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aDepartment of Chemistry, The University o

India. E-mail: pabitracc@yahoo.com; Fax:

ext. 424bMolecular Biology and Genetics Labora

University, India

† Electronic supplementary informacharacterization data, tables, gures10.1039/c4ra02378a

Cite this: RSC Adv., 2014, 4, 21471

Received 18th March 2014Accepted 22nd April 2014

DOI: 10.1039/c4ra02378a

www.rsc.org/advances

This journal is © The Royal Society of C

A FRET-based ‘off–on’ molecular switch: aneffective design strategy for the selective detectionof nanomolar Al3+ ions in aqueous media†

Buddhadeb Sen,a Siddhartha Pal,a Somenath Lohar,a Manjira Mukherjee,a

Sushil Kumar Mandal,b Anisur Rahman Khuda-Bukhshb and Pabitra Chattopadhyay*a

A new water-soluble rhodamine-based Al3+ ion-selective probe (L1) was synthesised and characterized by

physico-chemico and spectroscopic tools. In the presence of a large excess of other competing ions, L1

specifically binds Al3+ ions with a concurrent visually observable change from colorless to pink in

electronic spectral behavior, making it possible to detect the presence of Al3+ ions with the naked eye.

The addition of Al3+ ions to a solution of L1 in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 �C, results in

a decrease in the weak fluorescence intensity at lem ¼ 470 nm, while a new peak (at lem ¼ 588 nm)

increases gradually through a fluorescence resonance energy transfer process. This ratiometric

enhancement helps to detect Al3+ ions at a very low concentration of 33 nM. The detection limit of L1

for Al3+ ions was estimated to be 6.19 � 10�9 M using the 3s method. This probe is also useful for

imaging Al3+ ions in HeLa cells.

Introduction

Chemosensors for the selective detection of various biologicallyand environmentally relevant metal ions have attracted greatattention because of their potential use in medicine and inenvironmental research. Aluminum is the third most abundantelement on the Earth aer oxygen and silicon and there iswidespread exposure of humans to aluminum due to its usein food additives, aluminum-based pharmaceuticals and cook-ing utensils.1,2 Aer absorption, aluminum is distributedthroughout all living tissues in humans and animals andaccumulates in bone. Iron-binding proteins (e.g. transferrin C1and C2, ferritin) are the main carriers of Al3+ ions in plasma andAl3+ ions can enter into the brain and reach the placenta andfetus. Al3+ ions may persist for a very long time in various organsand tissues before excretion through urine. Aluminum salts areneurotoxic andmay be associated with Parkinson's disease3 andsenile dementia (commonly known as Alzheimer's disease4),microcytic anemia, dialysis dementia and osteomalacia, andhave even been shown to increase the risk of lung and bladdercancer.5–8 Aluminum should therefore be regarded as a toxic

f Burdwan, Golapbag, Burdwan 713104,

+91-342-2530452; Tel: +91-342-2558554

tory, Department of Zoology, Kalyani

tion (ESI) available: Schemes,, and some spectra. See DOI:

hemistry 2014

metal and its concentration in the environment should bemonitored.

In 1989 the WHO listed aluminum as a source of foodpollution and limited its concentration in drinking water to 200mg L�1 (7.41 mM). The FAO/WHO Joint Expert Committee onFood Additives recommended a maximum daily intake ofaluminum of 3–10 mg per day per kg body mass. It is believedthat almost 40% of the world's acid soils are affected byaluminum toxicity, which is a key factor affecting plant (i.e.crop) performance on acid soils.9,10 The detection of Al3+ ions istherefore crucial in controlling its concentration levels inenvironmental monitoring and its impact on human health.

Several conventional methods with moderate sensitivity forAl3+ ions have been developed with detection based on atomicabsorption spectrometry (AAS) and chromatographic and spec-trophotometric techniques.11 Of these methods, AAS requiresexpensive instrumentation and complicated sample preparationprocesses. Although other techniques have been developed fordetecting trace amounts of Al3+ ions, most of these involve theuse of harmful chemicals and the analytical procedures areeasily interfered with by variations in pH and the coexistence ofinterfering ions. However, spectrouorimetric methods havereceived considerable attention in recent years due to theirsimplicity, high sensitivity and real-time monitoring with a lowresponse time.12–14

There are several uorescent sensors for Al3+ ions with goodselectivity, but this approach has several disadvantages,including complicated synthetic procedures, poor water solu-bility, insensitivity to biological systems, interference fromother ions and variations with pH.15 The sensitive bioimaging of

RSC Adv., 2014, 4, 21471–21478 | 21471

Scheme 1 Synthesis of L1.

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Al3+ ions in cells is a prerequisite for understanding theunderlying mechanisms of how Al3+ ions cause aluminum-induced human diseases. Few reports have been published onchemosensors for the detection of Al3+ ions in aqueous mediaand most of these are based on chelation-enhanced uores-cence/photo-induced electron transfer.15 Very few publishedmethods have used the uorescence resonance energy transfer(FRET) mechanism.16 The sensors with uorescence enhance-ment (a ‘turn-on’ response) through FRET are of considerableinterest as FRET is a distance-dependent radiation-less transferof energy from an excited donor uorophore to a suitableacceptor uorophore which can be used to investigate molec-ular level interactions.17 Therefore the development of newratiometric FRET-based sensors for Al3+ ions with improveddetection limits in the presence of water is desirable.

We report here a newly designed ratiometric FRET-baseduorescent sensor (L1) which is highly selective for Al3+ ions inHEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 �C. On excitationat 350 nm, the L1 probe exhibits a uorescence maximum at470 nm, which decreases along with a gradual increase in anew peak at 588 nm due to the addition of Al3+ ions. Thisphenomenon is a result of the ring opening of the spirolactamsystem of rhodamine, which gives rise to a strong uorescenceemission and a visual color change of the solution from color-less to pink. Ratiometric responses are attractive because theratio between the two emission intensities can be used tomeasure the analyte concentration and the sensor moleculeconcentration, providing a built-in correction for environ-mental effects and giving stability under illumination.14,18

Interestingly, the presence of an excess of other metal ions, e.g.alkali (Na+, K+), alkaline earth (Mg2+, Ca2+), and transition metalions (Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+) andPb2+, does not affect the ‘switch-on’ behavior of the receptor L1

observed in the presence of Al3+ ions. Fluorescence microscopicstudies conrmed that L1 could also be used as an imagingprobe for the detection of the uptake of Al3+ ions in HeLa cells.

Experimental sectionMaterials and methods

High-purity HEPES, 8-aminoquinoline, 2-chloroacetyl chlorideand aluminum nitrate nonahydrate were purchased from SigmaAldrich (India) and rhodamine B and ethylene diamine (en)from E. Merck. The solvents used were of spectroscopic grade.All metal salts were used as either their nitrate or chloride salts.Other chemicals were of analytical-reagent grade and usedwithout further purication except where specied. Milli-Q,18.2 MU cm�1 water was used throughout all experiments. AShimadzu (Model UV-1800) spectrophotometer was used forrecording electronic spectra. FTIR spectra were recorded using aPerkin Elmer FTIR Model RX1 spectrometer using a KBr disk.1H-NMR spectra were obtained on a Bruker Avance DPX 500MHz spectrometer and a Geol 400 MHz spectrometer usingCDCl3 solution. Electrospray ionization (ESI) mass spectra wererecorded on a Qtof Micro YA263 mass spectrometer. ASystronics digital pH meter (Model 335) was used to measurethe pH of the solution and the pH was adjusted using either

21472 | RSC Adv., 2014, 4, 21471–21478

50 mM HCl or NaOH solution. Steady-state uorescence emis-sion and excitation spectra were recorded with a Hitachi 4500spectrouorimeter. Time-resolved uorescence lifetimemeasurements were obtained using a HORIBA JOBIN Yvonpicosecond-pulsed diode laser based time-correlated single-photon counting spectrometer from IBH (UK) at lex ¼ 377 nmwith a micro-channel plate photomultiplier tube (MCP-PMT) asa detector. Emission from the sample was collected at rightangles to the direction of the excitation beam maintainingmagic angle polarization (54.71). The full width at half-maximum of the instrument response function was 250 ps andthe resolution was 28.6 ps per channel. Data were tted tomulti-exponential functions aer deconvolution of the instru-ment response function by an iterative reconvolution techniqueusing IBH DAS 6.2 data analysis soware in which reduced w2

and weighted residuals served as parameters for goodness of t.

Synthesis of the L1 probe

The L1 probe was synthesised using a three-step reactionprocess (Scheme 1). Rhodamine B-en carboxamide (1) was rstprepared using a previously published method.19 Ethylenediamine (en, 4 mL) was added to a solution of rhodamine B (1 g,2.09 mmol) in ethanol (40 mL). The solution was reuxed for6 h. The reaction mixture was then evaporated under reducedpressure to give orange oil, which was subsequently recrystal-lized from methanol–water to give rhodamine B-en carbox-amide (1) as a light orange crystal (77%).

2-Chloro-N-(quinol-8-yl)acetamide (2) was then preparedfrom the reaction of 2-chloroacetyl chloride and 8-amino-quinoline. 2-Chloroacetyl chloride (5.31 mL) was dissolved inchloroform (5 mL) and then added dropwise to a cooled, stirredsolution of 8-aminoquinoline (2.88 g, 20 mmol) and Et3N(3.0 mL) in chloroform (10 mL) within 1 h. Aer stirring for 2 hat room temperature the mixture was removed under reducedpressure to obtain a white solid, which was then ltered out andextracted with dichloromethane to give compound 2 (ref. 20)with a yield of 82% (m.p. 133 � 2 �C).

In the nal step, rhodamine B-en carboxamide (1) was takenin dry acetonitrile with anhydrous K2CO3. 2-Chloro-N-(quinolin-

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8-yl)acetamide (2) in dry acetonitrile was then added dropwisewith stirring. The resulting reaction mixture was reuxed for 6h. The volume of the solution was reduced to obtain a solid andthen extracted with dichloromethane and nally puried bysilica gel column chromatography using dichloromethane asthe eluent. The yield was 73% (m.p. 103 � 2 �C).

C41H44N6O3. Anal. found: C, 73.39; H, 6.46; N, 12.83. Anal.calc.: C, 73.63; H, 6.63; N, 12.57. IR (cm�1): nNH, 3441; nC]C,2970; nC]O, 1681; nC]N, 1618.

1H-NMR (400 MHz, CDCl3): 10.91(s, 1-NH–CO), 8.43 (dd, 1H), 8.21 (dd, 1H), 8.12 (dd, 1H), 7.45(m, 2H), 7.08 (dd, 2H), 7.07 (m, 1H), 6.61 (dd, 2H), 6.06–6.14(m, 7H), 3.56 (s, 2H, –CH2–CO), 3.31 (q, 8H, 4CH2), 2.84 (t, 2H),2,49 (t, 2H), 1.06 (t, 12H, 4CH3).

13C-NMR (CDCl3): 166.18,165.02, 154.18, 153.72, 149.53, 149.26, 148.99, 136.39, 133.69,132.96, 131.60, 130.50, 129.32, 128.74, 127.59, 124.1, 123.57,122.03, 116.90, 109.01, 108.48, 103.98, 98.19, 67.11, 44.80,41.67, 40.54, 40.13, 13.04. ESI-MS m/z 669.0028 [M + H+, 60%],calc.: 669.347 [M + H+].

Synthesis of L–Al complex as [Al(L)(NO3)2]

A solution of aluminum nitrate was added dropwise to a 10 mLethanolic solution of L1 (0.01 mmol) and stirred for 4 h. Thesolvent was removed using a rotary evaporator and a blood redsolid was obtained (Scheme S1†).

[Al(L)(NO3)2]. Anal. found: C, 59.89; H, 5.16; N, 13.91. Anal.calc.: C, 60.14; H, 5.29; N, 13.68. IR (cm�1): nNH, 3437; nC]C,2845; nC]O, 1691; nC]N, 1612.

1H-NMR (500 MHz, CDCl3): 11.18(s, 1-NH–CO), 8.76 (dd, 1H), 8.52 (dd, 1H), 8.11 (dd, 1H), 7.93(dd, 1H), 7.87 (dd, 1H), 7.46 (dd, 2H), 7.39 (m, 1H), 7.08(dd, 2H), 6.36 (t, 1H), 6.22 (d, 1H), 6.19 (d, 1H), 6.15 (d, 1H), 3.89(s, 2H, –CH2–CO), 3.33 (q, 8H, 4CH2), 2.94 (t, 2H), 2.59 (t, 2H),1.09 (t, 12H, 4CH3).

13C-NMR (DMSO-d6): 169.73, 169.10,154.89, 154.50, 153.49, 150.12, 149.42, 134.44, 134.17, 130.23,129.42, 129.11, 128.91, 127.88, 124.46, 124.15, 123.53, 123.23,117.10, 109.15, 104.88, 104.68, 98.12, 47.02, 44.58, 38.64, 38.19,13.20. ESI-MS in methanol: [M + CH3OH + H]+, m/z, 851.4242(obs. with 11% abundance) (calc. m/z 851.85, where M ¼[Al(L)(NO3)2]).

General method of UV-visible and uorescence titration

The path length of the cells used for absorption and emissionstudies was 1 cm. For UV-visible and uorescence titrations, astock solution of L1 was prepared in HEPES buffer (1 mM, pH7.4, 2% EtOH) at room temperature. Working solutions of L1

and Al3+ ions were prepared from their respective stock solu-tions. Fluorescencemeasurements were performed using a 5 nm� 5 nm slit width. All the uorescence and absorbance spectrawere taken aer 30 minutes of mixing the Al3+ ions and L1 toacquire the optimized spectra.

A series of solutions containing L1 and Al(NO3)3 wasprepared such that the total concentration of L1 (10 mM)remained constant in all the sets. The mole fraction (X) of Al3+

ions was varied from 0.1 to 0.6. The absorbance at 561 nm wasplotted against the mole fraction of the probe for stoichiometricdetermination.

This journal is © The Royal Society of Chemistry 2014

Emission study

The organic moiety (L1) shows a very weak emission at 588 nmin HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 �C when excitedat 550 nm considering the absorption at 561 nm. Fluorescencequantum yields (F) were estimated by integrating the areaunder the uorescence curves using the equation:

Fsample ¼Fref �ODref � Asample � hsample

2

ODsample � Aref � href2

where A is the area under the uorescence spectral curve andOD is the optical density of the compound at the excitationwavelength of 550 nm and h is the refractive index of the solventused. The standard used for the measurement of uorescencequantum yield was rhodamine B (F ¼ 0.7 in ethanol).

Calculation of Forster distance (R0)

The Forster distance (R0) for the FRET process was calculatedfrom the following simplied equation:21,22

R0 ¼ 0:211½k2h�4FDJDA�1=6

¼ 0:211

�k2h�4FD

ðN0

IDðlÞ3AðlÞl4dl�1=6

where h is the refractive index (h ¼ 1.33 in water),23 FD is thequantum yield of the donor, k denotes the average squaredorientational part of a dipole–dipole interaction, typicallyk2 ¼ 2/3,24 JDA is the degree of spectral overlap between thedonor emission and the acceptor absorption, ID(l) is thenormalized uorescence spectra of the donor and 3A(l) is themolar absorption coefficient of the acceptor.

Preparation of cell and in vitro cellular imaging with L1

Human cervical cancer cells (HeLa cell line) were purchasedfrom the National Center for Cell Science, Pune, India and usedthroughout the study. Cells were cultured in Dulbecco's modi-ed Eagle's medium (DMEM; Gibco BRL) supplemented with10% fetal bovine serum (FBS) (Gibco BRL) and a 1% antibioticmixture containing penicillin, streptomycin and neomycin(PSN, Gibco BRL) at 37 �C in a humidied incubator with 5%CO2. For the experimental study, cells were grown to 80–90%conuence, harvested with 0.025% trypsin (Gibco BRL) and0.52 mM EDTA (Gibco BRL) in phosphate-buffered saline (PBS,Sigma Diagnostics), plated at the desired cell concentration andthen allowed to re-equilibrate for 24 h before treatment. Cellswere rinsed with PBS and incubated with DMEM containing L1

(10 mM, 1% DMSO) for 30 min at 37 �C. All experiments wereconducted in DMEM containing 10% FBS and 1% PSN antibi-otic. The imaging system consisted of a uorescence micro-scope (ZEISS Axioskop 2 plus) with an 10� objective lens.

Cell cytotoxicity assay

To test the cytotoxicity of L1, an MTT [3-(4,5-dimethyl-thiazol-2-yl)-2,S-diphenyl tetrazolium bromide] assay was performedusing a previously published procedure.25 Aer treatment of theprobe (5, 10, 20, 50 and 100 mM), 10 mL of MTT solution (10 mgmL�1 PBS) was added to each well of a 96-well culture plate and

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incubated continuously at 37 �C for 6 h. All media were removedfrom the wells and replaced with 100 mL of acidic isopropanol.The intracellular formazan crystals (blue-violet) formed weredissolved with 0.04 N acidic isopropanol and the absorbance ofthe solution was measured at 595 nm with a microplate reader.Data are given as the mean � S.D. of three independentexperiments. The cell cytotoxicity was calculated based on a cellviability of 100%.

Fig. 2 Naked eye visual and fluorescence color changes in (A) probe L1

only and (B) in the presence of Al3+ ions in HEPES buffer (1 mM, pH 7.4,2% EtOH).

Results and discussionSynthesis and characterization

Rhodamine B-en carboxamide (1) was obtained from rhodamineB using a previously published method19 and 2-chloro-N-(quino-lin-8-yl)acetamide (2) was prepared from the reaction of 2-chlor-oacetyl chloride and 8-aminoquinoline in chloroform in thepresence of NEt3 (Scheme 1). Probe L1 was then isolated from thereaction of rhodamine B-en carboxamide and 2-chloro-N-(qui-nolin-8-yl)acetamide in a dry acetonitrile solution in the presenceof anhydrous K2CO3. The formulation of L1 was conrmed byphysico-chemico and spectroscopic methods (Fig. S1A–D†). TheL–Al complex was obtained when aluminum nitrate was mixedwith an ethanolic solution of L1 with stirring. Aer removing thesolvent, a blood red solid was obtained (Scheme S1†). Theformulation of the L–Al complex was conrmed by physico-chemico and spectroscopic tools (Fig. S2A–D†).

UV-visible spectroscopic studies of L1

The UV-visible spectra of L1 recorded in HEPES buffer (1 mM,pH 7.4, 2% EtOH) at 25 �C show an absorption maximum at318 nm, which may be attributed to the intramolecular p–p*

charge transfer transition. On the stepwise addition of Al3+ ions(0–30 mM) to the solution of L1 in HEPES buffer (1 mM, pH 7.4,2% EtOH), the absorption intensity at 318 nm increased grad-ually and a new peak at 561 nm (Fig. 1) was generated due to theformation of a pink color from the colorless solution (Fig. 2).Considering the complexity of the intracellular environment, anadditional experiment was performed with the probe to deter-mine whether other ions were potential interferents. Metal ion

Fig. 1 UV-visible titration spectra of L1 (10 mM) with the incrementaladdition of Al3+ ions (0–30 mM) in HEPES buffer (1 mM, pH 7.4, 2%EtOH).

21474 | RSC Adv., 2014, 4, 21471–21478

selectivity assays were therefore performed while keeping theother experimental conditions unchanged. No signicantchange in the UV-visible spectral pattern was observed with theaddition of 10 equivalents excess of the relevant metal ions, i.e.Na+, K+, Ca2+, Mg2+, Cr3+, Mn2+, Fe3+, Co2+, Ni2+, Zn2+, Cd2+,Hg2+, Cu2+ and Pb2+.

The complex formed between L1 and Al3+ was found to be ofa 1 : 1 stoichiometry, as established using the absorbance datawith the help of Job's plot (Fig. S3†). Further conrmation of a1 : 1 stoichiometry with the probable formulation of an L–Alcomplex was established by the physico-chemical and spectro-scopic data of the L–Al complex isolated in the solid form. Themolecular ion peak in the ESI-MS spectra of L–Al3+ was observedat m/z 851.4242, evidence of the 1 : 1 stoichiometric species(Fig. S2B†).

Fluorescence spectroscopic studies of L1

To optimize the pH of the experimental conditions, a pHstudy was performed to control the efficiency of the probe(L1). In the absence of Al3+ ions, L1 showed a weak intensityuorescence and an interesting independence of pH in therange 6.0–10.0 (Fig. S4†). At low pH the probe showed a highemission intensity because, at these pH values, the spi-rolactam ring opens irrespective of the species of metal ionadded.18a,26 It was also noticed that the presence of Al3+ ionsenhances the emission intensity of L1 signicantly at pH 4.0–10.0. The emission spectrum of L1 excited at 350 nm shows auorescence maximum at 470 nm in HEPES buffer (1 mM, pH7.4, 2% EtOH) at 25 �C. The intensities at 470 nm weresignicantly decreased with a concomitant increase in theintensities at 588 nm through an isoemissive point at ca.553 nm when various concentrations of Al3+ ions (0–30 mM)were added (Fig. 3).

Ratiometric signaling of the uorescence output at twodifferent wavelengths plotted as a function of the concentrationof Al3+ ions indicates that the uorescence intensity ratio ofwavelengths 470 and 588 nm (I588/I470) gradually increases withan increase in the concentration of Al3+ ions (Fig. 4). L1 showedan almost 75-fold increase in its uorescence intensity with theaddition of only 3.0 equivalents of Al3+ ions.

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Fig. 3 Emission spectra of L1 (10 mM) in the presence of Al3+ ions(0–30 mM) at lex ¼ 350 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at25 �C.

Fig. 4 Ratiometric signaling of fluorescence output at two differentwavelengths plotted as a function of the concentration of Al3+ ions inaqueous HEPES buffer.

Fig. 5 (A) Fluorimetric titration spectra of L1 with Al3+ ions (0–30 mM)at lex ¼ 550 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 �C. (B)Fluorescence intensity as a function of Al3+ ion concentration atlex ¼ 550 nm in HEPES buffer (1 mM, pH 7.4, 2% EtOH) at 25 �C.

Scheme 2 Probablemechanism of FRET process induced by Al3+ ions.

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From the plot of the uorescence intensity at 588 nm (I588)versus [Al3+] it is suggested that L1 could be used to detect Al3+

ions as low as ca. 33 nM (Fig. S5†).27 To calculate the detectionlimit, a calibration graph (Fig. S6†) was obtained in the lowerregion. From the slope (S) of the graph and the standard devi-ation of seven replicate measurements at the zero level (szero),the detection limit was estimated using the equation 3s/S.17,28

This indicates that the detection limit of L1 for Al3+ ions is 6.19� 10�9 M, which is comparable with the previously reportedFRET-based Al3+ ion-selective uorescent sensor,16b althoughour sensor is superior to the one reported previously becauseour FRET process takes place in aqueous solution.

A metal ion selectivity study was performed using L1 underidentical experimental conditions to understand this phenom-enon. Interestingly, the introduction of other metal ions causesthe uorescence intensity to be either unchanged or weakened.Fluorescence enhancement of L1 (10 mM) was not observed withthe addition of an excess of 50 equivalents of biologically rele-vant metal ions, i.e. Na+, K+, Ca2+ and Mg2+ and 10 equivalentsexcess of several competitive metal ions (Cr3+, Mn2+, Fe3+, Co2+,Ni2+, Zn2+, Cd2+, Hg2+, Cu2+ and Pb2+) (Fig. S7†); also no colorchange was seen with visual naked eye detection (Fig. S8 andS9†). Almost no adverse effect on intensity was observed in the

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presence of a 10 times excess of various tested ions with L1 andAl3+ ions (Fig. S10†).

As the receptor L1 has two different uorophore units, weconsidered it appropriate to study the metal binding event of L1

at the two different excitation wavelengths corresponding to thexanthene unit (550 nm) and the quinoline unit (350 nm). Fig. 5shows that the excitation of L1 at 550 nm in the absence of Al3+

did not initially show any signicant emission in the range 550–700 nm, with a quantum yield of only 0.03. This supports thefact that the receptor remains in the spirolactam form in theabsence of metal ions; the non-existence of the highly conju-gated xanthene form results in the suppression of emission inthis range of wavelengths. However, the addition of Al3+ ions toL1 induces a signicant switch to an ON uorescence responsenear 588 nm, showing a visual display of reddish uorescencewith a quantum yield of 0.61 (�20-fold).

The switch to an ON response for the absorption spectralband at 561 nm and the emission band at 588 nm with bindingto Al3+ ions suggests the opening of the spirolactam ring of L1

when metal ion coordination occurs (Scheme 2). It is observedthat Al3+ ions bind to L1, inducing the ring opening of L1 and thegeneration of the xanthene moiety which is selective towardsAl3+ ions. No noticeable spectral change was seen for the othermetal ions tested.

The binding of Al3+ ions induces the opening of the spi-rolactam ring of L1 with an associated switch in the UV-visiblespectral response in the range 380–650 nm, which has asignicant spectral overlap with the emission spectrum of theN-(quinol-8-yl)-acetamide fragment (Fig. S11†). This suggests aplausible route for the non-radiative transfer of excitation

RSC Adv., 2014, 4, 21471–21478 | 21475

Fig. 7 Time-resolved fluorescence decay of L1 (10 mM) only and in thepresence of added Al3+ ions in HEPES buffer (1 mM, pH 7.4, 2% EtOH)at 25 �C using a nano-LED of 377 nm as the light source atlem ¼ 470 nm.

Table 1 Fluorescence lifetime (ns) of the corresponding of L1 and L–Alcomplexes at lem ¼ 470 nm

lem ¼ 470 nm sav (ns) c2 4 kr (108 s�1) knr (10

9 s�1)

L1 12.87 1.1 0.26 0.2020 0.0575L1 + Al3+ (1 : 0.5) 11.15 1.2 — — —L1 + Al3+ (1 : 1) 8.37 1.05 0.14 0.1673 0.1028

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energy from the donor quinoline to the acceptor xanthenemoiety within the Forster critical distance (R0), calculated to be20.03 A, and initiates an intramolecular FRET process (viz.Scheme 2). In the free state of L1 the FRET pathway is totallysuppressed and only an emission maximum near 470 nm wasobserved when L1was excited at 350 nm. Binding of the receptorto Al3+ ions induces the FRET process to produce an intenserhodamine-based reddish emission, i.e. energy transfer fromthe N-(quinol-8-yl)-acetamide moiety to xanthene is due to thering opening,29 resulting in an increase in the overlap integralbetween N-(quinol-8-yl)-acetamide and the xanthene moiety.Therefore, when titrated with Al3+ ions, the emission band at ca.470 nm begins to decrease, along with a concomitant genera-tion of a new uorescence band at ca. 588 nm. This change inuorescence was clearly visualised and the uorescence color issignicantly red (see Fig. 2).

The apparent binding constant (K) was determined using themodied Benesi–Hilderbrand method30 and was found to be5.81 � 106 M�1 (Fig. S12†). The ring-opening phenomenon isalso supported by the 13C-NMR analyses of L1 and the L–Alcomplex. In the 13C-NMR spectrum of L1 the signal at d¼ 67.114ppm, assignable to the tertiary carbon (sp3 hybridized) of thespirolactam ring in L1 (C6), is absent in the spectrum of the L–Alcomplex, but appears at d ¼ 134.44 ppm due to the conversionof C6 from sp3 to sp2 hybridized carbon; this feature supportsthe opening of the spirolactam ring (Fig. 6).31

In the 1H-NMR titration, a shi in the corresponding char-acteristic peaks into the downeld region was observed insupport of the chelation of L1 in the solution state (Fig. S13†).The peaks at d ¼ 3.56 ppm assignable to –CH2–CO protons andat d ¼ 10.91 ppm assignable to NH–CO protons in the spectrumof L1 (Fig. S1C†) are signicantly shied to d ¼ 3.89 ppm and11.18 ppm, respectively (Fig. S2C†).

The occurrence of the FRET process also agrees with theuorescence lifetime data (Fig. 7 and Table 1). In the uores-cence lifetime experiments (lem ¼ 470 nm), the average lifetimeof L1 was 12.87 ns. Aer the addition of Al3+ ions to the solutionof L1, the average lifetime (lem¼ 470 nm) of the complex speciesdecreased to 11.15 and 8.37 ns, respectively, when the

Fig. 6 13C-NMR spectra of L1 in CDCl3 and the L–Al complex inDMSO-d6.

21476 | RSC Adv., 2014, 4, 21471–21478

concentration of Al3+ ions was increased from 0.5 equivalent to1.0 equivalent with respect to L1. In contrast, the averagelifetime of the probe L1 increased from 1.60 to 9.77 ns(lem ¼ 588 nm) when Al3+ ions were added to the solution of L1,which resembles the FRET process induced by Al3+ ions(Fig. S14†).

The values of kr and knr for the organic moiety L1 and the L–Al species are listed in Tables 1and 2 according to the equa-tions32 s�1 ¼ kr + knr and kr ¼ Ff/s, where kr ¼ the radiative rateconstant and knr ¼ total non-radiative rate constant. The data inTable 2 suggest that kr has only changed slightly; the factor thatinduces uorescent enhancement is mainly ascribed to thedecrease in knr.

Biological studies of L1 in the presence of Al3+

To examine the utility of the probe in biological systems, itwas applied to the human cervical cancer HeLa cell line. Inthese experiments, both Al3+ ions and L1 were taken up by thecells and images of the cells were recorded by uorescencemicroscopy following excitation at�550 nm. Aer incubation

Table 2 Fluorescence lifetime (ns) of the corresponding L1 and L–Alcomplexes at lem ¼ 588 nm

lem ¼ 588 nm sav (ns) c2 4 kr (108 s�1) knr (10

9 s�1)

L1 1.6013 1.1 0.03 0.1873 0.60576L1 + Al3+ (1 : 0.5) 2.065 1.00 — — —L1 + Al3+ (1 : 1) 9.7729 1.05 0.61 0.6242 0.03990

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Fig. 8 Fluorescence image of HeLa cells after incubation with L1 for30 min followed by treatment with (1) 0 mM; (2) 3 mM; (3) 5 mM; and (4)10 mM Al3+ ions at 37 �C. The samples were excited at �550 nm.

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with L1 (10 mM) for 30 min, the cells displayed very faintintracellular uorescence. However, the cells exhibitedintensive uorescence when exogenous Al3+ ions were intro-duced into the cell via incubation with an Al salt (Fig. 8). Theuorescence responses of the probe with various addedconcentrations of Al3+ ions are clearly evident from thecellular imaging. In addition, the in vitro study showed that10 mM of L1 showed no cytotoxic effect on cells for up to 6 h(Fig. S15†). These results indicate that the probe has a hugepotential as a sensor for Al3+ ions in both in vitro and in vivoapplications.

Conclusions

In summary, we conclude that this newly designed uores-cent chemosensor (L1) behaves as a highly specic andselective FRET-based ratiometric uorescence probe towardsAl3+ ions, which can also be detected by the naked eye. Thisuorescence is due to the ring opening of the rhodaminespirolactam system. The detection limit of this L1 probe isvery low (6.19 � 10�9 M) and it is comparable with thepreviously reported FRET-based Al3+ ion-selective uorescentsensor. In this regard it may be considered as a superiorFRET-based chemosensor for Al3+ ions in aqueous sol-ution.16b This probe may be used as a biomarker for theimaging of Al3+ ions in living cells.

Acknowledgements

Financial assistance from CSIR, New Delhi, India is gratefullyacknowledged. B. Sen thanks UGC, New Delhi, India for offeringhim a fellowship. We sincerely acknowledge Professor SamitaBasu and Mr Ajay Das, Chemical Science Division, SINP, Kol-kata for enabling us to use the TCSPC instrument and we alsothank Mr Samya Banerjee, IISC, Bangalore and Dr B. Mukher-jee, IISER, Kolkata for recording the 1H-NMR, 13C-NMR andESI–MS spectra.

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