Dynamic Article LinksC<Analyst

Cite this: Analyst, 2012, 137, 3335

www.rsc.org/analyst PAPER

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online / Journal Homepage / Table of Contents for this issue

A ratiometric fluorescent chemosensor for iron: discrimination of Fe2+ andFe3+ and living cell application†

Supriti Sen,a Sandipan Sarkar,a Basab Chattopadhyay,b Anuradha Moirangthem,c Anupam Basu,c

Koushik Dharad and Pabitra Chattopadhyay*a

Received 22nd February 2012, Accepted 3rd May 2012

DOI: 10.1039/c2an35258c

A newly designed probe, 6-thiophen-2-yl-5,6-dihydrobenzo[4,5]imidazo-[1,2-c] quinazoline (HL1)

behaves as a highly selective ratiometric fluorescent sensor for Fe2+ at pH 4.0–5.0 and Fe3+ at pH 6.5–

8.0 in acetonitrile–HEPES buffer (1/4) (v/v) medium. A decrease in fluorescence at 412 nm and increase

in fluorescence at 472 nm with an isoemissive point at 436 nm with the addition of Fe2+ salt solution is

due to the formation of mononuclear Fe2+ complex [FeII(HL)(ClO4)2(CH3CN)2] (1) in acetonitrile–

HEPES buffer (100 mM, 1/4, v/v) at pH 4.5 and a decrease in fluorescence at 412 nm and increase in

fluorescence at 482 nm with an isoemissive point at 445 nm during titration by Fe3+ salt due to the

formation of binary Fe3+ complex, [FeIII(L)2(ClO4)(H2O)] (2) with co-solvent at biological pH 7.4 have

been established. Binding constants (Ka) in the solution state were calculated to be 3.88 � 105 M�1 for

Fe2+ and 0.21 � 103 M�1/2 for Fe3+ and ratiometric detection limits for Fe2+ and Fe3+ were found to be

2.0 mM and 3.5 mM, respectively. The probe is a ‘‘naked eye’’ chemosensor for two states of iron.

Theoretical calculations were studied to establish the configurations of probe–iron complexes. The

sensor is efficient for detecting Fe3+ in vitro by developing a good image of the biological organelles.

Introduction

Iron is the most abundant essential trace element in the human

body. Both Fe2+ and Fe3+ play vital roles in many biological

processes.1–3 In well-nourished people the total iron content is

�4 g (70% in Hgb, 25% in storage). Iron is indispensable in living

systems since it is the oxygen carrier in all tissues in the form of

hemoglobin and helps to transport electrons as cytochromes.

Deficiency of iron in primary stages can cause anemia, which can

harms or even kill by depriving organs of oxygen.

The development of selective and sensitive fluorescent probes for

detection of biologically relevant ions not only in vitro detection but

also for in vivo recognition has been the cynosure among the

chemists during recent years due to the ease of detection, sensitivity,

and tenability of fluorescence method over other techniques.4–9

Interestingly, various sensors specific for Fe3+ have been repor-

ted,10–15 but to the best of our knowledge, a ratiometric fluorescent

sensor specific to both Fe2+ and Fe3+ cations is still unexplored.

aDepartment of Chemistry, Burdwan University, Golapbag,Burdwan-713104, West Bengal, India. E-mail: pabitracc@yahoo.combDepartment of Solid State Physics, Indian Association for the Cultivationof Science, Jadavpur, Kolkata 700032, IndiacCytogenetics and Molecular Biology Laboratory, Department of Zoology,Burdwan University, Golapbag, Burdwan-713104, IndiadDepartment of Chemistry, Sambhu Nath College, Labpur, Birbhum-731303, West Bengal, India

† Electronic supplementary information (ESI) available. CCDCreference numbers 651981. For ESI and crystallographic data in CIFor other electronic format see DOI: 10.1039/c2an35258c

This journal is ª The Royal Society of Chemistry 2012

Moreover, the literature reports either single point CHEF (chela-

tion enhanced fluorescence)16–20 or quenching studies21 to sense only

Fe3+ except for one reporting a ratiometric sensor.22 Ratiometric

responses are more attractive because the ratio between the two

emission intensities can be used to measure analyte concentration

and provide built-in correction for environmental effects and

stability under illumination.23,24 So, it is a challenge for a chemist to

develop a ratiometric cell permeable, CHEF type fluorescent probe

for both Fe2+ and Fe3+ by overcoming the usual fluorescence

quenching nature of iron of paramagnetic behavior.

Here, we report a new simple, easy to make, small

probe, 6-thiophen-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c]quina-

zoline (HL1) which can detect Fe2+ and Fe3+ by the ‘‘naked eye’’ and

fluorimetrically. To the best of our knowledge so far, this is the

first report in which the probe can discriminate between the two

oxidation states (II/III) of iron depending on the pH of the

medium. Different types of fluorimetric and UV-vis studies were

performed to establish the applicability of this probe. Theoretical

calculations were also carried out to determine the structure of

iron complexes. Moreover, the probe (HL1) can detect iron not

only in an abiotic system but also in a cellular system.

Experimental

Materials and physical measurements

The elemental analyses (C, H, N and S) were performed on

a Perkin Elmer 2400 elemental analyzer and iron analyses were

Analyst, 2012, 137, 3335–3342 | 3335

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

done by Varian atomic absorption spectrophotometer (AAS)

model-AA55B, GTA using graphite furnace. Electronic

absorption spectra were recorded on a JASCO UV-vis/NIR

spectrophotometer model V-570. IR spectra (KBr discs, 4000–

300 cm�1) were obtained using a Perkin-Elmer FTIR model RX1

spectrometer. 1H NMR spectra were recorded on a Bruker

AC300 spectrometer using TMS as an internal standard in

CDCl3 solvents. Electron spray ionization (ESI) mass spectra

were recorded on a Qtof Micro YA263 mass spectrometer.

Molar conductance (LM) was measured in a Systronics conduc-

tivity meter 304 model using �10�3 mol L�1 solutions in meth-

anol. Fluorescence spectra of the titration of iron with organic

moiety (HL1) were recorded using a fluorimeter (Hitachi-2000).

The measurement of pH was carried out with the help of a digital

pH meter (Systronics, Model 335). Time resolved experiments

were carried out using Horiba Jobin Yvon single photon

counting set up. Fluorescence lifetimes were determined by time-

resolved intensity decay by the method of time-correlated single-

photon counting using IBM Decay Analysis Software for

Windows v6.1.98 and a nano LED at 372 nm as the light source.

X-ray crystal structure analysis of HL1 was carried out by

collecting the diffraction data using MoKa (l ¼ 0.71073 �A)

radiation at 150 K. Data analysis was carried out with the XDS

program.25 Structures were solved using direct methods with the

SHELXS97 program.26 Non-hydrogen atoms were refined with

anisotropic thermal parameters. Hydrogen atoms bonded to

carbon were included in geometric positions and given thermal

parameters equivalent to 1.2� those of the atom to which they

were attached. Hydrogen atoms attached to water molecules

were located on difference Fourier maps and refined with

distance constraints. An empirical absorption correction was

carried out on 1 using the DIFABS program.27 Refinement on all

four structures was carried out with a full matrix least squares

method against F2 using SHELXL97.

Synthesis of 6-thiophen-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-

c]quinazoline (HL1)

The organic moiety, 6-thiophen-2-yl-5,6-dihydro-benzo[4,5]imi-

dazo[1,2-c]quinazoline (HL1) was synthesized by mixing an

ethanolic solution of 2-(2-aminophenyl)-benzimidazole (2.09 g,

10.0 mmol) with thiophene-2-carboxylaldehyde (1.12 g, 10.0

mmol) in ethanol (25 mL) at room temperature (Scheme 1). This

mixture was then allowed to reflux for 6.0 h. The off-white

colored crystalline precipitate of compound (HL1) was obtained

from yellow colored solution by slow evaporation of the solvent.

C18H13N3S: Anal. found: C, 71.34; H, 4.43; N, 13.91; S,

10.66%; calc.: C, 71.26; H, 4.32; N, 13.85; S, 10.57%. m.p. 238� 1�C, EI-MS: [M + H]+, m/z, 304.16; IR (KBr, cm�1): nC]N, 1615,

nC–H, 2960; nN–H, 3180. 1H NMR (d, ppm in CDCl3): 8.201

Scheme 1 Synthetic pathway of the probe.

3336 | Analyst, 2012, 137, 3335–3342

(d, 1H, j¼ 6.2); 7.816 (d, 1H, j¼ 6.1); 7.286 (m, 1H); 7.124–7.174

(m, 3H); 7.097 (d, 1H, j ¼ 3.1); 7.023 (d, 1H); 6.844–6.891

(m, 3H); 6.773 (d, 1H, j ¼ 11.2); 5.447 (br, NH); yield: 90%.

Pale yellow colored single crystals of (HL1) suitable for X-ray

crystallography were obtained on slow evaporation of ethanolic

solution of off-white crystalline precipitate. Crystals were posi-

tioned at 70 mm from the image plate and 95 frames were

measured at 2� intervals with a counting time of 2 min.

Crystal structure determination of HL1

Crystal data. C18H13N3S, M ¼ 303.37, Monoclinic, a ¼8.3875(6) �A, b ¼ 12.9511(8) �A, c¼ 13.3838(9) �A; a ¼ g ¼ 90.00�,b ¼ 98.248(6)�, U ¼ 1438.81(17) �A3, T ¼ 150(2) K, space group

P21, Z ¼ 4, collected reflns ¼ 5286, independent reflns ¼ 1783,

reflns with I > 2s(I) ¼ 1735, F(000) ¼ 632, R1 [I > 2.0 s(I)] ¼0.0376, goodness-of-fit ¼ 0.680.

Synthesis of the complexes (1 and 2)

The preparation of the solid complexes of iron(II) and iron(III)

was carried out as follows (viz. Scheme 2).

[Fe(HL)(ClO4)2(CH3CN)2] (1). The solution of anhydrous

Fe(ClO4)2 (0.5 mmol, 127.5 mg) in acetonitrile was added into

the solution ofHL1 (0.5 mmol, 151.5 mg) in acetonitrile dropwise

under stirring conditions. The mixture was stirred for another

8.0 h in a dinitrogen inert atmosphere. The resulting solution

under inert atmosphere was kept aside at ambient temperature.

After a few days, a yellow colored iron(II) complex was obtained

by filtration followed by thorough washing with water and cold

ethanol and then dried in vacuo to perform the characterization.

[Fe(HL)(ClO4)2(CH3CN)2] (1): C22H19FeN5O8SCl2: Anal.

found: C, 41.25; H, 2.96; N, 10.93; S, 5.0; Fe, 8.75%; calc.: C,

41.12; H, 2.81; N, 10.73; S, 4.88; Fe, 8.64%. IR (cm�1): ns(ClO4),

1086, 1121, nas(ClO4), 625, nC]N, 1618; nC–H, 2921; nN–H, 3215.

Conductance (Lo, ohm�1 cm2 mol�1) in methanol: 49; ESI-MS in

methanol: [M + H]+, m/z, 641.33 (obsd with 11% abundance)

(calc. m/z, 641.21); where M ¼ Fw of 1; yield: 60–70%.

[Fe(L)2(H2O)(ClO4)] (2). To the solution of anhydrous

Fe(ClO4)3 (0.5 mmol, 177.0 mg) in acetonitrile was added the

solution ofHL1 (1.0 mmol, 303.0 mg) and the mixture was stirred

for 8.0 h to completion of the reaction. Here, the procedure as

above was carried out to obtain a dried green colored complex.

[Fe(L)2(H2O)ClO4] (2): C36H26FeN6O5S2Cl: Anal. found: C,

47.08; H, 2.83; N, 24.41; S, 6.9; Fe, 6.1%; calc.: C, 46.88; H, 2.74;

N, 24.21; S, 5.92; Fe, 5.98%. IR (cm�1): ns(ClO4), 1086, 1120,

nas(ClO4), 625, nC]N, 1618; nC–H, 2919. Conductance (Lo, ohm

�1

cm2 mol�1) in methanol: 45; ESI-MS in methanol: [M + Na]+,

m/z, 789.04 (obsd with 13% abundance) (calc.: m/z, 788.84);

where M ¼ Fw of 2; yield: 70–75%.

General method of absorption and fluorescence titration

Solution of HL1 in acetonitrile–water (1/4) (v/v) at 25 �C was

taken for absorption study and acetonitrile–HEPES buffer (1/4)

(v/v) solvent system was used for fluorometric titration. During

emission study initially HL1 concentration was 1 � 10�5 M in

acetonitrile–HEPES buffer (1/4) (v/v) solvent mixture then

This journal is ª The Royal Society of Chemistry 2012

Scheme 2 Probable mechanism of CHEF effect and structures of Fe(II) complex and Fe(III) complex.

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

concentration of perchlorate salt of iron(II/III) was varied.

Absorption study was also recorded varying metal salt strength

at constant [HL1] of 1 � 10�5 M. Fluorescence quantum yields

(F) were estimated by integrating the area under the fluorescence

curves with the equation:

Fsample ¼ ODstandard � Asample

ODsample � Astandard

� Fstandard

where A is the area under the fluorescence spectral curve and OD

is optical density of the compound at the excitation wavelength.

The standard used for the measurement of fluorescence quantum

yield was anthracene (F¼ 0.29 in ethanol). The binding constant

values were determined from the emission intensity data

following the modified Benesi–Hildebrand equations:28,29

1/DF ¼ 1/DFmax + (1/K[C]) (1/DFmax), DF ¼ Fx � F0,

DFmax ¼ FN � F0, for Fe2+

1/(Fx � F0) ¼ 1/(Fmax � F0) + (1/K[C]1/2) (1/(Fmax � F0), for Fe3+

where F0, Fx, and FN are the emission intensities of organic

moiety considered in the absence of iron ion, at an intermediate

iron concentration, and at a concentration of complete interac-

tion, respectively, and where K is the binding constant and [C] is

the iron concentration.

In vitro cell imaging with HL–iron

HeLa cells were procured from NCCS, Pune and grown in

Dulbecco’s Modified Eagle Medium (DMEM) supplemented

with 10% Fetal Bovine Serum (FBS), 1% L-glutamine–penicillin–

Streptomycin. The cells were maintained at 37 �C in a humidified

atmosphere of 5% CO2. Cells after reaching 80–90% confluence

in T25 flask, were trypsinised (with 0.25% trypsin–EDTA) and

plated on the cover slip in the 35 mm culture dish with seeding

density of 3 � 105 and allowed to grow for 60% confluence. The

media was replaced with fresh serum free media, supplemented

This journal is ª The Royal Society of Chemistry 2012

with FeCl3 (5 mM and 0.5 mM) to uptake Fe3+ by the growing

cells. For the control experiment, media was devoid of FeCl3.

After 3 h incubation, the media was removed and fresh serum

free media was added. HL1 (5 mM), dissolved in a HEPES buffer

(100 mM, DMSO : water ¼ 1 : 100 (v/v)) was added to media

to allow the cells to uptake. For the control experiment

DMSO : water (1/100, v/v) was added to the media instead of the

probe. A cover slip containing cells were mounted on a glass slide

and observed under fluorescence microscope (Lecia DM 1000)

using 40� objective with excitation at �365 nm and emission

filter around �476 nm. The fluorescence image of cells was

captured through an attached CCD camera using image acqui-

sition software.

In vitro cytotoxicity testing

The cytotoxic effect of HL was evaluated using HeLa cells, grown

as mentioned above in T25 flask. After reaching 80% confluence,

cells were seeded in the wells of 24 well-culture plate with seeding

density of 3 � 105 cells per well. After two days of incubation,

previous media was replaced by fresh media and HL (dissolved

DMSO : water) was added (50 mMand 5.0 mM) to all the wells and

incubated for 18 h. To all the wells, 50 ml of [3-(4,5-dimethylthiazol-

2-yl)-2,5-diphenyl tetrazolium bromide (MTT, 5 mg ml�1 in PBS)

was added and incubated for 3 h. Culture media was removed and

reduced formazon was dissolved by adding 350 ml of DMSO for 15

min to develop a soluble purple color and measured at 590 nm

(Shimazdu double beam spectrometer).

Results and discussion

Synthesis and characterization

Design of the probe (HL1) is one of the most important aspects in

developing chromogenic and fluorogenic sensors for target

analytes. Both Fe2+ and Fe3+ cations are relatively hard centres

over transition metal background. Here, the probe having

Analyst, 2012, 137, 3335–3342 | 3337

Table 1 Crystal data and details of refinements for HL1

HL1

Empirical formula C18 H13 N3 SFormula weight 303.37Crystal system MonoclinicSpace group P21a (�A) 8.3875(6)b (�A) 12.9511(8)c (�A) 13.3838(9)a (�) 90.00b (�) 98.248(6)g (�) 90.00Volume (�A3) 1438.81(17)Temperature, K 150(2)Z 4rcalc (g cm�3) 0.336F(000) 632q range (deg) 2.91–30.09m (MoKa) (mm�1) 0.224Collected reflns 5286Independent reflns 1783Reflns with I > 2s(I) 1735R1 [I > 2.0s(I)] 0.0376wR1 [I > 2.0s(I)] 0.1129Goodness-of-fit 0.680

Table 2 Selected bond distances (�A) and bond angles (�) for HL1

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

heterocyclic nitrogens of moderately hard donor centres was

chosen for chelation. The fluorescent probe, 6-thiophen-2-yl-5,6-

dihydro-benzo[4,5]-imidazo[1,2-c]quinazoline (HL1) was

synthesized by condensing an ethanolic solution of 2-(2-amino-

phenyl)benzimidazole, with thiophene-2-carboxaldehyde

(Scheme 1) in 1 : 1 mole ratio.HL1 was characterized by physico-

chemico and spectroscopic tools and finally the structure was

confirmed by single crystal X-ray crystallography. The molecular

view ofHL1 with atom labeling scheme is shown in Fig. 1 and the

crystallographic data and bond parameters are tabulated in

Tables 1 and 2. In solid state structure of HL1, the longer bond

distance of C11–N12 (1.444(3) �A) than that of N12–C13

(1.370(3) �A) and the bond angle of N12–C11–N27 (109.38(17)�)indicate the sp3-hybridised character of C11. HL1 undergoes

a solvent assisted 1,5-s tropic shift leading to a benzimidazole

derivative (HL) of more chelating environment in presence of

Fe2+ or Fe3+ (Scheme 2),7,30 and exhibit moderate fluorescence

intensity due to internal electron transfer process.

To establish formation of the iron(II/III) complexes (1 and 2),

the solid state complexes formulated as [Fe(HL)-

(ClO4)2(CH3CN)2] (1) and [Fe(L)2(H2O)(ClO4)] (2) were isolated

from the reaction of HL1 with anhydrous Fe(ClO4)2 and

Fe(ClO4)3, respectively, in acetonitrile stirring at ambient

temperature with the usual precautions. Complexes formed are

soluble in acetonitrile and methanol. Conductivity measurement

of the complexes in methanol at 300 K suggests that both

complexes (1 and 2) exist as non-electrolytes in solution state.

The ESI mass spectrum of 1 in methanol showed a peak at m/z

641.33 with 11% abundance) which could be assigned to [M +

H]+ (calculated value at m/z, 641.21; where M ¼ Fw of 1); and in

case of 2 in methanol, a peak at m/z, 789.04 with 13% abundance

attributable to [M + Na]+ (calc.: m/z, 788.84; where M ¼ Fw of 2)

was observed (Fig. S1A†). The IR stretching frequencies of the

complexes are comparable with the existence of the HL and L in

1 and 2 respectively. FTIR spectra of the complexes (1 and 2) also

confirm the binding of in situ formed HL with iron(II/III).

Stretching frequency at 3215 cm�1 for nN–H observed in HL1 was

absent in the spectrum of 2 due to the deprotonation of –NH

group, but it was present in the spectrum of 1. The –CH group

exhibited red shift (ca. 39 cm�1) due to the binding of imine–N

with Fe2+ and Fe3+. The observed characteristic stretching

frequencies around 1085, 1121 and 625 cm�1 are due to the

Fig. 1 A molecular view of HL1 with atom numbering scheme.

3338 | Analyst, 2012, 137, 3335–3342

binding of perchlorate with metal ion in coordination sphere.31

Here, in situ formed HL behaves as bidentate neutral ligand in 1

and as bidentate monobasic ligand in case of 2.

To clarify the configurations of the probe (Fig. S1B†) and

iron complexes, DFT calculations were performed using Dmol3

code32 in the framework of a generalized-gradient approxima-

tion (GGA). The energies of both HOMO and LUMO of HL1

are less stabilized than HL (Fig. S1C†) and it demonstrates the

facile conversion of HL1 to HL which is also in support of our

previous reports.7,30 HOMO and LUMO of Fe3+ complex are

less energetic than that of Fe2+ complexes (Fig. 2). Result of this

is narrowing of the energy gap between the HOMO and LUMO

due to s�p interaction, the red shift for the Fe3+ complex was

observed.

Bond distances (�A)

C34–S35 1.723(2)C11–N12 1.444(3)C11–N27 1.455(2)N12–C13 1.370(3)N20–C21 1.389(3)C26–N27 1.395(3)

Bond angles (�)

C64–S65–C61 92.62(12)N12–C11–N27 109.38(17)N12–C11–C31 110.86(18)N27–C11–C31 112.45(17)C13–N12–H12 118(2)C13–N12–C11 126.58(18)C19–N20–C21 104.94(17)C19–N27–C26 106.78(17)

This journal is ª The Royal Society of Chemistry 2012

Fig. 3 Excitation and emission spectra of HL (10 mM) in a HEPES

buffer (100 mM, acetonitrile : water ¼ 1 : 4 (v/v)) at 25 �C.

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

Emission study

Upon excitation at 365 nm, HL (formed in situ) exhibited

moderate emission at lem ¼ 412 nm with a quantum yield (Ff) of

0.19 in acetonitrile–HEPES buffer (100 mM) (1/4, v/v) at 25 �C(Fig. 3). Iron sensing ability of HL1 was investigated in aceto-

nitrile–HEPES buffer (1/4) (v/v) at different pH using 100 mM

HEPES buffer. This study showed that the probe selectively

sense Fe2+ in the range of pH 4.0–5.0 and Fe3+ in the pH range

6.0–8.2 (Fig. 4).

On fluorimetric titration, emission intensity at 412 nm due to

the probe gradually decreases with the addition of Fe2+ salt

solution and a new peak at 472 nm with isoemissive point at 436

nm is generated due to the formation of a mononuclear Fe2+

complex in acetonitrile–HEPES buffer (100 mM, 1/4, v/v) at pH

4.5 (Fig. 5, S2 and S3†). Similarly, during titration by Fe3+ salt

with similar co-solvent but at biological pH 7.4, a new peak

appeared at 482 nm with the isoemissive point at 445 nm due to

the formation of binary Fe3+ complex (Fig. 6, S4 and S5†).

Stoichiometry of the iron/HL in solution state was determined by

Jobs method which revealed 1 : 1 ratio for HL : Fe2+, whereas

2 : 1 ratio for HL : Fe3+ in acetonitrile–HEPES buffer (1/4) (v/v)

(Fig. S6†). The physico-chemical and spectroscopic data

supports the formation of complexes of Fe2+ and Fe3+ of HL

isolated as [FeII(HL)(ClO4)2(CH3CN)2] (1) and [FeIII(L)2-

(ClO4)(H2O)] (2) in solid state.

Addition of equimolecular concentrations of different metal

ions in the probe solution, only Fe2+ at pH 4.5 and Fe3+ at pH

7.4 induced more than an eight-fold increase of fluorescence

intensity, but during addition of other metal ions such as alkali

(Na+, K+), alkaline earth (Mg2+, Ca2+), and transition-metal ions

(Ni2+, Zn2+, Cd2+, Co2+, Cu2+, Cr3+, Hg2+) and Pb2+, Ag+ the

emission intensity of the probe remains entirely silent (Fig. 7).

This is due to in situ HL binding selectively Fe2+ at pH 4.5 and

Fe3+ at pH 7.4. As a result, the internal electron delocalization is

restricted and fluorescence intensity of the probe is enhanced by

CHEF mechanism (Scheme 2). Above pH 5.0, deprotonation of

the nitrogen atom of the N–H group of benzimidazole ring

(pKa ¼ 5.06 � 0.03209) (Fig. 8) was encouraged for binding the

Fig. 2 Energy level diagram for the frontier p MOs of iron complexes.

This journal is ª The Royal Society of Chemistry 2012

relatively hard Fe3+ centre with the deprotonated pyrolic–N

donor of increased electron density, but at less than pH 5.0 for

Fe2+ binding where deprotonation of N–H group had not taken

place.

To calculate the binding ability of iron with in situ formed HL,

modified Benesi–Hildebrand equations were applied (Fig. S7†).

The values of binding constants (Ka) in solution state were calcu-

lated as 0.21 � 103 M�1/2 for Fe3+ and 3.88 � 105 M�1 for Fe2+ and

these values clearly indicate the selective chelation enhanced fluo-

rescence (CHEF) in the presence of different cations. The selectivity

coefficients (k) of Fe2+ and Fe3+ over competent ions were deter-

mined (Table S1†) and the values were obtained to be in the range

from 534 to 1546. The selectivity of the probe towards Fe2+ and

Fe3+ over the mostly interfering ion, Cr3+ were found to be 534 and

541 fold, respectively, (Table S1†). The ratiometric detection limits

for Fe2+ and Fe3+ were calculated and these were found to be

2.0 mM and 3.5 mM, respectively, (Fig. S8†). This significant

observation indicates that the moiety, HL1 could be employed as

ratiometric CHEF probe for the discrimination of Fe2+ and Fe3+ of

micromolar concentration in acetonitrile–HEPES buffer (1/4) (v/v)

solution by tuning the pH of the medium.

The fluorescence life time (s) of the probe in acetonitrile–

HEPES buffer (1/4) (v/v) at pH 7.4 was determined by time

Fig. 4 Fluorescence response of the probe, the probe + Fe2+ and the

probe + Fe3+ ions at different pH (using 100 mM HEPES buffer).

Analyst, 2012, 137, 3335–3342 | 3339

Fig. 5 Emission spectra of HL (1.66 � 10�5 M) in presence of Fe2+

(1.66 � 10�6 to 8.33 � 10�5 M) in a HEPES buffer (100 mM, acetoni-

trile : water ¼ 1 : 4 (v/v), pH ¼ 4.5) at 25 �C.

Fig. 6 Emission spectra of HL (1.66 � 10�5 M) in presence of Fe3+

(3.33 � 10�6 to 3.33 � 10�4 M) in a HEPES buffer (100 mM, acetoni-

trile : water ¼ 1 : 4 (v/v), pH ¼ 7.4) at 25 �C.

Fig. 8 Fluorescence response to pH of HL in a 100 mM HEPES buffer

at 25 �C, pKa ¼ 5.06 � 0.03209.

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

resolved spectrum (Fig. 9) and it was found to be 7.53 ns.

According to the equations: s�1 ¼ kr + knr and kr ¼ Ff/s,33 theradiative rate constant, kr and total nonradiative rate constant

knr of organic moiety and iron complexes are given in Table 3.

The data suggest that kr has slightly changed but the factor that

Fig. 7 Metal ion selectivity of organic moiety in presence of different cations

and (b) pH 7.4.

3340 | Analyst, 2012, 137, 3335–3342

induces fluorescent enhancement is mainly ascribed to the

decrease of knr which was more than 13� that of Fe2+.

Absorption study

In another experiment, addition of Fe2+ and Fe3+ salts to the

colorless solution of HL1 in acetonitrile–water (1/4) (v/v) causes

instantaneous development of orange and green color, respec-

tively, (Fig. 10); this interesting observation indicates that the

probe can also serve as a selective ‘‘naked-eye’’ chemosensor for

the discrimination of Fe3+ and Fe2+ with the help of the spectral

features observed in UV-vis titration as a new peak appeared at

ca. 385 nm and ca. 368 nm due to the addition of Fe3+ and Fe2+

solution, respectively, (Fig. 11).

Cell imaging

To examine the utility of the probe in biological systems, it was

applied to human cervical cancer HeLa cells. Here, both the Fe3+

and HL taken up by the cells of interest and the images of the

cells were recorded by fluorescence microscopy following exci-

tation at �365 nm (Fig. 12). In addition, the in vitro study

showed that 50 mM of HL1 was not cytotoxic to cell up to 8.0 h

(Fig. S9†). These results indicated that the probe has potential

for both in vitro and in vivo application as a Fe3+ sensor, as well as

imaging live cells in the same manner as for fixed cells. An earlier

report34 showed that iron can promote neoplastic cell growth and

in HEPES buffer (100 mM, acetonitrile : water ¼ 1 : 4 (v/v)) at (a) pH 4.5

This journal is ª The Royal Society of Chemistry 2012

Fig. 9 Time-resolved fluorescence decay of HL (10 mM) in presence of

added Fe2+ (20 mM) only, and Fe3+(20 mM) in a HEPES buffer (100 mM,

acetonitrile : water¼ 1 : 4 (v/v)) using a nano-LED of 372 nm as the light

source at pH 4.5 (for Fe2+) and at pH 7.4 (for Fe3+).

Table 3 Fluorescence quantum yield (Ff) and life time (sf in ns) of thecorresponding singlet excited states

Ff sf (ns) kr(108 s�1) knr (10

9 s�1) c2

HL 0.19 7.53 0.252 0.107 1.22HL + Fe3+ 0.94 7.56 0.609 0.0720 1.19HL + Fe2+ 0.46 7.55 1.243 0.008 1.2

Fig. 10 The color of the solution having the probe in absence and in

presence of Fe(II) and Fe(III) salt in acetonitrile–water (1/4) (v/v).

Fig. 12 (A) Phase contrast; (B) fluorescence image of HeLa cells after

incubation with 5.0 mM HL1 for 30 min at 25 �C and washing with PBS;

cells were trypsinised (using 0.25% trypsin–EDTA) and rinsed with PBS

(pyr, 50 mM) in presence of sequentially increased concentrations of

added extracellular Fe3+ as (C) 1 mM and (D) 5 mM. In all images, the

samples were excited at �365 nm with emission at �476 nm by using

[40�] objective.

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

induce cancer cell proliferation due to excessive deposition of

iron at the site. As the sensor produces blue fluorescence when it

complexes with Fe3+ this probe can be used as an Fe3+ sensor in

neoplastic cell growth and as a quantitative indicator for iron

deposition.

Fig. 11 Absorption spectra of HL in the presence of different concentratio

(100 mM, acetonitrile : water ¼ 1 : 4 (v/v)).

This journal is ª The Royal Society of Chemistry 2012

Conclusion

A newly designed and one-pot synthesized fluorescent probe of

sufficiently low molecular weight (Fw ¼ 303.37), 6-thiophen-2-yl-

5,6-dihydro-benzo[4,5]-imidazo[1,2-c]quinazoline (HL1) is

employed as a highly specific ratiometric fluorescent sensor for

Fe2+/Fe3+ in acetonitrile–HEPES buffer (100 mM) (1/4) (v/v) at

two different pH. Interference due to biologically relevant and

other transition metal ions is insignificant, Fe3+ at pH 4.0–5.0

and Fe2+ at pH 6.5–8.0 do not interfere in the detection of Fe2+

and Fe3+, respectively. The probe therefore offers good potential

to discriminate between Fe2+ and Fe3+ at two different lem and

two different pH. The probe can also discriminate Fe2+/Fe3+ in

acetonitrile–water (1/4) (v/v) by the naked eye. The HL–Fe3+

complex can be used as a multi-modal imaging agent in cancer

affected cellular systems.

ns of (a) Fe2 + (0–1.5 equiv.) and (b) (0–2.0 equiv.) in a HEPES buffer

Analyst, 2012, 137, 3335–3342 | 3341

Publ

ishe

d on

07

June

201

2. D

ownl

oade

d by

UN

IVE

RSI

TY

OF

NE

BR

ASK

A o

n 26

/08/

2013

13:

09:2

2.

View Article Online

Acknowledgements

Financial assistance from Department of Science and Tech-

nology (DST), New Delhi (vide project no. SR/S1/IC-37/2008) is

gratefully acknowledged. A. Basu and A. Moirangthem are

thankful to DBT, New Delhi (project no. BT/PR11612/BRB/10/

671/2008) for the work in cancer cell.

References

1 E. Beutler, V. Felitti, T. Gelbart and N. Ho, Drug Metab. Dispos.,2001, 29, 495–499.

2 D. Touati, Arch. Biochem. Biophys., 2000, 373, 1–6.3 G. Cairo and A. Pietrangelo, Biochem. J., 2000, 352, 241–250.4 B. P. Esposito, S. Epsztejn, W. Breuer and Z. I. Cabantchik, Anal.Biochem., 2002, 304, 1–18.

5 B. Valeur and I. Leray, Coord. Chem. Rev., 2000, 205, 3–40.6 U. C Saha, K. Dhara, B. Chattopadhyay, S. K. Mandal, S. Mondal,S. Sen, M. Mukherjee, S. van Smaalen and P. Chattopadhyay, Org.Lett., 2011, 13, 4510–4513.

7 U. C. Saha, B. Chattopadhyay, K. Dhara, S. K. Mandal, S. Sarkar,A. R. Khuda-Bukhsh, M. Mukherjee, M. Helliwell andP. Chattopadhyay, Inorg. Chem., 2011, 50, 1213–1219.

8 S. Sen, T. Mukherjee, S. Sarkar, S. K. Mukhopadhyay andP. Chattopadhyay, Analyst, 2011, 136, 4839–4845.

9 K. Dhara, U. C. Saha, A. Dan, M. Manassero, S. Sarkar andP. Chattopadhyay, Chem. Commun., 2010, 46, 1754–1756.

10 (a) J. Zhan, L. Wen, F. Miao, D. Tian, X. Zhu and H. Li, New J.Chem., 2012, 36, 656–661; (b) S. L. Hu, N. F. She, G. D. Yin,H. Z. Guo, A. X. Wu and C. L. Yang, Tetrahedron Lett., 2007, 48,1591–1594.

11 (a) Y. Xiang and A. Tong, Org. Lett., 2006, 8, 1549–1552; (b) J. Mao,L. N. Wang, W. Dou, X. L. Tang, Y. Yan and W. S. Liu, Org. Lett.,2007, 9, 4567–4570.

12 X. B. Zhang, G. Cheng, W. J. Zhang, G. L. Shen and R. Q. Yu,Talanta, 2007, 71, 171–177.

13 O. Oter, K. Ertekin, C. Kirilmis, M. Koca and M. Ahmedzade, Sens.Actuators, B, 2007, 122, 450–456.

3342 | Analyst, 2012, 137, 3335–3342

14 G. E. Tumambac, C. M. Rosencrance and C. Wolf, Tetrahedron,2004, 60, 11293–11297.

15 J. L. Bricks, A. Kovalchuk, C. Trieflinger, M. Nofz, M. Buschel,A. I. Tolmachev, J. Daub and K. Rurack, J. Am. Chem. Soc., 2005,127, 13522–13529.

16 A. J. Weerasinghe, C. Schmiesing, S. Varaganti, G. Ramakrishna andE. Sinn, J. Phys. Chem. B, 2010, 114, 9413–9419.

17 N. C. Lim, S. V. Pavlova and C. Bruckner, Inorg. Chem., 2009, 48,1173–1182.

18 J. Mao, L. Wang, W. Dou, X. Tang, Y. Yan and W. Liu, Org. Lett.,2007, 9, 4567–4570.

19 Y. Xiang andA. Tong,Org. Lett., 2006, 8, 1549–1552 and refs therein.20 J. L. Bricks, A. Kovalchuk, C. Trieflinger, M. Nofz, M. Buschel,

A. I. Tolmachev, J. Daub and K. Rurack, J. Am. Chem. Soc., 2005,127, 13522–13529.

21 N. Li, Q. Xu, X. Xia, L. Wang, J. Lu and X. Wen, Mater. Chem.Phys., 2009, 114, 339–345.

22 M. Xu, S. Wu, F. Zeng and C. Yu, Langmuir, 2010, 26, 4529–4534.

23 Z. Xu, Y. Xiao, X. Qian, J. Cui and D. Cui, Org. Lett., 2005, 7, 889–892.

24 B. Valeur and I. Leray, Coord. Chem. Rev., 2000, 205, 3–40.

25 W. Kabsch, J. Appl. Crystallogr., 1988, 21, 916–924.26 G. M. Sheldrick, Shelxs97 and Shelxl97 Programs for

Crystallography, University of Gottingen, Germany, 1997.27 DIFABS: N.Walker and D. Stuart,Acta Crystallogr., Sect. A: Found.

Crystallogr., 1983, 39, 158–166.28 H. A. Benesi and J. H. Hildebrand, J. Am. Chem. Soc., 1949, 71,

2703–2707.29 I. Ravikumar and P. Ghosh, Inorg. Chem., 2011, 50, 4229–4231.30 H. Paul, T. Mukherjee, M. G. B. Drew and P. Chattopadhyay,

J. Coord. Chem., 2012, 65, 1289–1302.31 S. Dey, S. Sarkar, E. Zangrando, H. S. Evans, J.-P. Sutter and

P. Chattopadhyay, Inorg. Chim. Acta, 2011, 367, 1–8.32 B. Delley, Phys. Rev. B: Condens. Matter Mater. Phys., 2002, 66,

155125(1–9).33 N. J. Turro,Modern Molecular Photochemistry, Benjamin/Cummings

Publishing Co., Inc., Menlo Park, CA, 1978.34 E. D. Weinberg, Eur. J. Cancer Prev., 1996, 5, 19–36.

This journal is ª The Royal Society of Chemistry 2012