+) are (0.20 g, 0.4386 mmol) is dissolved in 10 mL ethanol, and
used as control. Fig. 7 indicates that RDHDTCA is able to added drop-wise to the ethanol solution of thiophene-2-car-
permeate the cell membrane and stain Cu2+ without any harm boxaldehyde (49 g, 0.4375 mmol in 10 mL) under stirring
(cells remain alive even after several hours of exposure to condition. The reaction mixture is refluxed for 6 hours
20 µM RDHDTCA).
(Scheme 1), filtered to remove suspended material and the
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In vitro cell imaging
Human breast cancer cell line MCF7 is grown in DMEM
(Sigma, St. Louis, USA) supplemented with 10% fetal bovine
serum (Sigma, St. Louis, USA), 2 mM glutamine, 100 U mL−1
penicillin–streptomycin solution (Gibco, Invitrogen, USA) in
the presence of 5% CO2 at 37 °C. For in vitro imaging studies,
the cells are seeded in 6 well culture plate with a seeding
density of 105 cells per well. After reaching 60%–70% conflu-
ence, the previous media is replaced with serum free media,
supplemented with Cu2+ and RDHDTCA at 50 µM and 20 µM,
and incubated for 2 h to facilitate metal ion or RDHDTCA
uptake by the cells. The cells are then observed under an
inverted microscope (Dewinter, Italy) at different magnifi-
cations to examine any adverse effect on cellular morphology.
RDHDTCA treated cells are then incubated with Cu2+ for
15–30 min and observed under an inverted fluorescence micro-
scope at different magnifications with blue filter. Images are
taken through an attached ccd camera with the help of Bio-
Wizard 4.2 software. Control experiment is done using
medium devoid of Cu2+ salt.
Scheme 1 Synthesis of RDHDTCA.
filtrate is kept undisturbed. After 3 days, pink color crystals
suitable for single crystal X-ray diffraction analysis are col-
lected. 1H NMR (300 MHz, CDCl3) (Fig. S9, ESI†): 1.13 (12H,
m, J = 6.9 Hz), 3.28 (8H, m), 6.23 (2H, d, J = 8.3 Hz), 6.45 (2H,
m), 6.89 (2H, m), 7.07 (1H, d, J = 2.8), 7.12 (1H, d, J = 6.7), 7.22
(1H, d, J = 4.5), 7.26 (1H, s), 7.48 (2H, s), 7.96 (1H, d, J = 2),
8.91 (1H, s); elemental analysis data as calculated for
C33H34N4O2S (%): C, 71.97; H, 6.22 and N, 10.17; S, 11.64;
Found (%): 72.02, 6.31, 10.14 and 11.53. QTOF-MS ES+
(Fig. S10, ESI†): [M + H]+ = 551.92; FTIR/cm−1 (Fig. S11, ESI†):
ν(CvO) 1721, ν(CvN) 1614.
Acknowledgements
S. Adhikari and A. Ghosh are thankful to UGC, New Delhi,
Department of Environment, Govt. W. B. and B. U. for provid-
ing fellowships. S. Lohar is thankful to CSIR for fellowship.
Authors sincerely acknowledge K. Chatterjee for recording
some data. We also acknowledge Indian Institute of Chemical
Biology (IICB), Jadavpur University and Burdwan University for
providing mass spectra, NMR and fluorescence microscope
facilities respectively.
Synthesis of [RDHDTCA–Cu2+] complex. In an ethanol solu-
tion of RDHDTCA (200 mg, 0.36 mmol), 2 mL solution of
Cu(NO3)2·3H2O (0.006 g, 0.024 mmol) in ethanol is added
drop-wise and stirred for 5 minutes. The solution is kept for
several days to finally obtain a deep pink compound. QTOF-MS
ES+ (Fig. S5, ESI†): [M + H]+ = 443.13; FTIR/cm−1 (Fig. S11,
ESI†), ν(CvO), 1585.
Single crystal X-ray structure analysis
Single crystal X-ray diffraction data for RDHDTCA are collected
at 100(2) K, on a Bruker SMART 1000 CCD diffractometer
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