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Sensors and Actuators B 183 (2013) 350– 355

Contents lists available at SciVerse ScienceDirect

Sensors and Actuators B: Chemical

journa l h om epage: www.elsev ier .com/ locate /snb

irst rhodamine-based “off–on” chemosensor with high selectivity andensitivity for Zr4+ and its imaging in living cell

jit Kumar Mahapatraa,∗, Saikat Kumar Mannaa, Subhra Kanti Mukhopadhyayb, Avishek Banikb

Department of Chemistry, Bengal Engineering and Science University, Shibpur, Howrah 711103, IndiaDepartment of Microbiology, The University of Burdwan, Burdwan, West Bengal, India

a r t i c l e i n f o

rticle history:eceived 15 January 2013eceived in revised form 2 April 2013ccepted 7 April 2013vailable online 15 April 2013

eywords:

a b s t r a c t

A new pyridine–thiophene appended rhodamine-based probe RhPT was synthesized as “off–on”chemosensor for Zr4+. Rhodamine spirolactam or spirolactone derivatives are nonfluorescent and col-orless, whereas ring-opening of the corresponding spirolactam/lactone gives rise to strong fluorescenceemission and a pink color. However, in the present case, chemosensor RhPT shows high sensitivity andselectivity toward Zr4+ ions by exhibiting both colorimetric and fluorescence responses in CH3OH–water(4:1, v/v, 10 �M HEPES buffer, pH 7.4). The selectivity of RhPT to the various metal ions was investi-gated. A color change and marked enhancement of fluorescence was found in the presence of Zr4+ due to

hodamineurn-onhemosensorirconiumaked eye

magingiving cells

the ring open reaction of rhodamine. The sensor shows extremely high fluorescence enhancement uponcomplexation with Zr4+ and it can be used as a “naked eye” sensor. The visual detection is possible by asharp change in color. In addition, the turn-on fluorescent probe upon the addition of Zr4+ was appliedin live cell imaging.

© 2013 Elsevier B.V. All rights reserved.

. Introduction

The recognition and sensing of various transition-metal ionsre vital for the biological and environmental important species1,2]. Among these is the zirconium ion, which is widely used forhe preparation of laboratory crucibles, metallurgical furnaces, as

refractory material, sintered, ceramic knife, jewelry and nuclearppliances. However, their frequent use can result in high levelsf residual zirconium ions, which may result in the contamina-ion of water systems and soil and therefore cause a health hazard.ome toxic effects of zirconium ion have been reported, withymptoms limited mostly to gastrointestinal complaints such asausea, abdominal pain and vomiting [3]. Inhalation of zirconiumompounds can cause pulmonary granulomas, skin and lung gran-lomas. However, we have little other knowledge of the role ofirconium in human metabolism due to the lack of effective toolsith which to study the mechanisms. Therefore, it is important toevelop highly sensitive and selective methods that would be veryseful for the real-time monitoring of the metal ions in environ-

ental and biological samples. In recent years, significant emphasis

as been placed on the development of new, highly selective flu-rescent chemosensors of different architectures for metal cations

∗ Corresponding author. Tel.: +91 33 2668 4561; fax: +91 33 2668 4564.E-mail address: mahapatra574@gmail.com (A.K. Mahapatra).

925-4005/$ – see front matter © 2013 Elsevier B.V. All rights reserved.ttp://dx.doi.org/10.1016/j.snb.2013.04.012

because of their potential applications in biochemistry and envi-ronmental research [4].

Fluorogenic methods in conjunction with suitable probes arepreferable approaches for the measurement of these analytesbecause fluorimetry is rapidly performed, nondestructive, highlysensitive and suitable for high-throughput screening applications[5]. In addition, colorimetric and/or fluorescent probes for thedetermination of transition metal cations have great popularitybecause they can monitor analytes both in solution by the naked eye[6] and in living cells by fluorescent microscopy [7]. To our knowl-edge, however, there is no report of a fluorescent probe used as aselective Zr4+ sensor. As the continuation of our work on the sensingof cations [8] and anions [9] of biological significance, herein, wereport the systematic investigations of rhodamine based novelchemosensor RhPT with different metal binder scaffold and sensingbehavior that combine a 6-thiophenyl-2-pyridinecarboxaldehydeand a rhodamine chromophore (Scheme 1). Rhodamine B is widelyused as a fluorescent probe [10] for the detection of cysteine [11]and metal ions [12], including Cu2+ [13], Cr3+ [14], Fe3+ [15] andHg2+ [16] due to the ring opening reaction of rhodamine. Rho-damine spirolactam or spirolactone derivatives are nonfluorescentand colorless, whereas ring-opening of the corresponding spirolac-

tam/lactone gives rise to strong fluorescence emission and a pinkcolor. However, in the present case, chemosensor RhPT shows highsensitivity and selectivity toward Zr4+ ions by exhibiting both col-orimetric and fluorescence responses in CH3OH–water (4:1, v/v,

A.K. Mahapatra et al. / Sensors and Ac

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Scheme 1. Synthetic route of RhPT.

0 �M HEPES buffer, pH 7.4). This rhodamine B derivative (RhPT)an selectively detect Zr4+ in the presence of other metal ions whenthylenediaminetetraacetic acid (EDTA) is used as a second ligand,nd thus be used as a Zr4+ sensor in living cells. As far as we areware, RhPT is the first chemosensor for zirconium based on rho-amine derivative.

. Experimental

.1. Materials and measurements

All the solvents were of analytic grade. All cationic compounduch as per chlorate of Hg2+, Cu2+, Cd2+, Fe2+, Co2+, Ni2+, Mn2+, Zn2+,b2+, nitrate of Y3+, Ag+, chlorides of Ti4+, Pd2+ and ZrOCl2·8H2Oere purchased from Sigma–Aldrich Chemical Co., stored in a des-

ccators under vacuum containing self-indicating silica, and usedithout any further purification. Solvents were dried according to

tandard procedures. Unless stated otherwise, commercial gradehemicals were used without further purification. All reactionsere magnetically stirred and monitored by thin-layer chromatog-

aphy (TLC) using Spectrochem GF254 silica gel coated plates.he 1H NMR spectra were recorded on Bruker-AM-400 spectrom-ter. The 1H NMR chemical shift values are expressed in ppmı) relative to CHCl3 (ı = 7.26 ppm). UV–visible and fluorescencepectra measurements were performed on a JASCO V530 and ahoton Technology International (LPS-220B). A 1.0 × 10−5 M solu-ion of the probe RhPT in MeOH/H2O (4:1, v/v) was prepared andtored in the dry atmosphere. Solutions of 2.0 × 10−4 M salts ofhe respective cation were prepared in analytic grade MeOH andere stored under a dry atmosphere. All experiments were carried

ut in CH3OH–H2O solution (CH3OH:H2O = 4:1, v/v, 10 �M HEPESuffer, pH = 7.4). Binding constant was calculated according to theenesi–Hildebrand equation. Ka was calculated following the equa-ion stated below: 1/(A − Ao) = 1/K(Amax − Ao)[Mn+] + 1/(Amax − Ao).ere Ao is the absorbance of receptor in the absence of guest,

is the absorbance recorded in the presence of added guestMn+), Amax is the maximum absorbance value that was obtainedt � = 563 nm during titration with varying (Mn+)and K is thessociation constant (M−1). The association constant (K) coulde determined from the slope of the straight line of the plotf 1/(A − Ao) against 1/[Mn+]. The binding constant value of Zr4+

ith receptor has been determined from the emission inten-ity data following the modified Benesi–Hildebrand equation./I − I0 = 1/K(Imax − I0)[Mn+] + 1/(Imax − I0), where I0, Imax and I rep-esent the emission intensity of free receptor, the maximummission intensity observed in the presence of added metal ion Zr4+

t 582 nm (�ext = 563 nm) and the emission intensity at a certainoncentration of the metal ion added, respectively.

.2. Cell culture and fluorescence imaging

Candida albicans cells (IMTECH No. 3018) and Pollen cellsrom exponentially growing culture in yeast extract glucose broth

edium (pH 6.0, incubation temperature, 37 ◦C) were centrifuged

tuators B 183 (2013) 350– 355 351

at 3000 rpm for 10 min, washed twice with 0.1 M HEPES buffer atpH 7.4. Then, cells were first incubated with 20 �M of sensor RhPT(in the MEM (modified Eagle’s medium) culture medium contain-ing 49:1, v/v, water–ethanol) for 30 min at 37 ◦C in two differentcover glass bottom dish containing 0.01% Triton X100 as perme-ability enhancing agent and then washed with PBS (containing 2.0%methanol) (0.1 M, pH = 7.4) three times to remove excess of RhPT.After incubation the cells, it was observed under a Leica DM 1000fluorescence microscope equipped with UV filter. Then Zr4+ (0, 20,50 mM) (aqueous solution) was added to the specimen at the pointof observation with the help of a micropipette at 37 ◦C. Cell imagingwas then carried out after washing cells with physiological saline.For the method of cell toxicity determination please see SI, pp.S12–S14.

2.3. Synthesis of chemosensor RhPT

A solution of rhodamine B hydrazide (1, 0.40 g, 0.88 mmol)and 6-thiophenyl-2-pyridinecarboxaldehyde (0.17 g, 0.89 mmol) in20 ml of dry methanol was refluxed for 12 h. After that, the solutionwas cooled (concentrated to 10 ml) and allowed to stand at roomtemperature overnight. The precipitate which appeared next daywas filtered and washed 4 times with 10 ml cold ethanol. After dry-ing under reduced pressure, the reaction afforded, 0.47 g RhPT aswhite solid. Yield: 85%; m.p. 214–216 ◦C. 1H NMR (CDCl3, 400 MHz)ı (ppm): 8.79 (s, 1H, CH N), 8.06 (d, 1H, J = 6.9 Hz), 7.58–7.53 (m,5H), 7.34 (d, 2H, J = 3.84 Hz), 7.07 (t, 2H, J = 5.4 Hz), 6.50 (dd, 4H),6.26 (s, 2H), 3.33 (q, 8H, NCH2CH3), 1.16 (t, 12H, NCH2CH3). 13CNMR (CDCl3, 400 MHz) ı (ppm): 12.71, 29.83, 44.56, 66.09, 98.21,104.63, 108.66, 118.21, 119.04, 124.11, 125.85, 125.79, 127.81,128.07, 128.38, 128.66, 129.42, 134.02, 137.01, 137.84, 144.56,152.17, 153.15, 154.21, 164.63. TOF MS ES+, m/z = 628.10 [M]+, calc.for C38H37N5O2S = 627.77. Anal. calcd. for C38H37N5O2S: C, 72.70;H, 5.94; N, 11.16; O, 5.09; S, 5.11 Found: C, 72.42; H, 5.67; N, 10.89;O, 4.81; S, 5.38.

3. Results and discussion

3.1. UV–vis and fluorescence spectra of chemosensor RhPT

As shown in Scheme 1, the rhodamine B derivative RhPT wasprepared in 85% yield as white solid by reacting 1 [17] with 6-thiophenyl-2-pyridinecarboxaldehyde in an equal molar ratio inmethanol. The structure of RhPT was verified by 1H NMR, 13C NMR,mass spectra and elemental analyses (see Supporting information,pp. S7–S9).

Chemosensor RhPT was designed to bind metal ions via thecarbonyl O, enamine N, pyridyl N and thiophene S as donors. Allthe spectroscopic studies were performed in 4:1 CH3OH–water inwhich chemosensor RhPT formed a colorless solution that was sta-ble for more than four month. The solution of compound RhPT wasvery weakly fluorescent in the absence of any analyte due to thepredominant ring-closed spirolactone and compound showing noabsorption at visible region. Absorption and fluorescence titrationsof RhPT were conducted in methanol–water (4:1, v/v) solution.The spectroscopic properties of the chemosensor RhPT toward themetal ions such as Zr4+, Y3+, Ti4+, Hg2+, Cu2+, Cd2+, Fe2+, Co2+, Ni2+,Mn2+, Zn2+, Pb2+, Pd2+and Ag+ were used in CH3OH–H2O solution(CH3OH:H2O = 4:1, v/v, 10 �M HEPES buffer, pH = 7.4). In this con-text, a higher content of water is seemed to be desirable for betterpractical applicability of the sensor. But we cannot use much more

water due to limited solubility of RhPT in water.

Emission and absorption titrations of RhPT were monitored inCH3OH–H2O (4:1, v/v, 10 �M HEPES buffer, pH = 7.4). Probe RhPTdisplays very weak spectral characteristics in emission spectrum

352 A.K. Mahapatra et al. / Sensors and Actuators B 183 (2013) 350– 355

Fig. 1. Fluorescence emission spectra of RhPT (c = 1.0 × 10−5 M) in aq. CH3OH(Z

(sragarbte

tRmicp1ttetodMsaSaaZbrnS

f(pR4t

Fig. 2. Fluorescence response of RhPT (c = 1.0 × 10−5 M) to 1.0 equiv. addition of Zr4+

and 100 equiv. of other metal ions (the black bar portion) and to the mixture of100 equiv. of other divalent metal ions with 1.0 equiv. of Zr4+ (the red bar portion).(For interpretation of the references to color in this figure legend, the reader isreferred to the web version of the article.)

new peak appeared at 563 nm with a shoulder at 520 nm, indicatingthat the formation of the spirolactam ring-opened RhPT. Moreover,the titration solution exhibited an obvious and characteristic colorchange from colorless to pink (Fig. 4), suggesting that sensor RhPT

CH3OH:H2O = 4:1, v/v, 10 �M HEPES buffer, pH = 7.4) upon addition of 1.0 equiv.r4+ (c = 2.0 × 10−4 M). Inset: change of emission intensity at 582 nm [�ext = 563 nm].

�ex = 563 nm). When Zr4+ was introduced to the solution of RhPT, aignificant enhancement of fluorescence with a strong orange fluo-escent emission band appeared at 582 nm, which was reasonablyssigned to the delocalized xanthene tautomer of the rhodamineroup. The titration curve showed a steady and smooth increase,nd about 1.0 equiv. of Zr4+ was required until a plateau waseached (Fig. 1 and its inset) with the quantum yield [18] as 0.58 inuffered MeOH/HEPES solution, based on optically matching solu-ions of rhodamine B standard (�F = 0.67 in methanol) [19] at anxcitation wavelength of 554 nm.

The recognition interaction was completed immediately afterhe addition of Zr4+ within 2–3 min (see SI, p. S6), and hence,hPT could be used in real-time determination of Zr4+ in environ-ental and biological conditions. There was a significant emission

ntensity enhancement with 1.0 equiv. of Zr4+ which indicates thatompound RhPT is an excellent turn-on sensor for Zr4+. The I/Io wasroportional to the amount of Zr4+ added with a detection limit of6.99 �M (see SI, p. S11). From the emission titration experiment,he association constant [20] (Ka) of RhPT with Zr4+ was estimatedo be 3.05 × 103 M−1 (see SI, p. S6). It is very important parameter tovaluate the performance of a good sensor system toward the selec-ivity of analyte over the other competitive species. The selectivityf RhPT to the various metal ions was investigated. On addition ofifferent metal ions, viz. Y3+, Ti4+, Hg2+, Cu2+, Cd2+, Fe2+, Co2+, Ni2+,n2+, Zn2+, Pb2+, Pd2+and Ag+ ions, to solution of RhPT, there is no

ignificant change in its fluorescence spectrum except in case ofddition of Zr4+ ions, while Cu2+ showed very weak responses (seeI, pp. S3–S4). As shown in Fig. 1, only the addition of Zr4+ resulted in

prominent enhancement of fluorescence at 582 nm (absorbancet 563 nm), which obviously implied the high selectivity of RhPT tor4+. The competition experiments (Fig. 2), which were carried outy adding Zr4+ to RhPT solution in the presence of other metal ions,evealed that the Zr4+ induced fluorescent responses were not sig-ificantly interfered by the commonly coexistent metal ions (seeI, p. S5).

Fig. 3 shows the result of absorption titration spectra obtainedrom the gradual addition of Zr4+ solution to the solution of RhPTc = 1.0 × 10−5 M) in CH3OH–H2O solution (4:1, v/v, HEPES buffer,H = 7.4). When no Zr4+ ions were added to the solution of RhPT, free

hPT, as expected, the absorption spectra showed no peak above00 nm, which was indicated that the spirolactam RhPT form washe predominant species. Upon the addition of 1.2 equiv. of Zr4+, a

Fig. 3. UV–vis absorption titration spectra of RhPT. Inset: Job’s plot that indicates1:1 stoichiometry.

Fig. 4. The visible color (top) and fluorescence changes (bottom) of receptor RhPTin CH3OH–H2O solution (CH3OH:H2O = 4:1, v/v, 10 �M HEPES buffer, pH = 7.4) uponaddition of various metal ions. (For interpretation of the references to color in thisfigure legend, the reader is referred to the web version of the article.)

nd Actuators B 183 (2013) 350– 355 353

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Fig. 5. Partial 1H NMR of (a) RhPT (3.46 × 10−3 M) and with (b) 0.5 equiv. and (c)1 equiv. amounts of Zr4+ in CDCl3 containing 2% d6-DMSO.

A.K. Mahapatra et al. / Sensors a

an serve as a “naked-eye” indicator for Zr4+ ions. Other metal ionsad little interference except Cu2+ ion (see SI, pp. S2–S3 and p. S5ar diagram). From the Job plot, we can observe that the absorbanceent through a maximum at a molar fraction of about 0.5, indicat-

ng that a 1:1 stoichiometry (inset of Fig. 3) was most possible forhe binding mode of Zr4+ and RhPT. From the absorption titrationxperiment, the association constant [21] for Zr4+ was estimated toe 2.16 × 103 M−1(see SI, p. S6). The corroborative evidence for theinding mode 1:1 also comes from the HRMS (EI) spectra (see SI,. S10) of the complex of chemosensor RhPT with Zr4+. The peak at/z = 718.5441 (calc. 718.9990) corresponds to [RhPT + Zr4+]. Thus,e propose that compound RhPT coordinates with Zr4+ with 1:1

toichiometry.Interestingly, a solution of RhPT (10 �M) in optimized

H3OH–H2O solution (4:1, v/v, HEPES buffer, pH = 7.4) is colorlessnd emits no yellow fluorescent light, but during the fluorometricitration of RhPT with Zr4+ ions the colorless solution of the recep-or became deep orange (Fig. 4). This orange fluorescent color isttributed to the opening of the spirolactam ring and generationf the delocalized xanthene moiety [22]. Whereas compound RhPThows obvious pink color in buffered MeOH/HEPES solution uponddition of Zr4+ under visible light. This was not observed withther metal ions except Cu2+ ion. In case of Cu2+ ion, the color-ess solution of RhPT turned into a very faint pink color (Fig. 4). Theesults support our expectation that RhPT could serve as a sensitiveaked-eye probe for Zr4+.

The reversibility is an important aspect of any probe to bemployed as a chemical sensor for detection of specific metal ions.ddition of 10 equiv. of Na2EDTA to a mixture of RhPT·Zr4+ results

n bleaching of the absorption band at 563 nm, which signifies theeneration of the spirolactam structure (see SI, p. S6).

.2. Sensing mechanism

In order to gain insight into the sensing mechanism, it is similaro most common ring-opening mechanism in the rhodamine-basedpirolactam chemosensors due to binding of metal ions. The for-ation ring opening was established from both the FTIR and 1HMR studies. In FTIR, the RhPT and RhPT–Zr4+ were then mea-

ured to confirm the binding mechanism. The peak at 1698 cm−1,orresponding to the characteristic lactam carbonyl absorption ofhPT, changed drastically to the lower frequency (1684 cm−1) uponddition of 1.0 equiv. of Zr4+ (see SI, p. S10). This supported that theactam carbonyl oxygen is actually involved in the coordination

rocess through spirolactam ring opening (Scheme 2). Further-ore, the sensing mechanism is found to be reversible as the purple

olor of the complex disappears with the addition of excess EDTAsee SI, p. S6).

Scheme 2. Proposed mechanism for the fluoresce

Fig. 6. Fluorescence intensity of free chemosensor RhPT and in the presence of5 equiv. of Zr4+ in aq. CH3OH (CH3OH:H2O = 4:1, v/v, 10 �M HEPES buffer) withdifferent pH conditions.

To get insight into the binding mode, the 1H NMR titrationexperiments were measured.

As shown in Fig. 5, the apparent downfield shift (�ıc = 0.54,�ıd = 0.50 and �ıb = 0.63 ppm) of the ring protons of rhodaminein the presence of Zr4+ suggested that ring participates in thebinding. Specially, the imine proton (He) at around ı 8.79 ppmwas considerably shifted downfield toward ı 9.58 ppm upon Zr4+

addition, indicating that a decrease in electron density at iminenitrogen resulting from direct coordination with Zr4+. The ‘Ha’ pro-ton showed an upfield shift of 0.37 ppm. This upfield movementis attributed to the increase in electron density arising from the

opening of the spirolactam ring. Taken together, based on the aboveresults, coordinating property of Zr4+, HRMS spectrum and the Job’splot, we proposed a plausible binding mode of the sensor RhPT withZr4+ as shown in Scheme 2.

nce changes of RhPT upon addition of Zr4+.

354 A.K. Mahapatra et al. / Sensors and Actuators B 183 (2013) 350– 355

Fig. 7. Fluorescence and bright field images of Candida albicans (Set I) and Pollen (Set II) cells: for Set I and Set II: (a) bright field images of respective cells treated withr t fielda erpreto

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eceptor RhPT (20 �M) for 30 min at 37 ◦C; (b) fluorescence images of (a); (c) brighfter addition of 10 and 20 �M Zr4+ solution for 30 and 45 min, respectively. (For intf the article.)

.3. Effect of pH

To study the practical applicability, the effects of pH on the fluo-escence response of RhPT in the absence and presence of Zr4+ ionsere evaluated. In the absence of Zr4+, almost no change in fluo-

escence intensity was observed in the free sensor over a wide pHange of 6.0–11.0, indicating that the free sensor RhPT was stablen the wide pH range, but at acidic conditions (pH < 5), an obviousff–on fluorescence appeared due to the formation of the open-ringtate because of the strong protonation.

In the pH range from 5.0 to 6.5, little fluorescence signalexcited at 563 nm) could be observed for free RhPT, suggest-ng that the molecules prefer the spirocyclic form. Upon theddition of Zr4+ ions, there was an obvious fluorescence off–onhange of RhPT under different pH values. Thus, the sensor RhPTas the maximal sensing response at physiological pH, indicat-

ng that the sensor RhPT is promising for biological applicationsFig. 6).

.4. Fluorescence image in living cells

To further demonstrate the practical biological application ofhe sensor RhPT, fluorescence imaging experiments were car-ied out in living cells. C. albicans cells (Set I) and Pollen (SetI) cells were first incubated with 20 �M of sensor RhPT (inhe MEM (modified Eagle’s medium) culture medium contain-ng 49:1, v/v, water–ethanol) for 30 min at 37 ◦C in two differentover glass bottom dish, and then washed with PBS (containing.0% methanol) (0.1 M, pH = 7.4) three times to remove excess ofhPT.

Microscopic images showed no intracellular fluorescencemages were detected on the cells, which indicated that sensorhPT was non-emissive (Fig. 7b). However, the cells loaded withr4+ (10–20 �M) displayed a significant red fluorescence from thentracellular area (Fig. 7d and e). Bright field measurements afterreatment with RhPT and Zr4+ confirmed that the cells remainediable throughout the imaging experiments (Fig. 7c) and it is indi-ating the subcellular distribution of Zr4+ that was internalizedithin the living cells from the growth medium. The results suggest

hat compound RhPT could be used for monitoring intracellularr4+ in living cells. To the best of our knowledge, this is the firstescription of a chemodosimeter suitable for monitoring Zr4+ in

iving cells. The results suggest that sensor RhPT is cell membraneermeable, viable throughout the imaging experiments and canlso be used for imaging of Zr4+ in living cells and potentially inivo.

images of (b) after addition of Zr4+ (20 �M); (d) and (e) fluorescence images of (b)ation of the references to color in the text, the reader is referred to the web version

4. Conclusions

In conclusion, we have synthesized rhodamine-based highlysensitive and selective chemosensor for the detection of Zr4+

through a change in structure between spirocyclic and ring-openforms of rhodamine. The probe switches to a highly fluorescentcomplex upon Zr4+ chelation under physiological conditions. Sen-sor RhPT showed high sensitivity and selectivity toward Zr4+ overother interference cations except Cu2+, which showed a signifi-cant but smaller effect. We can also use this probe as ‘naked-eye’chemosensor for zirconium ions. To the best of our knowledge, thiswork provides the first example of a fluorescent probe which sen-sitive to Zr4+. Furthermore, we have demonstrated that the sensorRhPT is applicable for Zr4+ imaging in the living cells.

Acknowledgments

We thank the DST-New Delhi [Project file no. SR/S1/OC-44/2012] for financial support. SKM thanks UGC, New Delhi, Indiafor a fellowship and also Bhaskar Pramanik for his support.

Appendix A. Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.snb.2013.04.012.

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Biographies

Ajit Kumar Mahapatra is a associate professor in Department of Chemistry, BengalEngineering and Science University, Shibpur, Howrah 711103, India. He obtainedhis Ph.D. degree in 2001 from Bengal Engineering and Science University, Shibpur,Howrah.

Saikat Kumar Manna obtained his B.Sc. degree in 2007 and M.Sc. degree in 2009,presently working for Ph.D. degree in Bengal Engineering and Science University,Shibpur, Howrah.

Subhra Kanti Mukhopadhyay is a assistant professor in Department of Microbi-

ology, The University of Burdwan, Burdwan, India. He obtained his Ph.D. degree in1997 from The University of Burdwan, Burdwan, India.

Avishek Banik obtained his B.Sc. degree in 2009 and M.Sc. degree in 2011, presentlyworking for Ph.D. degree in The University of Burdwan, Burdwan, India.