Zn2+ mediated solvent free solid state red emitting fluorescent complex formation in a mortar-pestle along with living cell imaging studies

Article abstract of DOI:10.1039/c5ra01059d

An acridone based highly Zn²⁺ selective, cell-permeable turn-on fluorescence probe (AAS) shows yellow fluorescence at 560 nm (λ_ex, 445 nm) in dry methanol/DMSO up to 100 μM Zn²⁺. At higher Zn²⁺ concentration (>1

Full text of DOI:10.1039/c5ra01059d

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Zn2+ mediated solvent free solid state red emitting
fluorescent complex formation in a mortar–pestle
along with living cell imaging studies†
Cite this: RSC Adv., 2015, 5, 33878
Susanta Adhikari, a Avijit Ghosh,a Sandip Mandal,b Animesh Sahanab and Debasis Dasb
*
An acridone based highly Zn2+ selective, cell-permeable turn-on fluorescence probe (AAS) shows yellow
fluorescence at 560 nm (lex, 445 nm) in dry methanol/DMSO up to 100 mM Zn2+. At higher Zn2+
concentration (>100 mM in dry methanol) AAS yields a red solid polymeric complex having strong
emission at 605 nm. Interestingly, the red solid polymer also appears at relatively lower Zn2+
concentration (>50 mM) in water. The lowest detection limit of AAS is 0.1 nM Zn2+. AAS is employed for
fluorescence bio-imaging of Zn2+ in human MCF 7 breast cancer cells and HeLa cells. Moreover, AAS
develops an orange colour detectable by the naked eye in the presence of trace Zn2+ in a mixture of
various common cations and anions in the solid state (requires no solvent). Hence, this method is
extremely green and interference free. AAS can be employed as a laboratory indicator for detection of
Zn2+ in real samples and live cells.
Received 19th January 2015
Accepted 30th March 2015
DOI: 10.1039/c5ra01059d
www.rsc.org/advances
an antagonist for treatment of estrogen receptor (ER)-positive
breast cancer. It has been found that Zn2+ mediates
1. Introduction
Zinc plays an important role in cell growth,1 apoptosis and
metabolism,2 gene expression, signal transduction,3,4 regulation
of endocrine,5 and immune and neuronal functions implicated
in the pathophysiology of depression.6 Severe Zn deciencies
cause developmental anomalies in human and animals while
an increased Zn level causes high cytotoxicity in the context of
acute brain injury.7 The role of Zn2+ in prostate cancer cells,
ER-positive MCF-7 breast cancer cells, human melanoma cells
and many other important biological processes demands its
accurate trace level measurement in biological systems.
Evidence for a zinc uptake transporter in human prostate
cancer cells has already been established.8 Expression of the
zinc transporter ZnT4 decreases in the progression from early
prostate disease to invasive prostate cancer.9 In the prostate,
zinc is believed to help with reproductive functions by aiding in
the accumulation of citrate, a component of semen and has a
protective role against development and progression of prostate
cancer.10,11 However, recent observations indicate that high
supplemental zinc intake is associated with an increased risk of
advanced prostate cancer (PCa).11–14 Tamoxifen, a non-steroidal,
selective estrogen receptor modulator (SERM) has been used as
tamoxifen-induced autophagy and cell death in MCF-7 breast
cancer cell line.15 Several probes can monitor low mobile Zn2+
concentration for in vivo testing via living cell-imaging.16,17 As
Zn2+ concentration varies with physiological environments, viz.
12 mM in serum and 0.1–0.5 mM in gray matter and the brain,
the development of a nontoxic interference free Zn2+ sensor
with concentration tunable emission properties is highly
demanded. Visible light excitation minimizes photo damage
and background interference from auto-uorescence of bio-
logical samples.18,19 Zn2+ sensors are primarily based on uo-
rescein, coumarin, di-picolyl amine etc.20–25 Substituted
benzoxazole derivative sensing Zn2+ via excited-state intra-
molecular proton transfer (ESIPT) is also reported.26 Acridone
alkaloids having varieties of biological activities viz. anticancer,
antimicrobial, antiviral and anti-parasitic properties27 have
been used for labelling and uorescence-based assays for their
chemical inertness and resistance to photo-bleaching. Acri-
dones can also detect bio-molecules and, are used to measure
Lewis acidity of cations28 and monitoring of enzyme activity.29
Recently, we have reported heme-interacting acridone deriva-
tive that can prevent free heme-mediated protein oxidation and
degradation, markers for heme-induced oxidative stress.27 In
contrast to uorescein, dansyl, anthracene and rhodamine
moieties, mostly used in chemosensors, acridone is highly
resistant to photo-bleaching. Our ongoing effort for improved
aDepartment of Chemistry, University of Calcutta, 92, A.P.C. Road, Kolkata 700 009,
West Bengal, India. E-mail: adhikarisusanta@yahoo.com; Fax: +91 33 23519755;
Tel: +91 33 23509937
sensors16,30 have resulted
a
new acridone derivative,
bDepartment of Chemistry, The University of Burdwan, Burdwan 713104, West Bengal,
(E)-4-(2-hydroxybenzylideneamino)acridin-9(10H)-one (AAS) for
selective sensing of Zn2+ under solvent free condition. To the
best of our knowledge, this is the rst report on visible light
India
† Electronic supplementary information (ESI) available. See DOI:
10.1039/c5ra01059d
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excitable, Zn2+ assisted polymeric complex formation which Hz, d), 7.23 (m, 2H, e), 7.44 (m, 1H, f), 7.53 (d, 1H, J ¼ 7.5 Hz, g),
gives intense solid state red emission (orange in naked eye) of 7.69 (m, 1H, h), 7.89 (d, 2H, J ¼ 8.1 Hz, i), 8.11 (d, 1H, J ¼ 9.9 Hz,
AAS. Naked eye detection of a colorless d10 cation (Zn2+) under j), 8.19 (d, 1H, J ¼ 8.1 Hz, k), 8.98 (s, 1H, c), 10.87 (s, 1H, b), 11.53
solvent free and interference free condition is a signicant and (s, 1H, a); 13C NMR (Fig. S2†) (125 MHz, DMSO-d6) d 118.96,
challenging contribution to green chemistry. Cd2+ interferes in 121.36, 122.59, 123.85, 124.44, 126.09, 126.52, 127.43, 128.35,
dry test for Zn2+ detection in solid state.31 Visible light excitation 129.31, 131.22, 137.30, 138.69, 139.13, 140.65, 144.73, 146.15,
and strong red emission are highly desirable in vivo application. 164.91, 168.56, 182.07; HRMS (Fig. S3†), m/z (M + H)+ calculated
We have been successful to cluster all the desired properties in a for C20H14N2O2: 315.3453, found: 315.3453. IR (neat) lmax
single probe, AAS. Moreover, AAS–Zn2+ system having solid state 3392.78, 1598.98, 1521.83, 1452.39, 1332.81, 1274.94, 744.52
red uorescence may be useful in optoelectronic applications.
(Fig. S4†).
2. Experimental section
2.1 General information of materials and methods
2.3 Synthesis of 2-(2-aminophenylamino)benzoic acid (1)
A mixture of 1,2-phenylenediamine (2 g, 18.50 mmol), 2-chloro
benzoic acid (2.89 g, 13.08 mmol), powdered Cu (200 mg), Cu2O
(200 mg), trans 1,2-cyclohexyldiamine (10 mol%) and K2CO3
(3.873 g, 27.75 mmol) in diglyme (30 mL) were heated under
reux for 8 h. Excess diglyme was removed by distillation and
the mixture poured into 1 L of hot water. Then 10 mL of 6(N)
HCl was added to the mixture. The bluish black solid formed
was ltered, washed and collected. The crude acid was dissolved
in aqueous KOH, boiled in presence of activated charcoal and
ltered. On acidication of the ltrate with HCl, a bluish black
solid precipitate of 1 was obtained 1.26 g (yield ꢁ 30%). Rf: 0.41
All metal cation were used as either their nitrate or their chlo-
ride salts and the anions as their Na salts. Other chemicals were
of analytical reagent grade and used without further purica-
tion except when specied. Milli-Q Milipore 18.2 MU cmꢀ1
water was used throughout all experiments. A JASCO (model
V-570) UV-vis spectrophotometer was used for recording UV-vis
spectra. FTIR spectra were recorded on a JASCO FTIR spectro-
photometer (model FTIR-H2O). Mass spectra were carried out
using a QTOF Micro YA 263 mass spectrometer in ES positive
mode. 1H NMR spectra were recorded using a Bruker Avance
300 (300 MHz) inDMSO-d6 whereas 13C NMR spectra were
recorded using a Bruker Avance 500 (125 MHz) in DMSO-d6.
Melting point was measured with a VEEGO digital melting point
apparatus. Elemental analysis was performed using a Perkin-
Elmer CHN-Analyzer with the rst 2000-Analysis kit. Steady-
state uorescence emission and excitation spectra were recor-
ded with a Hitachi-Hitachi F-4500 spectrouorometer. A
Systronics digital pH meter (model 335) was used to measure
the solution pH. Either 50 mM HCl or KOH was used for pH
adjustment.
1
ꢂ
(2% methanol in dichloromethane); mp: 125–127 C; H NMR
(Fig. S5†) (300 MHz, DMSO-d6) d 6.67 (m, 1H), 6.76 (m, 1H), 7.19
(d, 2H, J ¼ 7.2 Hz), 7.32 (d, 2H, J ¼ 7.2 Hz), 7.39 (d, 1H, J ¼ 6.9
Hz), 7.89 (d, 1H, J ¼ 6.6 Hz), 9.18 (s, 1H); HRMS (Fig. S6†) m/z
(M
+
H)+ calculated for C13H12N2O2N: 229.2545, found:
229.2543.
2.4 Synthesis of 4-amino acridone (2)
400 mg (1.75 mmol) of 1 was taken in a round bottom ask to
which 5 mL of Eaton's reagent was added. The mixture was
heated under stirring condition in nitrogen atmosphere for 2 h,
whereby the mixture turned to yellow colour. Then the reaction
mixture was poured into 500 mL cold water and made alkaline
by liquor ammonia. The greenish yellow precipitate formed was
ltered, washed with water and dried. The crude solid was
puried by silica gel column chromatography using 15% MeOH
in dichloromethane as eluent to obtain 2 (238 mg, yield ꢁ 65%)
as light green solid. Rf: 0.3 (10% methanol in dichloromethane);
2.2 Synthesis of ligand AAS
Scheme 1 presents the synthetic protocol of AAS. 4-Amino
acridone, the signalling unit is synthesized following our pub-
lished procedure.27 The acridone based ligand AAS can be syn-
thesised by the Schiff's base formation between 4-amino
acridone (2) with salicylaldehyde.
320 mg of 2 (1.508 mmol) and 0.116 mL of salicylaldehyde
(1.508 mmol) were taken in 10 mL of dry methanol and reuxed
under argon for 6 h. A yellow solid precipitate was obtained.
Filtered the solid and washed with cold methanol, dried in
vacuum to obtain 375 mg (yield ꢁ 80%) of AAS as a yellow solid.
1H NMR (Fig. S1†) (300 MHz, DMSO-d6): d 6.97 (d, 2H, J ¼ 8.1
ꢂ
mp: 300 C; IR (neat) nmax 3410.26, 1680.05, 1506.46, 1452.45,
1230.63, 1128.39; 1H NMR (Fig. S7†) (300 MHz, DMSO-d6) d 6.90
(m, 2H), 7.10 (m, 1H), 7.40 (m, 1H), 7.60 (m, 2H), 8.11 (dd, 1H,
J ¼ 8.25, 1.05 Hz), 10.522 (bs, 1H); HRMS (Fig. S8†) m/z (M + H)+
calculated for C13H10N2O: 211.2393, found: 211.2365.
An alternative route for compound 1 following two steps
reaction sequences via 2-((2-nitrophenyl)amino)benzoic acid
(1A) was also adopted (Scheme S1, ESI†). In this route, 2-nitro-
aniline was condensed with 2-chlorobenzoic acid using Ullmann
reaction to obtain 1A and subsequent reduction of the nitro
group in presence of SnCl2 in methanol resulted compound 1 in
good yields. 1H NMR, 13C NMR, TOF-MS and ATIR (Fig. S1–10†)
are used for its structure elucidation (Scheme 2).
Scheme 1 Synthetic protocol of AAS.
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Scheme 2 Synthetic route of 4-amino acridone (2).
3. Results and discussion
3.1 Effects of Zn2+ on spectroscopic properties of AAS
Emission properties of AAS have been observed in HEPES buff-
ered aqueous methanol (0.1 M, water–methanol, 97.5 : 2.5, v/v,
pH 7.4). The pattern is almost same to that of dry methanol;
however emission intensities and wavelengths are slightly
different (Fig. S20, ESI†). In presence of Zn2+, the emission band
of AAS is red shied in MeOH, DMSO and mixed solvents (water–
Fig. 1 Changes in the fluorescence spectra of AAS (10 mM) upon
gradual addition of Zn2+ (0.0001, 0.0005, 0.001, 0.005, 0.01, 0.05, 0.1,
methanol/DMSO, 97.5 : 2.5, v/v). In dry methanol, the red shi is 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 6.0, 8.0, 10.0, 15.0, 25.0, 30.0, 40.0, 50.0, and
about 18 nm (AAS: lem, 542 nm; AAS–Zn2+ adduct: lem, 560 nm).
100.0 mM) in dry methanol. Inset: lower concentration (0.0001 mM to
1.0 mM).
Additionally, at higher Zn2+ concentration, AAS forms a red
precipitate in dry methanol which shows solid state red uo-
rescence (lem, 605 nm). The red polymer gradually forms upon
(Fig. S30, ESI†). The naked eye yellow colour changes are
addition of water in methanol solution of the Zn2+–AAS adduct.
Solvent and pH, two inuential parameters have been optimized
to study the photo-physical properties of AAS and its Zn2+ adduct
shows the emission intensities of the systems is highly solvent
dependent (Fig. S11, ESI†). Interestingly, emission intensity of
free AAS increases with increasing water content up to ꢁ35%
(v/v) followed by gradual decrease to the minimum at 97.5% (v/v,
lex ¼ 445 nm, Fig. S12, ESI†). On the other hand, emission
intensity of AAS–Zn2+ system decreases gradually with increasing
water percentage (Fig. S13, ESI†). However, AAS–Zn2+ system
have always higher emission intensity than free AAS. Fluores-
cence titrations have been performed both in dry and aqueous
methanol (97.5 : 2.5, v/v) to have deeper insight of role of water
on AAS–Zn2+ interaction. Changes in the uorescence spectra of
AAS upon addition of Zn2+ in dry methanol are presented in
Fig. 1. Lowest detection limit of AAS is 0.0001 mM of Zn2+. The
emission intensities gradually increase with increasing Zn2+
(Fig. S18, ESI†), the linear region of the plot, (inset, Fig. S18,
ESI†) is useful for determination of unknown Zn2+ concentra-
tion. In presence of Zn2+, emission intensity of free AAS
enhances ꢁ41.36 fold in dry methanol whereas in 97.5% water–
methanol (v/v), enhancement is ꢁ8.42 fold. Fig. S19 (ESI†) shows
the colour changes of AAS upon gradual addition of Zn2+ in dry
methanol whereas Fig. S21 (ESI†) shows the colour changes of
AAS in presence of Zn2+ in HEPES buffered media under hand
held UV lamp. Here, the linearity is observed up to 10 mM Zn2+
(Fig. S22, ESI†).
observed consistently upon addition of Zn2+ (Fig. S31, ESI†). On
the other hand, in aqueous methanol, the band at 409 nm
gradually decreases with the appearance of three new bands at
348 nm, 482 nm and 517 nm respectively due to complexation
between AAS and Zn2+ (Fig. S32, ESI†). Corresponding naked eye
colour changes due to gradual addition of Zn2+ and time
dependent colour changes of the AAS–Zn2+ complex under
UV-lamp are shown in Fig. 3.
3.2 Effects of pH on spectroscopic properties of AAS
The emission intensities of free AAS and AAS–Zn2+ systems are
almost equal at lower pH (up to pH 4.0) (Fig. S14, ESI†). Free
AAS shows slight uorescence enhancement above pH 4.0 and
gradually decreases above pH 6.0. Whereas the emission
intensity of AAS–Zn2+ system gradually increases with pH,
reaching maximum in the pH range 7.0 to 9.0 and nally
The absorbance of free AAS (at 409 nm) decreases with
increasing amount of water (Fig. S28, ESI†). In presence of Zn2+,
absorbance of AAS decreases at 414 nm, and increases at
522 nm with increasing water content (Fig. S29, ESI†). In pres-
ence of Zn2+ (in methanol), the absorbance of AAS is red shied
from 409 nm to 460 nm along with two isosbestic points at
424 nm and 265 nm (Fig. 2).
Fig. 2 Changes in the absorbance of AAS (10 mM) upon gradual
addition of Zn2+ in dry methanol (free AAS, 0.0005, 0.005, 0.05, 0.5,
1.0, 3.0, 5.0, 8.0, 10.0, 15.0, 30.0, 40.0, 50.0, and 100.0 mM Zn2+, from
bottom to top).
Plot of absorbance vs. concentration of Zn2+ have yielded a
sigmoidal curve with a linear region up to 10 mM Zn2+
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Furthermore AAS is also highly Zn2+ selective in presence of
common anions (viz. PO43ꢀ, AsO43ꢀ, AcOꢀ, Fꢀ, Clꢀ, Brꢀ, Iꢀ,
NO3ꢀ, NO2ꢀ, SO42ꢀand SCNꢀ) and no anion interference was
observed during detection of Zn2+ (Fig. S17 and inset of Fig. S17
in ESI†).
3.5 Solid state uorescence property of AAS–Zn2+ complex
The AAS–Zn2+ adduct have a strong solid state red uorescence
at 605 nm. In dry methanol, the yellow colour AAS turns red in
presence of high Zn2+ concentration (>100 mM) while the same
phenomenon is observed in aqueous methanol (water–meth-
anol, 97.5 : 2.5, v/v) at >50 mM Zn2+. In solid state, AAS and
AAS–Zn2+ adduct have emissions at 545 nm and 605 nm
respectively (Fig. S25, ESI†). Naked eye and UV irradiated
colours of solid AAS and AAS–Zn2+ adduct are shown in Fig. S26
(in ESI†). Mortar–pestle grinding of solid AAS with traces of Zn2+
salt also generates visibly orange colour complex having red
uorescence (Fig. 5).
Fig. 3 Naked eye color change of AAS (10 mM) in water, HEPES buffer
(0.1 M, pH 7.4) upon gradual addition of Zn2+ (mM) at room tempera-
ture (top) and formation of red color AAS–Zn2+ complex as a function
of time (min) in water–methanol (97.5 : 2.5, v/v, [Zn2+] ¼ 100 mM, [AAS]
¼ 10 mM) under UV-lamp.
decreases above pH 9.0. Probably, at higher pH, precipitation of
Zn2+ occurs while at lower pH, H+ competes with Zn2+ for
binding with AAS.
Thus AAS is an excellent ligand for detection of Zn2+ in real
sample by Mortar–pestle grinding or by a dipstick probe for the
detection of Zn2+ in water, completely avoiding organic solvents.
For this purpose AAS was adsorbed on alumina, a slurry of which
was coated on a thermoplastic stick as support. Solid state
detection of Zn2+ also performed also in presence of various
common anions (Fig. S27 in ESI†). Thus, AAS can be employed as
a laboratory indicator for detection of Zn2+ in real sample.
3.3 Determination of the binding constant of the AAS–Zn2+
complex
Job's plot (Fig. S23, ESI†) indicates 2 : 1 (AAS : Zn2+, mol ratio)
stoichiometry of the AAS–Zn2+ adduct. In dry methanol, the
equilibrium binding constant of AAS for Zn2+ is 3.049 ꢃ 105 Mꢀ1/2
as measured using Benesi–Hildebrand32 equation (Fig. S24, ESI†).
3.4 Zn2+ binding selectivity of AAS
3.6 Zn2+ binding modes of AAS
Emission properties of AAS with common cations viz. Na+, K+,
Ca2+, Mg2+, Mn2+, Ni2+, Cr3+, Fe3+, Al3+, Zn2+, Cu2+, Co2+, Hg2+,
Pb2+ and Cd2+ have been investigated both in dry methanol
(Fig. 4) and HEPES buffered (0.1 M, pH, 7.4) water–methanol
(97.5 : 2.5, v/v) media. Fig. 1 reveals that emission intensity of
AAS signicantly enhanced in presence of Zn2+. Colours of AAS
in presence of different cations under UV light as well as
through naked eye are shown in Fig. S15 (ESI†). No signicant
interferences have been found from common cations as
mentioned supra (Fig. S16, in ESI†).
Solvent dependent and Zn2+ concentration regulated differen-
tial interaction between AAS and Zn2+ is also nicely demon-
1
strated by H NMR titration in DMSO-d6 (Fig. 6) which reveals
0.02 ppm (d ¼ 11.531 to 11.552) downeld shi of –NH of
acridone, 0.016 ppm downeld shi of –OH and 0.034 ppm
upeld shi of imine proton upon addition of 2.5 equivalent
Zn2+ to AAS indicating interaction of these donor sites with Zn2+
leading to chelation enhanced uorescence (CHEF). Upon
Fig. 4 Fluorescence spectra of AAS (10 mM) in presence of common Fig. 5 (a) Naked eye colour of free AAS and (b) after mortar–pestle
cations (100 mM) in dry methanol: (1) free AAS, (2) Na+, (3) K+, (4) Ca2+
,
grinding with Zn2+ in presence of other common cations, (c) colour of
(5) Mg2+, (6) Mn2+, (7) Ni2+, (8) Cr3+, (9) Fe3+, (10) Al3+, (11) Cu2+, (12) AAS in presence of Zn2+ under UV light and (d) colour of AAS under UV
Co2+, (13) Hg2+, (14) Pb2+, (15) Cd2+ and (16) Zn2+; lex ¼ 445 nm. Inset: light with Zn2+ in presence of other common cations. AAS shows slight
respective emission intensities.
yellowish-green colour under UV light (Fig. S26(c) in ESI†).
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Fig. 8 Energy optimised structures of AAS and AAS–Zn2+
.
(Fig. S37 and S38, ESI†) are distinctly different. Loss of H2O
between 102.8 ꢂC and 168.0 ꢂC corresponding to ꢁ4.64% weight
loss clearly establishes our proposed AAS–Zn2+ adduct (Fig. S38,
ESI†). Since EDTA is a good Zn2+ chelator, decomplexation of
the red solid would be expected upon washing with EDTA
solution but the red solid remain unchanged by EDTA solution.
Thus we expected the red solid may be a polymeric complex.
The plausible binding mechanism of AAS with Zn2+ has been
shown in Fig. 7. AAS is weak emitter due to ICT process. Addi-
tion of Zn2+ in dry methanol inhibits ICT to produce CHEF.
Upon addition of water to the system, an intermediate state is
observed where imine bond breaks and imine N centre gets
protonated. When amount of water is relatively high, then
complete proton transfer from –OH to imine –N centre occurs34
(Fig. S39, ESI†). At higher Zn2+ or water concentration, depro-
tonation of OH leading to formation of C]O occurs and
subsequently the red complex polymer forms (Fig. 7). Fig. S33
(ESI†) reveals that the polymeric AAS–Zn2+ complex have no
–OH or acridone–NH protons. So, it is electrically neutral and
gets precipitated in polar solvent. Life time measurement
experiment of AAS, [AAS–Zn2+] and [AAS–Zn2+–Water] with
addition of water also supports the involvement of ESIPT
process (Fig. S40 in ESI†).
Fig. 6 1H NMR spectral changes of AAS after interacting with Zn2+: (I)
free AAS; (II) AAS + 0.5 equivalent Zn2+; (III) AAS + 2.5 equivalent Zn2+
;
(IV) AAS + 2.5 equivalent Zn2+ + 0.1 mL D2O; (V) AAS + 2.5 equivalent
Zn2+ + 0.3 mL D2O. 1H NMRs are recorded in 0.5 mL DMSO-d6. “S” is
the new ‘NH’ proton.
addition of water to the Zn2+–AAS complex or at higher Zn2+
concentration, the keto form predominates, responsible for red
shi of the emission band from 560 nm (dry methanol) to
565 nm (aqueous methanol). Addition of 0.1 mL D2O to 0.5 mL
DMSO-d6 solution of AAS–Zn2+ adduct results a new peak at
10.12 ppm, assigned to a new –NH proton(s) arising due to
keto–enol tautomerism. Thus the imine bond is lost leading to
free rotation feasible. The –NH proton of acridone further
downeld shied to 11.72 ppm. Further addition of 0.2 mL D2O,
complete the appearance of the new proton. 1H NMR of the
isolated red complex (DMSO-d6, in spite of its very poor solu-
bility) shows only one –NH proton at 9.21 ppm (Fig. S33, ESI†),
demonstrating that –NH proton of acridone also takes part into
keto–enol tautomerism (acridine). These type of molecules have
the molecular structure to give ESIPT.33 Mass spectrum also
support the adduct formation involving two AAS units with one
Zn2+ in dry methanol. In presence of water, one H2O acts as a
linker between two AAS–Zn2+ units (Fig. S34 and S35 respec-
tively, ESI†), which support the polymer formation. Thermog-
ravimetric analyses (TGA) of AAS and AAS–Zn2+ complex
3.7 DFT calculation and theoretical study
DFT calculations are performed to better understand the
possible binding modes of AAS with Zn2+. The optimized
geometries of AAS (Fig. 8) and AAS–Zn2+ adduct (Fig. 9) are
Fig. 9 Fluorescence microscope images of (I) human breast cancer
MCF7 cell and (II) HeLa cell: (a) and (b) are bright field and fluorescence
images of cells after 30 min incubation with Zn2+ (50 mM); (c) and (d)
are bright field and fluorescence images of Zn2+ (50 mM) incubated
cells after 2 h treatment with AAS (10 mM).
Fig. 7 Plausible binding mechanism of AAS with Zn2+
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generated using 3-21G/B3LYP basis sets respectively of fellowships. S. Adhikari acknowledges the SERB-DST (project
Gaussian 09 soware.35 In free AAS the HOMO lies in acridone no. SR/S1/OC-101/2012) for nancial support.
ring and the LUMO spreads over in the salicylaldehyde imine
part (Fig. S41†). On complexation the energy of individual
HOMO and LUMO decreases and the energy gap between the
highest occupied molecular orbital (HOMO) and lowest unoc-
Notes and references
cupied molecular orbital (LUMO) also decreases, thus there is
an increase in the stability of the whole system (Fig. S41†).
Hence, the observed red shi in the absorption spectra of
AAS–Zn2+ can be explained in the terms of decreased band gap
of HOMO and LUMO.
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´
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To further demonstrate the practical application of the probe, we
carried out experiments in living cells (detail in ESI†). AAS is very
efficient to detect intracellular Zn2+ in human breast cancer cell,
MCF7 and HeLa cells under normal and uorescence micro-
scope (Fig. 9). Incubation of cells with Zn2+ (50 mM) for 30 min. at
37 ꢂC was followed by the addition of AAS (10 mM) aer washing
three times with media and then was incubated further for
another 2 h. The enhancement of uorescence was observed
(Fig. 9). The results suggest that AAS can penetrate the cell
membrane and can be used for imaging of Zn2+ in living cells.
Furthermore, we have investigated the cell permeability of AAS
in presence and absence of an intracellular zinc chelator TPEN,
the results shows that AAS is cell permeable and can be used to
detect intracellular zinc ion concentration (Fig. S42, in ESI†). In
presence of TPEN, the uorescence of [AAS–Zn2+] completely
diminished due to the unavailability of Zn2+ ion in cells.
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3.9 Cytotoxicity of AAS
Further, the cytotoxicity of AAS on MCF7 and HeLa cells was
determined by a conventional MTT assay (details in ESI,
Fig. S43†), which revealed that, upon exposure to a 10 mM
concentration of AAS (a concentration that was comparable to
that used for confocal imaging studies; Fig. 8) for 12 h, ꢁ90% of
the cells remained viable. This nullied the possibility of any
signicant cytotoxic inuence of the reagent AAS on HeLa cells.
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viable chemosensor for Zn2+ in biological samples.
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4. Conclusion
Thus, an acridone based probe generates solid state red uo-
rescence in presence of trace level Zn2+ having naked eye orange
color in presence of all the other cation and anion including
biologically important ones. Interference free detection of Zn2+
in real sample and live cells is possible. The polymeric red
emitting AAS–Zn2+ complex may be useful as optoelectronic
materials.
Acknowledgements
24 S. Goswami, A. K. Das, K. Aich, A. Manna, S. Maity, K. Khanra
and N. Bhattacharyya, Analyst, 2013, 138, 4593–4598.
Authors acknowledge Dr R. Banerjee (IICT, Hyderabad) for cell
imaging studies. A. Ghosh and S. Mandal are thankful to UGC, 25 S. Goswami, A. K. Das, B. Pakhira, S. B. Roy, A. K. Maity,
New Delhi and A. Sahana to CSIR, New Delhi for providing
P. Saha and S. Sarkar, Dalton Trans., 2014, 43, 12689–12697.
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