RSC Advances

PAPER

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article OnlineView Journal

aDepartment of Chemistry, Burdwan Unive

Bengal, India. E-mail: pabitracc@yahoo.combDepartment of Chemistry, Guru Nanak DevcCytogenetics and Molecular Biology Labora

Kalyani, Kalyani 741235, India

† Electronic supplementary informationand crystallographic data in CIF or10.1039/c3ra43305f

Cite this: DOI: 10.1039/c3ra43305f

Received 29th June 2013Accepted 12th August 2013

DOI: 10.1039/c3ra43305f

www.rsc.org/advances

This journal is ª The Royal Society of

A cell permeable Cr3+ selective chemosensor and itsapplication in living cell imaging†

Manjira Mukherjee,a Buddhadeb Sen,a Siddhartha Pal,a Maninder S. Hundal,b

Sushil Kumar Mandal,c Anisur Rahman Khuda-Bukhshc and Pabitra Chattopadhyay*a

An efficient fluorescent Cr3+ receptor, 2-(5,6-dihydro-benzo-[4,5]imidazo[1,2-c]quinazolin-6-yl)-quinolin-8-

ol (H2L1) was synthesized and characterized by physico–chemico and spectroscopic tools along with single

crystal X-ray crystallography. This probe (H2L1) behaves as a highly selective fluorescent sensor for Cr3+ ions

at biological pH in ethanol–water (1 : 5, v/v) HEPES buffer (0.1M, pH 7.4) at 27 �C.Metal ions, viz. alkali (Na+,

K+), alkaline earth (Mg2+, Ca2+), and transition-metal ions ((Mn2+, Fe3+, Co3+, Ni2+, Cu2+, Zn2+) and Pb2+, Ag+

did not interfere. The lowest detection limit for Cr3+ was calculated to be 3.6 � 10�7 mol L�1 within a very

short responsive time (15–20 s) in ethanol–water (1 : 5, v/v) HEPES buffer (0.1M, pH 7.4) at 27 �C. The sensoris efficient for detection of Cr3+ in vitro, developing a good image of the biological organelles.

Introduction

Chromium(III) is essential for many biochemical and physio-logical functions in organisms ranging from bacteria tomammals due its role in cholesterol and glucose metabolism,1

but can oen be toxic to certain biological systems when thelevels of Cr3+ exceed cellular needs.2 The release of aqueouschromium as trivalent or hexavalent salt to the subsurface atnumerous sites including drinking water supply systemsthrough a variety of industrial processes such as metallurgical,chemical industries, electroplating, refractories, pigments,tanning industries, oxidative dying and cooling water towers.3

Usually low chromium levels are determined aer separation bysorption, liquid–liquid extraction or co-precipitation4 followedby analysis by potentiometry,5 spectrophotometry,6 uorim-etry,7 ame atomic absorption spectrometry,8 graphite furnaceatomic absorption spectrometry,9 inductively coupled plasmaatomic emission spectroscopy,10 X-ray uorescence spectrom-etry11 or by electroanalytical techniques.12

Fluorescence techniques have become powerful tools forsensing and imaging metal ions in trace amounts because ofsimplicity, high sensitivity and real-time monitoring with ashort response time. Sensitive and selective uorogenicmolecular sensors are good tools for evaluating and dynami-cally mapping the intracellular uctuations of metal ions by

rsity, Golapbag, Burdwan-713104, West

University, Amritsar-143005, India

tory, Department of Zoology, University of

(ESI) available. CCDC 942239. For ESIother electronic format see DOI:

Chemistry 2013

using microscopy techniques to allow real-time local imaging.13

Due to the paramagnetic nature of Cr3+ ion for uorescencequenching of uorophore via enhancement of spin–orbitcoupling, a few cell permeable uorescent turn-on probe for thedetection of Cr3+ are available in the literature.14–17 Reports ofthe rhodamine based15 or dansyl-based16 or coumarin based17

Cr3+ selective chemosensor are available. However, to the best ofour knowledge, 2-(2-amionophenyl)benzimidazol based organicmoiety, 2-(5,6-dihydro-benzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-quinolin-8-ol (H2L

1) as a chemosensor for Cr3+ ion in aqueoussolution is still unexplored.

Herein, we report the preparation and characterization of anovel chemosensor for Cr3+ ion in ethanol–water (1 : 5, v/v)HEPES buffer (0.1 M, pH 7.4) at 27 �C. This is based on theselective uorescence enhancement through chelation (CHEF)with Cr3+ ion in the physiological pH range. The proposedsensor has signicant advantages as it offers a very low detec-tion limit (18.72 ppb of Cr3+ ion), covers a wider workingconcentration range (3.6� 10�7 to 4.5� 10�5 mol L�1), displaysa faster response time (15–20 s) and exhibits a higher selectivityin the presence of several common interfering ions.

ExperimentalMaterials and physical measurements

All of the solvents were of analytical grade. The elemental anal-yses (C, H and N) were performed on a Perkin Elmer 2400 CHNelemental analyzer. A Shimadzu (model UV-1800) spectropho-tometer was used for recording electronic spectra. IR spectrawere recorded using Perkin Elmer FTIRmodel RX1 spectrometerpreparing KBr disk. 1H NMR spectrum of organic moiety wasobtained on a Bruker Avance DPX 300 spectrometer usingDMSO-d6 solution. Electrospray ionization (ESI) mass spectra

RSC Adv.

Table 1 Crystal data and details of refinements for H3L1$NO3$CH3OH

Empirical formula C24H21N5O5

RSC Advances Paper

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article Online

were recorded on a Qtof Micro YA263 mass spectrometer. Molarconductance (LM) was measured in a Systronics conductivitymeter 304 model using �10�3 mol L�1 solutions in methanol. ASystronics digital pHmeter (model 335) was used tomeasure thepH of the solution and the adjustment of pH was done usingeither 50 mM HCl or KOH solution. Steady-state uorescenceemission and excitation spectra were recorded with a PerkinElmer LS-55 spectrouorimeter. The uorescence spectra of thetitration of chromium ion with organic moiety were obtained atan emission wavelength of 426 nm in the uorimeter. Time-resolved uorescence lifetime measurements were performedusing a HORIBA JOBIN Yvon picosecond pulsed diode laser-based time-correlated single-photon counting (TCSPC) spec-trometer from IBH (UK) at lex ¼ 370 nm and MCP-PMT as adetector. Emission from the sample was collected at a right angleto the direction of the excitation beammaintaining magic anglepolarization (54.71). The full width at half-maximum (FWHM) ofthe instrument response function was 250 ps, and the resolutionwas 28.6 ps per channel. Data were tted to multiexponentialfunctions aer deconvolution of the instrument response func-tion by an iterative reconvolution technique using IBH DAS6.2 data analysis soware in which reduced w2 and weightedresiduals serve as parameters for goodness of t.

The uorescence property of the sensor was investigated inethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 27 �C.pH study was done in ethanol–water (1 : 5, v/v) HEPES buffer(0.1 M) by adjusting pH with HCl or NaOH at 27 �C. In vivo studywas performed at biological pH� 7.4 taking ethanol–water (1 : 5,v/v) HEPES buffer (0.1 M, pH 7.4) at 27 �C. The stock solutions(�10�2 M) for the selectivity study of the probe (H2L) towardsdifferent metal ions were prepared taking nitrate salts ofsodium(I), potassium(I), copper(II), chromium(III), silver(I);acetate salt of manganese(II), zinc(II); chloride salts of nickel(II),cobalt(II), mercury(II), calcium(II), magnesium(II), iron(III); iron(II)sulphate; in HEPES buffer (0.1 M). In this selectivity study theamount of these metal ions was a hundred times greater thanthat of the probe used. Fluorescence titrationwas performedwiththe solution of [Cr(H2O)6](NO3)3$3H2O in ethanol–water (1 : 5,v/v) HEPES buffer (0.1M, pH 7.4) varying themetal concentration0 to 100 mM and the probe concentration was 16.6 mM at 27 �C.

Formula weight 459.46Crystal system MonoclinicSpace group P21/ca (A) 12.4864(7)b (A) 17.5092(11)c (A) 10.6207(5)a ¼ g 90�

b 108.458(2)�

Volume (A3) 2202.5(2)Temperature (K) 296(2)Z 4rcalc (g cm�3) 1.386m (mm�1) 0.100F(000) 960Crystal size (mm3) 0.18 � 0.16 � 0.10q range (deg) 1.72–24.82Reections collected/unique 3774/2539Final R indices [I > 2s(I)] 0.0856R indices (all data) 0.0524Goodness-of-t on F2 1.028

Preparation of 2-(5,6-dihydro-benzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-quinolin-8-ol (H2L

1)

2-(2-Aminophenyl)benzimidazole (2.09 g, 10.0 mmol) and8-hydroxy quinolin-2-carboxyl-aldehyde (1.73 g, 10.0 mmol)were mixed in dry C2H5OH (25 ml) at room temperature underdinitrogen atmosphere. Then the reaction mixture wascontinued to reux for 6.0 h. The yellow coloured precipitate ofthe compound (H2L) was obtained from the solution throughslow evaporation of the solvent. The compound was recrystal-lized from the methanol. C23H16N4O: anal. found: C, 75.14; H,4.29; N, 15.56; calcd: C, 75.81; H, 4.43; N, 15.38. ESI-MS: [M +H]+, m/z, 364.90 (100%) (calcd: m/z, 364.40; where M ¼ molec-ular weight of H2L

1); IR (KBr, cm�1): nOH, 3437; nNH, 3231;nCH]N, 1619.

1H NMR (d, ppm in DMSO-d6): 9.551 (s, 1H); 8.197(d, 1H, J¼ 8.6); 7.979–7.928 (m, 2H); 7.677 (d, 1H, J¼ 7.5); 7.514

RSC Adv.

(d, 1H, J ¼ 7.8); 7.428 (t, 1H, J ¼ 7.9); 7.302 (d, 1H, J ¼ 8.1);7.252–7.204 (m, 2H); 7.172–7.107 (m, 2H); 7.001 (d, 1H, J ¼ 8.5);6.933 (d, 1H, J ¼ 8.1); 6.827 (t, 1H, J ¼ 7.5); yield: 90%.

Preparation of the chromium(III) complex [Cr(L)(NO3)(H2O)]

To a methanolic solution of H2L1 (363.0 mg, 1.0 mmol) solid

chromium(III) nitrate nonahydrate (400.2 mg, 1.0 mmol) wasadded at a time and the reaction mixture was stirred at ambienttemperature for 6.0 h. The solution thus obtained was then keptaside for slow evaporation at room temperature. Aer a fewdays, deep green crystalline complex were collected by washingwith water and methanol, and then dried in vacuo.C23H16CrN5O5: anal. found: C, 55.69; H, 3.18; N, 14.77; Cr,10.19; calcd: C, 55.88; H, 3.26; N, 14.17; Cr, 10.52. IR (cm�1):nC]N, 1618.30; conductivity (Lo, ohm

�1 cm2 mol�1) in meth-anol: 51. ESI-MS in methanol: [M + Na]+, m/z, 517.39 (obsdwith 30% abundance) (calcd: m/z, 517.40; where M ¼[Cr(L)(NO3)(H2O)]). Yield: 75%.

X-ray data collection and structural determination

The single crystals were obtained from the solution of thecompound (H2L

1) in methanol mixed with a few drops ofaqueous sodiumnitrate solution on slow evaporation. X-ray datawere collected on a Bruker's Apex-II CCD diffractometer usingMo Ka (l ¼ 0.71069). The data were corrected for Lorentz andpolarization effects and empirical absorption corrections wereapplied using SADABS from Bruker. A total of 13 333 reectionswere measured out of which 3774 were independent and 2539were observed [I > 2s(I)]. The structure was solved by directmethods using SIR-92 (ref. 18) and rened by full-matrix leastsquares renement methods based on F2, using SHELX-97.19 Allnon-hydrogen atoms were rened anisotropically. All calcula-tions were performed using Wingx20 package. Important crystaland renement parameters are given in Table 1.

This journal is ª The Royal Society of Chemistry 2013

Fig. 1 An ORTEP view of H3L1$NO3$CH3OH with atom numbering scheme.

Paper RSC Advances

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article Online

Preparation of cell and in vitro cellular imaging with H2L1

Human cervical cancer cell, HeLa cell line was purchased fromNational Center for Cell Science (NCCS), Pune, India and wasused throughout the study. Cell were cultured in Dulbecco'smodied Eagle's medium (DMEM, Gibco BRL) supplementedwith 10% FBS (Gibco BRL), and 1% antibiotic mixture con-taining penicillin, streptomycin and neomycin (PSN, GibcoBRL), at 37 �C in a humidied incubator with 5% CO2. Forexperimental study, cells were grown to 80–90% conuence,harvested with 0.025% trypsin (Gibco BRL) and 0.52 mM EDTA(Gibco BRL) in PBS (phosphate-buffered saline, Sigma Diag-nostics) and plated at desire cell concentration and allowed tore-equilibrate for 24 h before any treatment. Cells were rinsedwith PBS and incubated with DMEM-containing H2L

1 (10 mM,1% DMSO) for 15 min at 27 �C. All experiments were conductedin DMEM containing 10% FBS and 1% PSN antibiotic. Theimaging system was composed of a uorescence microscope(ZEISS Axioskop 2 plus) with an objective lens [10�].

Cell cytotoxicity assay

To test the cytotoxicity of H2L1, MTT [3-(4,5-dimethyl-thiazol-2-

yl)-2,S-diphenyl tetrazolium bromide] assay was performed bythe procedure described earlier.21 Aer treatments of the probe(5, 10, 25, 50, and 100 mM), 10 ml of MTT solution (10 mg ml�1

PBS) was added in each well of a 96-well culture plate andincubated continuously at 27 �C for 8 h. All mediums wereremoved from wells and replaced with 100 ml of acidic iso-propanol. The intracellular formazan crystals (blue-violet)formed were solubilized with 0.04 N acidic isopropanol and theabsorbance of the solution was measured at 595 nm wavelengthwith a microplate reader. Values are means � S.D. of threeindependent experiments. The cell cytotoxicity was calculatedas percent cell cytotoxicity ¼ 100% cell viability.

Results and discussionSynthesis and characterization

The organic moiety (H2L1) was synthesized by condensing an

ethanolic solution of 2-(2-aminophenyl)benzimidazole with8-hydroxy quinolin-2-carboxylaldehyde in equimolar ratio(Scheme 1). It was characterized by physico–chemico andspectroscopic tools and nally structure was conrmed by

Scheme 1 Synthetic routes of the compounds.

This journal is ª The Royal Society of Chemistry 2013

single crystal X-ray crystallography. The probe is soluble incommon polar organic solvents and sparingly soluble in water.Microanalytical and spectroscopic data conrm the composi-tion of the compound as shown in Scheme 1. The peaksobtained in 1H NMR spectrum of H2L

1 have been assigned andthese are in accordance with structural formula of the H2L

1 inthe solution state (Fig. s1†). The ESI mass spectrum of thecompound in methanol shows a peak at m/z 364.90 with 100%abundance assignable to [M + H]+ (calculated value at m/z,364.40) where M¼molecular weight ofH2L

1 (Fig. s2†). All thesedata support the formulation of H2L

1 obtained from the X-raycrystallographic study. An ORTEP view of the nitrated form ofthe probe H2L

1 formulated as H3L1$NO3$CH3OH with the atom

numbering scheme is illustrated in Fig. 1. The crystallographicdata and the bond parameters (selected bond distances andangles) are listed in Tables 1 and 2, respectively. The bondlengths reported in Table 2 indicate that C17–N4 bond distance(1.329 A) is very close to that of C17–N2 (1.335 A) but both valuesare signicantly shorter than that of either C18–N4 (1.396 A) orC23–N2 (1.396 A). This resemblance of both N2 and N4 may bedue to the bonding of one hydrogen atom (H41) to N4 atom.Here, the resulting positive charge of the organic moiety is

Table 2 Selected bond distances (A) and bond angles (�) for H3L1$NO3$CH3OH

Bond distances (A)C1–O1 1.354(3) C11–N3 1.387(4)C6–N1 1.370(3) C17–N4 1.329(3)C9–N1 1.314(3) C17–N2 1.335(3)C10–N3 1.436(4) C18–N4 1.396(3)C10–N2 1.476(3) C23–N2 1.396(3)Bond angles (�)C1–O1–H1 116.0(3) N3–C10–N2 106.6(2)C9–N1–C6 118.0(2) N3–C10–C9 113.9(2)N2–C10–C9 111.2(2) C11–N3–C10 121.6(2)C17–N2–C23 109.0(2) C11–N3–H31 116(2)C17–N2–C10 123.3(2) C10–N3–H31 115.6(2)C23–N2–C10 127.5(2) C17–N4–C18 109.0(2)C17–N4–H41 120.7(2) C18–N4–H41 129.2(2)

RSC Adv.

Fig. 2 Fluorescence spectra of the ligand (H2L1) (10 mM) as a function of

externally added [Cr3+] [0–30 mM] in ethanol–water (1 : 5, v/v) HEPES buffer(0.1 M, pH 7.4) at 27 �C [lem: 426 nm, lex: 370 nm].

RSC Advances Paper

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article Online

balanced by a nitrate anion, as counter anion.H2L1 undergoes a

solvent assisted 1,5-s tropic shi leading to a benzimidazolederivative (H2L) of more chelating environment in presence ofCr3+ ion (Scheme 1)13h,k,22 and exhibit moderate uorescenceintensity due to internal electron transfer process.

To establish the fact of the formation of the chromium(III)complex, the solid state complex was obtained from the reactionof onemolar chromium(III) nitrate with onemolar of the organicmoiety in the methanol medium in stirring condition. Thecomplex is soluble in methanol, DMSO, acetonitrile. Theconductivity measurement of the complex in methanol at 300 Ksuggests that the complex exists as nonelectrolytes in solutionstate. The IR stretching frequencies of the complex are compa-rable with the existence of the L in 1. FTIR spectrum of thecomplex conrms the binding of in situ formed H2L with Cr3+

ion. Stretching frequencies at 3437 cm�1 and 3231 cm�1 for nOHand nNH, respectively observed in H2L

1 (Fig. s3†) were absent inthe IR-spectrum of the chromium(III) complex (Fig. s4†) due tothe deprotonation of both –OH and –NH groups. The ESI massspectrum of the complex in methanol shows a peak at m/z,517.39 with 30% abundance, assignable to [M + Na]+ (calculatedvalue at m/z, 517.40) where M ¼ [Cr(L)(NO3)(H2O)] (Fig. s5†).Here, the organic moiety (H2L) behaves as tetradentate dibasicligand. All these data conrm the composition of chromium(III)complex as [Cr(L)(NO3)(H2O)] (1).

Fig. 3 Change of relative fluorescence intensity profile of organic moiety inpresence of different cations ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH7.4) at 27 �C (lex ¼ 370 nm) [black: only probe; red: probe + Cr3+; blue: probe +Cr3++ other metal ion; green: resulting interference].

Fig. 4 Binding constant (K) value of 4.55 � 104 M�1 determined from theintercept/slope of the plots.

Spectral characteristics

Emission study. The uorescence emission spectra of H2L1

at 426 nm (lex ¼ 370 nm) (Fig. s6†) was very weak with aquantum yield ofF¼ 0.014 but the emission intensity graduallyincreases with increase of added [Cr3+]. Adding up of Cr3+

(10 mM) to H2L1 (10 mM) the emission band of enhanced

intensity with a quantum yield of almost 3.5 times (F ¼ 0.0464)in ethanol–water (1 : 5, v/v) was obtained (Fig. s7). Fluorescencequantum yields (F) were estimated by integrating the area underthe uorescence curves with the equation:

Fsample ¼ Fref �ODref � Asample

ODsample � Aref

where A is the area under the uorescence spectral curve andOD is the optical density of the compound at the excitationwavelength. The reference used for the measurement of uo-rescence quantum yield was anthracene (F ¼ 0.29 in ethanol).

To unfold the properties of H2L1 as a receptor for Cr3+, a

titration of the receptor was performed with increasingconcentration of Cr3+. As depicted in Fig. 2, the uorescenceintensity of a 10 mM solution of H2L

1 was enhanced withincremental addition of Cr3+ ion, which also conrmed thatreceptor H2L

1 exhibited a high sensitivity toward Cr3+ with nearabout 6-fold increase of its uorescence. In absence of Cr3+ ionthe uorescence intensity of H2L

1 is very low; but in presence ofCr3+ the uorescence intensity greatly increased due to therigidity of the resulting chelated complex.

There was almost no interference for the detection of Cr3+

even in the presence of 500 equivalent concentration of alkaliand alkaline earth metal ions (Na+, K+, Mg2+, Ca2+), and 100

RSC Adv.

equivalent concentration of several transition metal ions (Mn2+,Fe3+, Co3+, Ni2+, Cu2+, Zn2+) (Fig. 3). Job's plot analysis (Fig. s8†)revealed that in situ formed H2L yield a 1 : 1 (L : Cr3+) chro-mium(III) complex. The binding constant value of 4.55 �104 M�1 was determined from the emission intensity data(Fig. 4) following the modied Benesi–Hildebrand equation.23

This journal is ª The Royal Society of Chemistry 2013

Fig. 5 Time-resolved fluorescence decay of H2L1 (10 mM) in the absence and

presence of added Cr3+ (5 mM and 10 mM) (at lex ¼ 376 nm) in ethanol–water(1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 27 �C [lem: 426 nm, lex: 370 nm].

Fig. 6 Changes in the absorption spectra of H2L1 (10 mM) upon addition of 0–

30 mM of Cr3+ in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 27 �C.

Paper RSC Advances

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article Online

1/(Fx � F0) ¼ 1/(Fmax � F0) + (1/K[C])(1/(Fmax � F0)

where F0, Fx, and FN are the emission intensities of organicmoiety considered in the absence of Cr3+ ion, at an intermediateCr3+ concentration, and at a concentration of complete inter-action, respectively, and where K is the association constant and[C] is the Cr3+ concentration.

The uorescence average lifetime measurement of organicmoiety (H2L

1) in presence and absence of Cr3+ ion in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 27 �C indicatesthe gradual increase with increase of Cr3+ ion concentration(Fig. 5). The average lifetimes were calculated to be 1.21 nsfor only H2L

1, 4.71 ns for the mixture of H2L–Cr3+ (1 : 0.5) and

5.50 ns H2L–Cr3+ (1 : 1) (see ESI, Table s1†). The strong binding

of Cr3+ with organic moiety reected from the binding constantvalue, has played a key role for the selective chelation enhanceduorescence (CHEF) in the presence of Cr3+ ion. According tothe equations: s�1 ¼ kr + knr and kr ¼ Ff/s,24 the radiativerate constant kr and total non-radiative rate constant knr ofthe organic moiety, H2L

1 and chromium(III) complex,[Cr(L)(NO3)(H2O)] were listed in Table 3. The data suggest thatthe uorescent enhancement is ascribed to the decrease of theratio of knr/kr from 70.92 for H2L

1 to 20.74 for chromium(III)complex.

Absorption study. The UV-Vis spectrum of the probe showedthe characteristic absorption bands at ca. 291 nm (3, 19 600),301 nm (3, 21 280) and 346 nm (3, 9440) attributable to intra-molecular p–p* and n–p* transitions. In UV-Vis titration,addition of the solution of Cr3+ ion to the colourless solution of

Table 3 Fluorescence quantum yield (Ff) and life time (sav in ns) of the corre-sponding singlet excited states

Parameter H2L1 H2L

1 + Cr3+

Ff 0.014 0.046sav (ns) 1.209 5.500kr (10

9 s�1) 0.0115 0.00836knr (10

9 s�1) 0.8156 0.1734knr/kr 70.92 20.74c2 1.001 1.011

This journal is ª The Royal Society of Chemistry 2013

H2L1 in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at

27 �C, a new peak appeared in the visible region (ca. 405 nm)due to the formation of bluish-green of the resulting solution(Fig. s9†). The peak in UV region at 346 nm obtained in theabsorption spectrum of the probe, gradually decreases with theaddition of Cr3+ ions and, a new peak generates at around 405nmwith a 60 nm red shi through an isosbestic point at 377 nm(Fig. 6) due to the formation of the colored chromium(III)complex of the probe in the solution state.

Selectivity

The uorescent response of organicmoiety towards the differentmetal ions were investigated with 100 times concentration ofalkali (Na+, K+), alkaline earth (Mg2+, Ca2+), and transition-metalions (Ni2+, Zn2+, Cd2+, Co2+, Cu2+, Fe2+/3+, Cr3+, Hg2+) and Pb2+,Ag+ (Fig. 3 and s10†) in ethanol–water (1 : 5, v/v) HEPES buffer(0.1 M, pH 7.4) at 27 �C. It reveals that organic moiety has anexcellent selectivity and specicity to Cr3+ ion over other cations.

Effect of pH

The uorescence intensity of organic moiety was measured atvarious pH values in HEPES buffer (0.1 M) at 27 �C by adjusting

Fig. 7 Fluorescence response to pH of H2L1 (10 mM) in absence and in presence

of Cr3+ (one equivalent) at different pH in ethanol–water (1 : 5, v/v) HEPES buffer(0.1 M) at 27 �C.

RSC Adv.

RSC Advances Paper

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article Online

the pH using HCl or NaOH, in presence and absence of Cr3+ ion.In the absence of Cr3+ ion, organic moiety exhibited weakuorescence intensity and showed pH independency over thepH range 6.0 to 10.0 (Fig. 7). It indicates that the uorescenceintensity almost does not vary in the pH range of 6.0–10.0 anduorescence intensity of the organic moiety in the presence ofCr3+ ion is higher than that in the absence of Cr3+ ion. This isdue to the formation of complex through the deprotonation ofthe oxygen atom of the phenolic –OH group of the quinolinering and of the nitrogen atom of –NH of the imidazole ring ofH2L

1. As a result, the studies were carried out at pH 7.4 inethanol–water (1 : 5, v/v) HEPES buffer (0.1 M) at 27 �C.

Analytical gure of merit

The detection limit and the sensitivity of the sensor towards theCr3+ ion have been checked in ethanol–water (1 : 5, v/v) HEPESbuffer (0.1 M, pH 7.4) at 27 �C. The graph concentration of Cr3+

versus emission intensity (Fig. 8) indicates that this probe iseffective to detect Cr3+ ion of very low concentration and it wasestimated to be 3.6 � 10�7 mol L�1 (18.72 ppb) of Cr3+ ion. Tocheck the linearity of the experiments for performing the

Fig. 8 Detection limit of Cr3+ in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M,pH 7.4) at 27 �C.

Fig. 9 Fluorescence image of HeLa cells (A) cells were incubated with 0 mM Cr3+;(B) cells incubated with 5 mM Cr3+; (C) cells after incubated with 10 mM Cr3+

solution. All the samples were excited at 370 nmwith emission 425 nm by using a[10�] objective.

RSC Adv.

detection of Cr3+ ion in the solution, a calibration graph(Fig. s11†) has been obtained and this graph was linear over theconcentration range of 3.6 � 10�7 to 4.5 � 10�5 mol L�1 of Cr3+

ions within a very short responsive time (15–20 s).

Cell imaging

To examine the utility of the probe in biological systems, it wasapplied to human cervical cancer HeLa cell. Here, Cr3+ andH2L

1

were allowed to uptake by the cells of interest and the images ofthe cells were recorded by uorescence microscopy followingexcitation at �370 nm (Fig. 9). The HeLa cells rinsed with PBSand incubated with DMEM-containing H2L

1 (10 mM, 1% DMSO)displayed almost no uorescence (shown in Fig. 9A); aeradding Cr3+ to the cells, they displayed strong intracellularuorescence (viz. Fig. 9B and C). This observation demonstratesthat the probe is cell permeable.

In addition, the in vitro study showed that 50 mM ofH2L1 was

not cytotoxic to cell up to 8.0 h (Fig. s12†). These results indicatethat the probe has a huge potentiality for both in vitro and invivo application as Cr3+ sensor as well as imaging in differentways as same manner for live cell imaging can be followedinstead of xed cells.

Conclusion

In conclusion, a new uorescent chemosensor for Cr3+ ion hasbeen developed and it is highly selective with almost no inter-ference. The probe, 2-(5,6-dihydro-benzo[4,5]imidazo[1,2-c]qui-nazolin-6-yl)-quinolin-8-ol (H2L

1) behaves as a highly selectiveuorescent sensor for Cr3+ ions at biological pH in ethanol–water (1 : 5, v/v) HEPES buffer (0.1 M, pH 7.4) at 27 �C. Thisphenomenon has been accounted for the formation of mono-nuclear [Cr(L)(NO3)(H2O)] through a solvent assisted 1,5-stropic shi in H2L

1 to transform to a benzimidazole derivative(H2L) in presence of Cr3+ and hence, uorescence intensityarises due to internal electron transfer process. The complexhas been characterized spectroscopically aer the isolation ofthis coordination complex. The sensor H2L

1, containing quin-oline moiety exhibits high selectivity for Cr3+ ions in abioticsystems, presumably due to the CHEF effect during the chela-tion of H2L toward the Cr3+ ion in a 1 : 1 complex mode.

Acknowledgements

Financial support from Council of Scientic and IndustrialResearch (CSIR), New Delhi, India is gratefully acknowledged.M. Mukherjee wishes to thank to UGC, New Delhi, India foroffering the fellowship. We sincerely acknowledge Prof. SamitaBasu and Mr Ajay Das, Chemical Science Division, SINP, Kol-kata, for enabling TCSPC instrument.

Notes and references

1 (a) J. O. Nriagu and E. Nicboer, Chromium in the Natural andHuman Environment, New York, Wiley, 1988, p. 571; (b)K. Govindaraju, T. Ramasami and D. Ramaswamy, J. Inorg.Biochem., 1989, 35, 137.

This journal is ª The Royal Society of Chemistry 2013

Paper RSC Advances

Publ

ishe

d on

13

Aug

ust 2

013.

Dow

nloa

ded

by U

nive

rsity

of

Gla

sgow

Lib

rary

on

26/0

8/20

13 1

7:05

:17.

View Article Online

2 V. Gomez and M. P. Callao, TrAC, Trends Anal. Chem., 2006,25, 1006–1015.

3 (a) Z. T. Ma and D. Janke, Steel Res. Int., 2003, 74, 99; (b)Y. Harada, M. Ohmori, F. Yoshida and R. Nowak, Mater.Lett., 2003, 57, 1142; (c) A. P. Greeff, C. W. Louw andH. C. Swart, Corros. Sci., 2000, 42, 1725.

4 S. Pramanik, S. Dey and P. Chattopadhyay, Anal. Chim. Acta,2007, 584, 469 and reference therein.

5 S. S. M. Hassan, M. N. Abbas and G. A. E. Moustafa, Talanta,1996, 43, 797.

6 M. A. Zayed, B. N. Barsoum and A. E. Hassan, Microchem. J.,1996, 54, 72.

7 (a) N. Jie and J. Jiang, Analyst, 1991, 116, 395; (b) J. Yang andJ. Guo, Anal. Lett., 1992, 25, 1447.

8 A. Tunceli and A. R. Turker, Talanta, 2002, 57, 1199.9 (a) A. Xue, S. Qian, G. Huang and L. Chen, J. Anal. At.Spectrom., 2000, 15, 1513; (b) F. Shemirani and M. Rajabi,Fresenius' J. Anal. Chem., 2001, 371, 1037.

10 M. Sugiyaura, O. Fujino, S. Kihara and M. Matsui, Anal.Chim. Acta, 1986, 181, 159.

11 S. Peraniemi and M. Ahlgre, Anal. Chim. Acta, 1995, 315,365.

12 (a) R. K. Sharma and A. Goel, Anal. Chim. Acta, 2005, 534,137; (b) L. Lin, N. S. Lawrence, S. Thongngamdee, J. Wangand Y. Lin, Talanta, 2005, 65, 144.

13 (a) H. Komatsu, T. Miki, D. Citterio, T. Kubota, Y. Schindo,Y. Kitamura, K. Oka and K. Suzuki, J. Am. Chem. Soc., 2005,127, 10798; (b) M. Taki, M. Desaki, A. Ojida, S. Iyoshi,T. Hirayama, I. Hamachi and Y. Yamamoto, J. Am. Chem.Soc., 2008, 130, 12564; (c) Q. He, E. W. Miller, A. P. Wongand C. J. Chang, J. Am. Chem. Soc., 2006, 128, 9316; (d)H. Yang, Z. Zho, K. Huang, M. Yu, F. Li, T. Yi andC. Huang, Org. Lett., 2007, 9, 4729; (e) E. M. Nolan andS. J. Lippard, Acc. Chem. Res., 2009, 42, 193; (f) J. S. Kimandand D. T. Quang, Chem. Rev., 2007, 107, 3780; (g) K. Dhara,U. C. Saha, A. Dan, M. Manassero, S. Sarkar andP. Chattopadhyay, Chem. Commun., 2010, 46, 1754; (h)U. C. Saha, B. Chattopadhyay, K. Dhara, S. K. Mandal,S. Sarkar, A. R. Khuda-Bukhsh, M. Mukherjee, M. Helliwelland P. Chattopadhyay, Inorg. Chem., 2011, 50, 1213; (i)

This journal is ª The Royal Society of Chemistry 2013

U. C. Saha, K. Dhara, B. Chattopadhyay, S. K. Mandal,S. Mondal, S. Sen, M. Mukherjee, S. van Smaalen andP. Chattopadhyay, Org. Lett., 2011, 13, 4510; (j) S. Sen,T. Mukherjee, S. Sarkar, S. Mukherjee andP. Chattopadhyay, Analyst, 2011, 136, 4839; (k) S. Sen,S. Sarkar, B. Chattopadhyay, A. Moirangthem, A. Basu,K. Dhara and P. Chattopadhyay, Analyst, 2012, 137, 3335;(l) S. Sen, T. Mukherjee, B. Chattopadhyay,A. Moirangthem, A. Basu, J. Marek and P. Chattopadhyay,Analyst, 2012, 137, 3975; (m) S. Sen, K. Chakrabarty,I. Hauli, S. K. Mukhopadhyay and P. Chattopadhyay, Org.Biomol. Chem., 2013, 11, 1537.

14 M. Sarkar, S. Banthia and A. Samanta, Tetrahedron Lett.,2006, 47, 7575.

15 (a) J. Mao, L. Wang, W. Dou, X. Tang, Y. Yan and W. Liu, Org.Lett., 2007, 9, 3187; (b) K. Huang, H. Yang, Z. Zhou, M. Yu,F. Li, X. Gao, T. Yi and C. Huang, Org. Lett., 2008, 10, 2557;(c) Z. Zhou, M. Yu, H. Yang, K. Huang, F. Li, T. Yi andC. Huang, Chem. Commun., 2008, 3387.

16 H. Wu, P. Zhou, J. Wang, L. Zhao and C. Duan, New J. Chem.,2009, 33, 653.

17 S. Guha, S. Lohar, A. Banerjee, A. Sahana, I. Hauli,S. K. Mukherjee, J. S. Matalobos and D. Das, Talanta, 2012,91, 18.

18 A. Altomare, G. Cascarano, C. Giacovazzo and A. Guagliardi,J. Appl. Crystallogr., 1993, 26, 343.

19 G. M. Sheldrick, Acta Crystallogr., Sect. A: Found. Crystallogr.,2008, 64, 112.

20 L. J. Farrugia, J. Appl. Crystallogr., 1999, 32, 837.21 J. Ratha, K. A. Majumdar, S. K. Mandal, R. Bera, C. Sarkar,

B. Saha, C. Mandal, K. D. Saha and R. Bhadra, Mol. Cell.Biochem., 2006, 290, 113.

22 H. Paul, T. Mukherjee, M. G. B. Drew and P. Chattopadhyay,J. Coord. Chem., 2012, 65, 1289.

23 (a) A. Mallick and N. Chattopadhyay, Photochem. Photobiol.,2005, 81, 419; (b) H. A. Benesi and J. H. Hildebrand, J. Am.Chem. Soc., 1949, 71, 2703.

24 N. J. Turro, Modern Molecular Photochemistry, Benjamin/Cummings Publishing Co., Inc., Menlo Park, CA, 1978,p. 246.

RSC Adv.