Journal of Luminescence 143 (2013) 374–381

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Journal of Luminescence

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journal homepage: www.elsevier.com/locate/jlumin

Förster Resonance Energy Transfer (FRET) from Triton X-100to 4-benzothiazol-2-yl-phenol: Varying FRET efficiency with CMCof the donor (Triton X-100)

Bijan Kumar Paul a,n,1, Aniruddha Ganguly a, Saswati Karmakar b, Nikhil Guchhait a,n

a Department of Chemistry, University of Calcutta, 92 A.P.C. Road, Calcutta 700009, Indiab Department of Chemistry, Sree Chaitanya College, Habra, North 24 Parganas, India

a r t i c l e i n f o

Article history:Received 5 November 2012Received in revised form11 April 2013Accepted 3 May 2013Available online 23 May 2013

Keywords:Förster resonance energy transferTriton X-100CMC4-benzothiazol-2-yl-phenolEnergy transfer efficiencyLong-range dipole–dipole interaction

13/$ - see front matter & 2013 Elsevier B.V. Ax.doi.org/10.1016/j.jlumin.2013.05.015

viations: FRET, Förster Resonance Energy Tra-benzothiazole-2-yl-phenol; CMC, Critical MicR, Nuclear Magnetic Resonance; TLC, Thin Larrelated Single Photon Counting; LED, Light Eyclohexane; ACN, Acetonitrile; THF, Tetrahydesponding authors. Tel.: +91 33 2350 8386; faail addresses: bijan.paul.chem.cu@gmail.com (ait@yahoo.com (N. Guchhait).esent address: Department of Chemistry ando, Boulder, CO 80309, United States.

a b s t r a c t

A heterocyclic compound viz., 4-benzothiazol-2-yl-phenol (4B2YP) has been synthesized and itsphotophysics have been examined through steady-state absorption, emission and time resolved emissionspectroscopic techniques, in brief. Then 4B2YP has been exploited as an acceptor in the FörsterResonance Energy Transfer (FRET) process from photoexcited benzene aromatic nucleus of TritonX-100 (TX-100) surfactant. Dependence of the energy transfer efficiency on the donor concentrationwith respect to its critical micelle concentration (CMC) is clearly reflected in the study. High values ofStern–Volmer constant (KSV) for quenching of the donor fluorescence in the presence of the acceptorsuggest the operation of long-range dipole–dipole interaction in the course of energy transfer process,while the inference is aptly supported from time resolved fluorescence decay results. Experimentalresults show maximum FRET efficiency at the CMC of the donor (TX-100).

& 2013 Elsevier B.V. All rights reserved.

1. Introduction

Almost six decades have passed since the technique of FörsterResonance Energy Transfer (FRET) was first brought to light [1],and while having gained the significant nick name “SpectroscopicRuler”, it still continues to fascinate the researchers from variousfields not only because of its elating fundamental photophysics butalso due to the vast range of applications. FRET is unique in itscapacity to provide spatial measurements and to detect molecularcomplexes over distances from 10 to 100 Å with a satisfactorydegree of accuracy which makes this technique to be enormouslyexploited for studying various biological [2–8] and bichromophoricmolecular systems [9].

FRET is primarily described as a distance dependent electro-dynamic phenomenon that involves the interaction between two

ll rights reserved.

nsfer; TX-100, Triton X-100;ellar Concentration; IR, Infra-yer Chromatography; TCSPC,mitting Diode; MCH,rofuranx: +91 33 2351 9755.B.K. Paul),

Biochemistry, University of

chromophores in different electronically excited states.

Dn þ A-Dþ An

here Dn is the excited donor chromophore that transfers thephoton to the acceptor chromophore A in the ground-state andthereby extinguishes the emission of photon from the donormolecular system [1,4,10,11]. The rate of excitation energy transferfor the above process depends on: (i) quantum yield of Dn emission,(ii) the extent of overlap between donor emission spectrum andacceptor absorption spectrum, (iii) light absorbing ability of A, and(iv) relative orientation of the donor and acceptor transition dipoles.An elegant theory on the mechanism of FRET was first put forwardby Förster [1]. Later on some theoretical studies focusing on thelimitation or validity of Förster theory have also appeared [12–14].

Energy transfer phenomena have profuse application in energyconversion process like photosensitization, an ever persuasiveexample of which being photosynthesis [15]. The technique of FREThas been immensely employed in almost all applications of fluor-escence spectroscopy, including medical diagnostics, DNA analysis,optical imaging and so forth [4]. Also FRET occupies a commendableposition in biochemical and biophysical research because of itscompetence in studying protein folding [16], measurement of thedistances between fluorescent tags in biological macromolecules,e.g., protein [2,17]. Investigation of kinetics of conformationalchanges in nucleic acid by time resolved FRET [18], application ofFRET in photodynamic therapy in cancer treatment [4,10],

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381 375

determination of the proximity of a guest molecule to tryptophanmoiety in a proteinous environment [5–8,19] are few examples ofthe enormously important applications of this technique. Theacceptor molecule chosen in the present work is composed of abenzothiazole nucleus. Recent time has witnessed a burgeoningthrust of interest surrounding benzothiazole derivatives in bio-chemical and medicinal research avenues. A large number oftherapeutic agents are synthesized with the help of benzothiazolenucleus. During recent years there have been some interestingdevelopments in the biological activities of benzothiazole derivatives.These compounds have special significance in the field of medicinalchemistry due to their remarkable pharmacological potentialities.A wide variety of benzothiazole compounds has been shown to beprospective candidates in analgesic, anthelmintic, antibacterial, antic-onvulsant, antifungal, antimicrobial activities and so forth [20–22].

Owing to such great potential and vast arena of applications ofFRET, quite a large volume of work has been devoted to the study ofdifferent aspects of this process in organized environments formed bysurfactants i.e., micelles and reverse micelles [23–26]. Most of thesestudies are found to restrict their focus in taking extrinsic donor andacceptor molecules solubilized in these environments. However, theapplicability of the surfactant molecule itself as a participatingcounterpart in the FRET process has rather remained only sporadicallyvisited in the literature. The present study is designed to cast light inthis perspective. Use of non-ionic surfactant, Triton X-100 (TX-100),rules out any possibility of electrostatic interaction between the donorand the acceptor molecules in the course of energy transfer process.The acceptor unit, 4-benzothiazole-2-yl-phenol (4B2YP) is also aneutral molecule. The present work covers premicellar, micellar andpostmicellar environments in order to follow the energy transferprocess as a function of micellization on the donor counterpart. Thusapart from the spectroscopic characterization of the pharmacologicallyimportant benzothiazole derivative (4B2YP), its spectroscopic signa-tures during excitation via resonance energy transfer from TX-100 in abiomimicking microheterogeneous micellar environment is alsoexplored in the present program. We are optimistic that the presentstudy will enrich the understanding of the photophysics of suchpharmaceutically important benzothiazole compounds, in additionto throwing light on the possibility and prospect of their implementa-tion as potent biological photosensitizer. Moreover, the present studyalso draws relevance to the context of the promising prospects oforganized assemblies, such as micelles, on biological, photochemical,and photophysical processes [4,23–26]. Micelles are among suchpromising organized assemblies that have formed the nucleus ofmany-faceted research activities over the past few years. Micelles arehighly cooperative, thermodynamically stable, dynamic nanostruc-tures formed out of amphiphilic surfactant molecules above a criticalconcentration (precisely a narrow concentration range) known as thecritical micellar concentration (CMC). The interactive nature of innu-merous fluorophores in micellar systems has been quite extensivelyaddressed in the literature mainly with a view to the ability of suchdynamic nanostructures to mimic biological membranes in a muchsimpler model, given that the general principle underlying theformation of micelles (that is, the hydrophobic effect) is, in essence,common to such related assemblies like liposomes, and biologicalmembranes [4,23–26].

S

N

OH

O(CH2CH2O)n H (n = 9.5)

Scheme 1. Schematic structures of (a) TX-100 and (b) 4B2YP. .

2. Experimental

2.1. Synthesis and characterization of 4-benzothiazol-2-yl-phenol(4B2YP)

For the synthesis of 4-benzothiazol-2-yl-phenol (4B2YP) (cf.Scheme 1) [27], a mixture of 2-aminothiophenol, 4-hydroxybenzaldehyde and Dowex 50W (10 mol%) were stirred in water at 70 1C

for 10 h. The reaction mixture was cooled, diluted with ethylacetate, filtered to remove Dowex 50W and extracted the aqueouspart with ethyl acetate. The combined organic layers were washedwith brine, dried over anhydrous Na2SO4 and concentrated invacuo. The crude product was purified by crystallization from ethylacetate and petroleum ether (60–80 1C) to obtain the title com-pound. M.p.: 225–226 1C (observed); 227–228 1C (reported);

IR (KBr): 3436, 2366, 1601, 1479, 1308, 1256, 1171, 1025, 832 and760 cm−1;

1H-NMR (DMSO-d6, 300 MHz) δ: 10.23 (brs, 1H, OH), 8.07 (brd,J¼7.9 Hz, 1H, C4–H), 7.98 (brd, J¼8.1 Hz, 1H. C7–H), 7.94 (d,J¼8.6 Hz, 2H, C2–H and C6–H), 7.50 (brt, J¼7.1 Hz, 1H, C5–H), 7.40(brt, J¼7.6 Hz, 1H, C6–H), 6.94 (d, J¼8.6 Hz, 2H, C3–H and C5–H);13C NMR (DMSO-d6, 75 MHz), δ: 167.9, 161.0, 154.2, 134.6, 129.5,126.9, 125.4, 124.5, 122.8, 122.6, 116.6 (numbering of atomsaccording to Scheme 1).

For spectroscopic measurements the compound 4B2YP wasrecrystallized from EtOH before use and the purity was establishedon TLC plate.

2.2. Materials

Spectroscopic grade solvents such as methylcyclohexane(MCH), acetonitrile (ACN), tetrahydrofuran (THF), iso-prpanol(iPrOH), methanol (MeOH) were purchased from Spectrochem(India) and were used after proper distillation. Triton X-100(cf. Scheme 1) was purchased from Spectrochem (India) and usedas received. KBr and DMSO-d6 for IR and NMR spectral measure-ments, as mentioned above, were purchased from Sigma ChemicalCo., USA and used as received. Triply distilled deionized water wasused throughout the study. Freshly prepared micellar solutionswere only used for experiments to avoid aging of the micelles dueto degradation of the surfactant [28].

All solvents appeared visually transparent and the purity wasalso tested by running their fluorescence spectra in the studiedwavelength range within the same experimental window.All experiments have been carried out at an ambient temperature(∼27 1C).

In order to establish the purity of TX-100 used in the presentstudy the critical micellar concentration (CMC) determinationmethod has been exploited since the CMC of a surfactant is knownto be sensitive to the presence of impurities [29]. Firstly, a conducto-metric titration has been performed with TX-100/water binarysolution as a function of TX-100 concentration in which the variationof conductivity of water has been observed with increasing TX-100concentration. As seen in Fig. 1a the conductivity of the mediumincreases with added TX-100 surfactant and when the concentrationof the surfactant is high (CMC or higher) it favors self-association ofTX-100 molecules to form the micellar units resulting in changes inthe observed conductivity of the TX-100/water binary system.Consequently, the ‘conductivity–surfactant concentration’ plot allowsthe determination of CMC of the surfactant (TX-100) throughintersection of the two straight lines in Fig. 1a. The as-determinedCMC of TX-100, i.e.,∼0.20 mM is found to be in excellent juxtaposition

Fig. 1. (a) Overlap between emission spectrum of TX-100 (dashed line) and absorption spectrum of 4B2YP (solid line) in aqueous medium. (b) Representative emissionprofile of TX-100 ([TX-100]¼0.2 mM) with incremental addition of 4B2YP (λex¼270 nm). Curves (i) ® (x) correspond to [4B2YP]¼0, 0.12, 0.27, 0.41, 0.54, 0.81, 0.95, 1.10, 1.22,1.35 μM. Inset shows the fluorescence excitation profile monitored at λem¼377 nm in the presence of the donor (TX-100) and the acceptor (4B2YP).

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381376

with literature reports [29]. As a further test of the CMC of theTX-100 surfactant the fluorometric method has been employed inwhich 4B2YP functions as the extrinsic probe molecule. The variationof fluorescence intensity of the probe in aqueous phase has beenmonitored as a function of the concentration of TX-100 added.The break-point, Fig. 1b, in the trend of variation of fluorescenceintensity of the probe (4B2YP) leads to determination of CMC of thesurfactant (TX-100) and the result (∼0.23 mM) is strikingly similar tothat obtained from the probe-independent conductometric titrationmethod. This finding also advocates for the fact that addition of theextrinsic probe (4B2YP) can hardly perturb the micellar propertieswithin the present experimental window. However, this postulate isfurther substantiated from the result that the hydrodynamic dia-meter of the micellar unit as obtained from dynamic light scatteringmeasurement is only nominally influenced by the addition of themaximum concentration of the probe molecule used in subsequentexperiments, Fig. 1c (hydrodynamic diameter of TX-100 micelle,dh¼8.572 nm, [TX-100]¼0.20 mM [29]).

2.3. Instrumentations and methods

2.3.1. Steady-state spectral measurementsThe absorption and emission spectra were recorded on

a Hitachi UV–vis U-3501 spectrophotometer and a Perkin-ElmerLS-50B fluorimeter, respectively. All spectra have been appropri-ately background subtracted.

2.3.2. Time resolved fluorescence decay measurementThe time resolved fluorescence decays were acquired on

FluoroCube-01-NL spectrometer based on the time-correlatedsingle photon counting (TCSPC) technique using a laser diode at375 nm as the light source for 4B2YP and during analysis of FRET

the donor fluorescence (phenyl moiety of TX-100) was triggeredby a nanoLED at 290 nm and the signals were collected at themagic angle of 54.71 to eliminate the contribution from anisotropydecay [4]. The decays were deconvoluted and analyzed on DAS-6decay analysis software [4].

From the deconvoluted fluorescence lifetime data the intensity-weighted mean (average) fluorescence lifetime (oτi04) wascalculated from the following equation [4]:

⟨τi0⟩¼∑αiτ

2i

∑αiτið1Þ

and amplitude-weighted mean (average) fluorescence lifetime(oτa04) was calculated using the following equation [4]:

⟨τa0⟩¼∑aiτi ð2Þ

in which αi is the relative amplitude corresponding to the ith decaycomponent having the characteristic decay time constant τi. Theacceptability of the fits was judged from the χ2 criterion and theresidual of the fitted functions to the actual data [4].

2.3.3. Dynamic light scatteringDynamic light scattering (DLS) measurements have been car-

ried out on a Malvern Nano-ZS instrument employing a 4 mWHe−Ne laser (λ¼632.8 nm) and equipped with a thermostattedsample chamber. The sample was poured into a DTS0112 lowvolume disposal sizing cuvette of 1.5 mL (path-length 1 cm). Theoperating procedure was programmed by the DTS software in afashion that there was an average of 25 runs, each run beingaveraged for 15 s, and then a particular hydrodynamic diameterand size distribution was extracted using the DTS software.

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381 377

2.3.4. Conductometric titrationThe conductivity measurements were performed on Systronics

304 Digital conductivity meter (employing platinum electrode)using triply distilled deionized water.

2.3.5. Fluorescence quantum yield calculationFluorescence quantum yield (Φf) was determined using Bovine

Serum Albumin as the secondary standard (λabs¼280 nm, Φf¼0.15in Tris–HCl buffer, pH¼7.03 [4,5]) in the following equation.[4–7,19]:

ΦS ¼ΦRAS

AR� ðAbsÞR

ðAbsÞS� η2S

η2Rð3Þ

where A terms denote the fluorescence area under the curve, “Abs”denote absorbance, n is the refractive index of the medium andΦ is the fluorescence quantum yield and subscripts “S” and “R”stand for denoting respective parameters for the studied sampleand reference respectively.

3. Results and discussion

3.1. Förster Resonance Energy Transfer (FRET) from Triton X-100to 4B2YP

Before going to the details of the present study it is necessary topresent an overview of the photophysical behavior of the synthe-sized compound (4B2YP), which has been used as the acceptorcounterpart during the FRET studies. Fig. 2a presents the absorp-tion spectra of 4B2YP recorded in organic solvents of varyingpolarities. In all the solvents assayed 4B2YP is found to exhibit aquite solvent polarity independent broad, structureless band at∼315 nm. In comparison to other similar studied systems [30] thisband is ascribed to the ππn transition of the chromophoric unit

Fig. 2. Representative (a) absorption, (b) emission (λex¼270 nm) and (c) excitation spectpurple; iPrOH: orange sphere, MeOH: −▼−, pink; Water: −▲−, green). (For interpretationversion of this article.)

in 4B2YP. Following photoexcitation at the corresponding λabsmax

in different solvents at room temperature, 4B2YP displays broademission band peaking at λem∼375 nmwhich too reveals negligiblesensitivity towards medium polarity (vide Fig. 2b). The emissionspectral properties thus seem to be attributable to the emissionfrom the locally excited-state of 4B2YP [5–7]. The absorption andemission spectral parameters are summarized in Table 1.The excitation spectra of 4B2YP monitored in various solventsare found to juxtapose reasonably well with the correspondingabsorption spectral parameters (cf. Fig. 2c and Table 1) indicatingthat the observed emission of the studied compound originatesthrough photoexcitation of the ground state species and thus inturn substantiates our previous inference on the purity of thecompound as judged from other methods e.g., melting pointdetection, observation from thin layered chromatography (videSection 2.1). Also for the sake of completeness the fluorescencedecay behavior of 4B2YP has been monitored in various bulksolvents. The typical time resolved decay profiles are depicted inFig. 3 with the respective fitting parameters being compiled inTable 2. However, the compound 4B2YP is found to exhibitcomplex fluorescence decay behavior in all the solvents assayedwhen a complicated triexponential decay function was required toadequately describe the data (cf. Table 2). It is also pertinent tonote that the overall fluorescence decay behavior of 4B2YP iscomprised of major contributions from ultrafast decay component(in ps time regime) with relatively shorter contribution froma slower decay component in the ns time regime. However, weare yet to specifically rationalize the origin of such complicateddecay behavior of the heterocyclic compound (4B2YP) even in bulkhomogeneous solvents, though it is reported in the literature formany other fluorophores also [31–33]. It is not unlikely to considerat this juncture that 4B2YP containing so many heteroatoms willobviously comprise of an unsymmetrical charge distribution overthe molecular framework which in turn might lead to unequalsolvation.

ral profiles of 4B2YP in different solvents (MCH: −○−, cyan; ACN: −■−, red; THF: −�−,of the references to color in this figure legend, the reader is referred to the web

Table 1Spectroscopic parameters of 4B2YP in various bulk solvents.

Solvent λabs (nm) λex (nm) λem (nm) Quantum yield (Φf)

MCH 308 300 365 0.011ACN 312 310 370 0.046THF 314 312 373 0.081iPrOH 319 316 377 0.321MeOH 316 314 377 0.124Water 313 315 388 0.024

Fig. 3. Typical time resolved fluorescence decay profiles of 4B2YP in various bulksolvents as specified in the figure legend. The sharp gray profile on the extreme leftrepresents the instrument response function (IRF).

Table 2Time resolved fluorescence decay parameters of 4B2YP in various bulk solvents.

Solvent τ1 (ps) τ2 (ps) τ3 (ns) α1(%)

α2(%)

α3(%)

χ2 ⟨τi0⟩(ns)

ACN 10.471.57 897743 2.8670.11 18 70 12 1.07 1.59iPrOH 14.979.39 821711 4.1670.06 33 45 22 1.06 3.19MCH 66.973.16 898712 4.4870.04 36 50 14 1.05 2.92MeOH 27.671.87 69476.4 3.8570.05 28 60 12 0.99 2.33THF 125711 84676.1 3.4270.06 13 80 7 1.00 1.49Water 93.371.95 431711 3.2170.06 38 43 19 1.02 2.46

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381378

As long as the absorption and emission spectral features of thedonor unit, TX-100 is concerned, they are well documented as thebenzene aromatic nucleus present in TX-100 absorbs appreciably at∼270 nm and yields a broad fluorescence profile with λem∼300 nm.The absorption profile of 4B2YP actually prompted us to couple themolecule with TX-100 to form a good pair of donor–acceptor systemfor FRET studies since 4B2YP absorbs negligibly at 270 nm andappreciably beyond 300 nm (λabs∼315 nm, cf. Fig. 2a). This is,indeed, evidenced by observing the extent of overlap betweendonor emission and acceptor absorption spectral profiles as illu-strated in Fig. 4a. In an aqueous solution of TX-100 (in pre-micellar,micellar and post-micellar environments), the incremental additionof 4B2YP is found to impart a progressive decrease of fluorescenceintensity of the benzene nucleus in TX-100 (λem∼300 nm) alongwith a concomitant increment of the same for 4B2YP (λem∼377 nm)with an isoemissive point at 350 nm (vide Fig. 4b). However, a closeperusal of the emission spectral profile of 4B2YP (cf. Fig. 4b) revealsthat the emission wavelength of 4B2YP in the presence of thesurfactant is somewhat blueshifted with respect to that in the bulkaqueous medium (λem∼388 nm in aqueous medium vs. λem∼377 nmin the presence of TX-100). This is, however, not surprising giventhe varied polarity of the bulk aqueous medium in the presence ofthe surfactant. A discernible blueshift in the emission wavelength ofthe fluorophore (4B2YP) in the presence of the surfactant unveils thesignature of reduced polarity in the vicinity of the fluorophore [34–36].

This is also evidenced on the lexicon of spectroscopic properties of4B2YP recorded in various bulk homogeneous solvents (cf. Table 1)which reveal a blueshift of the emission wavelength followinglowering of the medium polarity on moving from water(λem∼388 nm) to nonpolar solvent MCH (λem∼365 nm).

At this point it is imperative to establish that the fluorescenceof 4B2YP in the presence of TX-100 originates through FRET andnot from direct excitation of the fluorophore and the initialconfirmation for the operation of FRET in the present choice ofdonor–acceptor couple was derived from comparing the totalfluorescence (in terms of area under the fluorescence curve)coming from the mixtures of donor and acceptor to that fromthe blank experiments with the same concentration of theacceptor alone. For λex¼270 nm, the fluorescence yield of theacceptor is found to be greater in the presence of the donorcompared to that in absence of the latter. The inset of Fig. 4bshows that the fluorescence excitation profile monitored atλem∼377 nm (emission wavelength of 4B2YP in the presence ofthe donor) appears with distinct feature of excitation/absorptioncorresponding to the benzene nucleus of TX-100 (donor) apartfrom the transition corresponding to the ππn band of 4B2YP(acceptor). This observation in turn provides strong evidence forthe occurrence of FRET in the present case of study [4,11,23,37–39].Additionally, a clear indication of Förster type resonance energytransfer from TX-100 donor to 4B2YP acceptor is obtained fromthe excellent consistency with some earlier reports of Lakowicz[4], De et al. [23], Sengupta et al. [37,38], Das et al. [39] and ourown work [5–7,11].

It is pertinent to mention at this stage that the absorptionprofile of TX-100 when titrated with 4B2YP displays no additionalband other than the individual bands of TX-100 and 4B2YPimplying the inoperativeness of any ground-state complex forma-tion between the two concerned partners [22,39–41]. Similarly,fluorescence spectral profile of the mixture of the donor (TX-100)and acceptor (4B2YP) is also found to yield no new band at longerwavelength region and thereby negates the possibility of exciplexformation between the photo-excited donor and acceptor mole-cules. These observations offer further support for the transfer ofenergy from the photoexcited donor to the acceptor via nonradia-tive routes [4,11,39].

It is interesting to note that the present study demonstratesthat in order for FRET to occur with commendable efficiency therequired concentration of the acceptor (4B2YP) in the medium isquite low (cf. Fig. 4b). This can, however, be rationalized from highquantum yield of 4B2YP (cf. Table 1). Now, remembering theimperative role of FRET on the enormously significant subjects ofphotosensitization, energy conversion processes [15] etc., it seemslogical to think that the present investigation points towards theefficiency of the heterocyclic molecular system (4B2YP) togenerate light at longer wavelength through FRET with reasonableefficiency. At the same time, the energy transfer efficiency in thiscase leaves ample scope of tunability. Thus the application of theheterocyclic system, 4B2YP for study on the important issue ofFRET, photosensitization in different environments could beintriguing.

The quenching of the donor (TX-100) fluorescence intensity asa function of increasing concentration of the acceptor (4B2YP)under various conditions (viz., pre- and post-CMC conditions ofthe surfactant TX-100) was followed on the well-known Stern–Volmer relation:

I0I¼ 1þ KSV Q½ � ð4Þ

where I0 designates the original fluorescence intensity, I designatesthe quenched intensity, [Q] represents the molar concentration of thequencher (here 4B2YP) and KSV is the Stern–Volmer constant [4].

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381 379

Fig. 5a represents the Stern–Volmer plots for donor quenching underdifferent circumstances viz., pre- and post-CMC domains. Thelinearity of the plots in all the cases indicates the operation ofonly one type of quenching mechanism (dynamic or static) [4]. Asalready inferred from the aforementioned discussion that theabsence of any additional absorption band in the donor–acceptormixture rules out the probability of ground-state complex forma-tion, the actuating mechanism behind the observed fluorescencequenching could be an excited-state affair, which is rationalized inthe context of the energy transfer process. The Stern–Volmerquenching constant values (KSV) obtained from the slopes ofthe linear plots at different conditions are comprised in Table 3and the values are found to be in the order of magnitude higherthan that observed for a normal diffusion controlled quenchingprocess [4,11,39–41]. Also this observation is found to reasonablycomply with other similar studied systems [4,23,37–39]. Drawingon these studies, the present findings suggest that the dominantmechanism of fluorescence quenching (cf. Fig. 4b) is the resonanceenergy transfer through long-range dipole–dipole interactionrather than simple diffusion limited process between the exciteddonor and ground-state acceptor molecules.

Table 3Energy transfer efficiency (E) and KSV values at different concentrations of TX-100.

[TX-100]70.02 (mM) E (75%) (%) KSV �10−5 (M−1)

0.05 16.42 1.6370.080.1 26.22 2.3670.060.2 (CMC) 29.84 3.3370.070.5 19.24 1.8170.101.0 8.88 1.6070.15

3.1.1. Efficiency of energy transferOnce the occurrence of FRET between the chosen pair of

molecular systems have been established, it is pertinent toestimate the energy transfer efficiency (E) between the donor(TX-100) and the acceptor (4B2YP) counterparts under differentcircumstances viz., pre- and post-micellar conditions so that anidea can be derived about the variation of E as with micellization.

Fig. 4. Overlap between emission spectrum of TX-100 (dashed line) and absorption spectTX-100 ([TX-100] = 0.2 mM) with incremental addition of 4B2YP (λex¼270 nm). Curves (Inset shows the fluorescence excitation profile monitored at λem¼377 nm in the presen

Fig. 5. (a) Stern–Volmer plots for quenching of TX-100 fluorescence by 4B2YP in pre-m(0.5 mM; −�−, green) domains. (b) Variation of energy transfer efficiency (E in %) as a fuprofile reveals only a visual guide to the pattern of variation. (For interpretation of the refarticle.)

According to Förster theory [1,4] the efficiency of energy transferprocess (E) is given as [4]

E¼ R60

R60 þ r6

¼ 1−II0

ð5Þ

where I and I0 are the fluorescence intensities of the donor in thepresence and absence of the acceptor, respectively and R0 isthe critical energy transfer distance and r is the distance betweenthe donor and acceptor molecules. Table 3 collectively presents thevariation of E in different conditions for a given acceptor concen-tration ([4B2YP]¼1.35 μM) and reveals that the energy transferefficiency (E) attains its maximum value at CMC of TX-100 anddecreases at both pre- or post-micellar conditions forming anapproximately bell shaped profile as depicted in Fig. 5b.

An increase in the energy transfer efficiency in the micellarenvironment compared to that in the pre-micellar environmentcan be rationalized from the consideration of micellar aggregates.Before CMC, the surfactant molecules remain in rather a scatteredand unorganized pattern, so that attainment of proper orientation

rum of 4B2YP (solid line) in aqueous medium. (b) Representative emission profile ofi) ® (x) correspond to [4B2YP]¼0, 0.12, 0.27, 0.41, 0.54, 0.81, 0.95, 1.10, 1.22, 1.35 μM.ce of the donor (TX-100) and the acceptor (4B2YP).

icellar (0.1 mM; −○−, purple), micellar (0.2 mM; orange sphere) and post-micellarnction of donor (TX-100) concentration passing through the CMC value. The dottederences to color in this figure legend, the reader is referred to the web version of this

Table 4Time resolved fluorescence decay parameters of TX-100 (donor) in the presence ofvarious concentrations of 4B2YP (acceptor).

[4B2YP] (lM) τ1 (ns) τ2 (ns) α1 (%) α2 (%) χ2 ⟨τa0⟩ (ns)

0 2.2670.08 6.4570.01 23 77 0.99 5.490.12 1.7770.04 6.1970.01 36 64 1.00 4.60.27 1.5470.025 6.0170.009 47 53 1.03 3.910.41 1.3470.018 5.8470.01 56 44 1.03 3.320.54 1.2770.015 5.6970.011 62 38 1.05 2.950.81 1.1270.013 5.4670.011 68 32 1.08 2.510.95 1.0170.010 5.2770.011 72 28 1.05 2.201.10 0.9470.009 5.0870.011 75 25 1.00 1.971.35 0.8870.008 4.9870.012 77 23 1.06 1.82

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381380

between the donor and acceptor dipoles could be a bit difficult insuch circumstances, but this difficulty is overcome by formation ofmicellar aggregates which ensures a better organization of thedonor dipoles, as manifested in enhanced energy transferefficiency (cf. Table 3, Fig. 5b).

With an estimate of the energy transfer efficiency (E) and R0,the distance between the donor and acceptor dipoles (r) could becalculated. An uncertainty, however, will creep in the calculationdue to the orientation factor [1,4,11,19]. Additionally, since R0 isexpressed as [4]:

R0 ¼ 8:8� 10−25k2n−4ΦDJ ð6Þ

the determination of R0 and hence r will require the involvementof donor quantum yield (ΦD) which incorporates further uncer-tainty in the calculation in our present case due to remarkabledependence of the intrinsic fluorescence quantum yield of TX-100on the concentration of the surfactant in the medium [42,43].Calculation of r with such uncertainty will thus hardly makea sense. However, an excellent review by Chandrasekhar [44]points towards the possibility of calculating the distance theore-tically from the dimensions and concentrations of the acceptormolecules using nearest neighbor distribution.

3.2. Time resolved fluorescence decay

Though the aforementioned discussions based on steady-statespectral data support the operation of FRET from TX-100 to 4B2YP,the most convincing evidence is, however, deduced from timeresolved fluorescence decay measurements. In order to deriveconclusive evidence for the occurrence of FRET from TX-100 to4B2YP, the fluorescence decay of the donor (TX-100) has beeninvestigated in the presence of increasing acceptor (4B2YP) con-centrations as displayed in Fig. 6 with the corresponding datebeing comprised in Table 4. As an illustrative example here, theconcentration of the donor has been kept at [TX-100]∼0.2 mM i.e.,the CMC of the surfactant. The fluorescence decay profile of thedonor (TX-100) in the absence of the acceptor (4B2YP) is found tobe adequately described by a biexponential function (vide Fig. 6,Table 4) [45] acquiescing to an amplitude-weighted average life-time ⟨τa0⟩¼5.45 ns (cf. Table 4). The acceptor (4B2YP)-inducedchanges in the fluorescence decay profile of TX-100 are found toconform to a gradual decrease of ⟨τa0⟩ as a function of 4B2YPconcentration reaching to 1.82 ns in the presence of 15 mM 4B2YP(cf. Table 4). In this context, the bimolecular rate constant for thequenching process (kq) has been estimated from the following

Fig. 6. Typical time resolved fluorescence decay profiles of the donor (TX-100) inthe presence of increasing concentration of the acceptor (4B2YP). Curves (i)-(ix)correspond to [4B2YP]¼0.0, 0.12, 0.27, 0.41, 0.54, 0.81, 0.95, 1.10, 1.35 mM. The sharpgray profile on the extreme left represents the instrument response function (IRF).

equation [4]:

kq ¼KSV

τ0ð7Þ

in which τ0 represents the fluorescence lifetime of TX-100 in theabsence of the quencher (here 4B2YP) and KSV designates theStern–Volmer quenching constant as obtained from steady-statefluorescence measurements (cf. Table 3, Fig. 5). The as-calculatedvalue of kq amounts to kq¼6.11�1013 M−1 S−1 using ⟨τa0⟩ (in caseof energy transfer phenomenon decrease of amplitude-weightedaverage lifetime (⟨τa0⟩) of the donor in the presence of the acceptorshould provide the signature for operation of FRET [4], henceemphasis is given on the parameter ⟨τa0⟩ for the analysis offluorescence lifetime in the context of FRET). These quenchingrate constants are several orders of magnitude higher than themaximum threshold for diffusion-controlled process as expectedfrom the Smoluchowski equation (kdiff¼8RT/3η) [46], and hencesuch unrealistically large quenching rate constant cannot berationalized from Stern–Volmer equation in the context of onlydynamic quenching process as the actuating mechanism. Further,the progressive decrease of fluorescence lifetime of the donor(TX-100) with increasing acceptor (4B2YP) concentrations (videTable 4) is consistent with the normal expectation that addition of4B2YP should lead to an enhancement in the energy transferprocess whence the fluorescence lifetime of TX-100 shoulddecrease.

4. Conclusion

A new heterocyclic molecular system viz., 4B2YP has beensynthesized and its photophysical behavior is investigated spectro-scopically. The compound 4B2YP is then employed as the acceptorunit in the study of FRET from TX-100 donor unit. A combinedapplication of steady state and time resolved fluorescence spectro-scopic techniques has been employed as the actuating tools tosubstantiate the occurrence of long-range dipole–dipole interac-tion in the context of energy transfer process. The study revealsthat the energy transfer efficiency is remarkably higher at CMCcompared to that at pre- and post-CMC domains of TX-100(donor). Also another important aspect of the phenomenon ofenergy transfer observed in the present work is its susceptibilitytowards environmental effects leading to the possibility of tuningthe energy transfer efficiency.

Acknowledgments

AG acknowledges CSIR, India for Junior Research Fellowship.This work is supported by Grants from DST, India (Project no. SR/S1/PC/26/2008), UPE Laser group and UGC, India (Project no.

B.K. Paul et al. / Journal of Luminescence 143 (2013) 374–381 381

PSW-194/11-12 (ERO)). The authors convey sincere thanks toDr. Chhanda Mukhopadhyay of Department of Chemistry of ourUniversity for her kind gift of the compound 4B2YP.

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