AnalyticalMethods

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School of Chemistry, Madurai Kamaraj Un

dcmku123@gmail.com; Fax: +91 452 24591

† Electronic supplementary informa10.1039/c3ay42057d

Cite this: Anal. Methods, 2014, 6, 2343

Received 19th November 2013Accepted 9th January 2014

DOI: 10.1039/c3ay42057d

www.rsc.org/methods

This journal is © The Royal Society of C

Pyrene based selective–ratiometric fluorescentsensing of zinc and pyrophosphate ions†

Gandhi Sivaraman, Thangaraj Anand and Duraisamy Chellappa*

A simple pyrene-based receptor has been synthesized. It shows a colorimetric and ratiometric fluorescent

detection of zinc in aqueous solution. The ratiometric fluorescent change has been accounted to the

conformational change of the probe. The ratiometric fluorescence change has been further supported

by DFT/TDDFT calculations. The PDP-1 + Zn2+ complex is also further successfully utilized for

ratiometric fluorescence detection of pyrophosphate anions in buffered aqueous solution. The

applicability of these probes for real sample analysis for the detection of Zn(II) and pyrophosphate ions

has been also studied.

Introduction

Zinc ion (Zn2+) is the second most abundant transition metalion in the human body aer iron. Many studies have revealedthat Zn2+ is involved in a number of biological processes, suchas brain function and pathology, gene transcription, immunefunction, and mammalian reproduction.1 Increased levels ofzinc are known to play a key role in potential neurologicaldisorders such as Alzheimer's and Parkinson's disease2 howevera detailed understanding of the role played by the Zn(II) ion incell homeostasis, signal transduction, and translocation stillremains a challenge. Within the past decade, pioneeringresearch in PET,3 ICT4 and FRET5 based uorescent chemo-sensors have made signicant progress in understanding thebioinorganic chemistry and coordination properties of Zn(II).The development of highly selective and ratiometric uorescentchemosensors for Zn2+ ions is still an important task,6–9

although some uorescent sensors for Zn2+ by the ratio of theemission intensity changes at two wavelengths have beenreported in the literature.10–13 For the construction of efficientoptical chemosensors, uorescence is particularly attractive.Pyrene subunits are widely employed due to their well-knownphotophysical properties as well as their characteristic andenvironment-sensitive monomer or excimer emissions. Partic-ularly, the introduction of two pyrene moieties can be situatedclosely enough to yield excimer emission. Upon coordinationwith a specic guest ion, the resulting compound could be ne-tuned to yield monomer and/or excimer emissions dependingon the orientation of the two pyrene moieties.14–18 Furthermore,only a few ratiometric uorescent probes with pyrene for Zn2+

iversity, Madurai-625021, India. E-mail:

81; Tel: +91 452 2456614

tion (ESI) available. See DOI:

hemistry 2014

have been found in the literature, involving multistepsynthesis.19–21 Phosphates act as substrates or inhibitors byreversibly coordinating to Zn(II) ions in the enzymes. Suchcompounds were initially designed as small molecule models ofenzyme active sites.22 They play numerous important roles incell biology.22 ATP is also known as the ‘energy currency’ ofliving cells.23 Phosphates are involved in the modulation of ionchannels,24 and apart from these it is also involved in DNAreplication and transcription.25,26 The enhanced binding abilityof phosphates with zinc means that the utilization of a zinc ioncomplex as a binding site for phosphates has become the mostpopular approach.27,28 Due to the simplicity and high sensitivityof uorescence methods when compared to other methods,they are very attractive for the detection of phosphate ions.29

Despite this, there have been few reports of effective ratiometricuorescent chemosensors for phosphate ions.30 Herein wereport a simple, convenient, rapid single step synthesis of aprobe which shows selective, ratiometric, uorescent switchingbehaviour towards zinc ions by the excimer/monomer emissionof the pyrene moiety. During the addition of phosphate anionsthe PDP-1 + Zn2+ complex showed a ratiometric uorescencechange in buffered aqueous solution.

Results and discussion

PDP-1 was synthesized by a single step reaction between1-pyrene carboxaldehyde and cystamine dihydrochloride in thepresence of triethylamine (Scheme 1). PDP-1 was characterizedby NMR and mass spectral techniques (Fig. S1–S3 in ESI†).

Spectroscopic measurements were carried out in aqueousHEPES buffer (10 mm HEPES pH 7.4) containing 70% CH3CN.The absorption spectrum of PDP-1 displays the characteristicspectral features of pyrene, with absorption bands at 278, 332 and348 nm. Upon addition of Zn2+ ions to the PDP-1 there wassignicant change in the absorption bands around 390–400 nm

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Scheme 1 Synthesis of PDP-1.

Fig. 1 UV-vis absorption spectra of PDP-1 (10 mM) in the presence ofFe3+, Fe2+, Ag+, Ca2+, Cd2+, Co2+, Cu2+, Hg2+, K+, Mg2+, Mn2+, Na+,Ni2+, Pb2+ and Zn2+ (10 mM) in aqueous HEPES buffer (10 mM HEPES,pH ¼ 7.4, H2O–CH3CN ¼ 30 : 70).

Fig. 2 Fluorescence spectra of PDP-1 in the presence of Fe3+, Fe2+,Ag+, Ca2+, Cd2+, Co2+, Cu2+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, Pb2+ andZn2+ (10 mM) in aqueous HEPES buffer (10 mm HEPES pH 7.4) con-taining H2O–CH3CN (30 : 70). Excitation was performed at 350 nm.

Fig. 3 Fluorescence emission spectrum of PDP-1 (1 mM) upon additionof Zn2+ (0–1 equiv.) in aqueous HEPES (10 mMHEPES, pH¼ 7.4, H2O–CH3CN ¼ 30 : 70).

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(Fig. 1). This is also responsible for the perceptible deepening ofthe color frompale yellow toadarker shade.This isdirectly relatedto an increase in the molecular weight of chromogens that is dueto the formation of the PDP-1 + Zn2+ complex. The absorptionspectrawithseveralmetal cations (Fe3+, Fe2+, Ag+,Ca2+,Cd2+,Co2+,Cu2+, Hg2+, K+, Mg2+, Mn2+, Na+, Ni2+, and Pb2+) did not produceany appreciable change in the UV-vis spectra31 (Fig. 1).

The uorescence spectra of PDP-1 (Fig. 2) shows a strongexcimer emission at 515 nm and a weak monomer emission at390 and 412 nm (excitation wavelength 340 nm), with anintensity ratio of monomer to excimer emission (IM/IE z I412/I515) ¼ 0.21. The formation of an excimer band at 515 nmindicates a strong face to face p–p stacking between the twopyrene units. The incremental addition of zinc ions leads to thecomplete disappearance of the excimer band at 515 nm and theconcomitant increase in the uorescence intensity at 412 nm(Fig. 3). In PDP-1, the ratio of monomer to excimer emission isbarely changed with the change in the concentration, indicatingthat the excimer emission results from an intramolecular exci-mer but not from intermolecular interactions. The green uo-rescence of PDP-1 is attributed to the stacked conformation oftwo pyrene units; whereas the change in the uorescence aeraddition of Zn2+ is due to the conformational change fromstacked pyrene into the non-stacked conformation. There is a

2344 | Anal. Methods, 2014, 6, 2343–2348

very weak change in the emission band at 475 nm which may bedue to the possibility of intermolecular excimer formation inHEPES-buffered acetonitrile solution. Upon addition of Zn2+ tothe PDP-1 solution, the emission intensities at around 374–400nm increased signicantly, but the peak at 515 nm blue shiedto 470 nm (Fig. 2) and the blue shi is attributed to the rigidityof the pyrene conformers. To study the selectivity of PDP-1 forZn2+, its uorescent properties in the presence of variouscations were examined in HEPES buffer. The biologicallysignicant metal ions, such as Ag+, Ba2+, Ca2+, Cd2+, Co2+, Cu2+,Fe3+, Fe2+, Hg2+, K+, Mg2+, Na+, Ni2+ and Pb2+, did not produceany change in the uorescence behaviour of PDP-1 (Fig. S6 inESI†). The interference of other metal ions (Ag+, Ba2+, Ca2+,Cd2+, Co2+, Cu2+, Fe3+, Fe2+, Hg2+, K+, Mg2+, Na+, Ni2+ and Pb2+)in the detection of Zn(II) was studied by competition experi-ments using mixed solutions containing equimolar solution ofZn(II) and the solution of PDP-1 indicates that the metal ionsdoes not interfere the detection of zinc(II) ions (Fig. 4). In order

This journal is © The Royal Society of Chemistry 2014

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to understand the binding stoichiometry of Zn2+ with PDP-1,the plot was constructed using Job's continuous variationmethod by measuring the uorescence intensity ratios atdifferent mole fraction of Zn2+.

The Figure shows a maximum at the 0.5 mol fraction of Zn2+,indicating the formation of a 1 : 1 complex between PDP-1 andthe Zn2+ ion (Fig. S7 in ESI†). This was further supported by thepeak at m/z 691.25 in the ESI-MS spectrum corresponding tomolecular weight of [PDP-1 + Zn2+ + MeOH + H2O � H+] (Fig. S4in ESI†). There is a good linear relationship between the ratioof excimer and monomer uorescence intensity andthe concentration of Zn2+. The detection limit was found to be8.548 � 10�8 M, (Fig. S9 in ESI†) which was sufficiently low forthe detection of Zn2+ in many chemical and biological systems.The association constant (Ka) of PDP-1 with Zn2+ was deter-mined by using nonlinear least-squares t analysis32 of the ratioof the emission intensity at selected wavelengths in bufferedsolutions. It was found to be 4.4 � 104 mol L�1. The 1H NMRspectrum Of PDP-1 in the presence of Zn(II) in DMSO-d6 wasmeasured (Fig. S8 in ESI†). Aer the addition of zinc ions, mostof the protons of the pyrene rings shied downeld and themethylene protons were split into four different signals. Thisclearly signies the breaking of the stacked pyrene rings and thechange in the conformation aer complexation of zinc ions.33

The inuence of pH for the uorescence of PDP-1 was studied.At acidic pH (pH 1–5), the complete disappearance of theemission band at 515 nm with a sharp increase in emission at435 nm clearly indicates the disturbance of p–p stacking,whereas in neutral and basic pH there is no appreciable changein uorescence of PDP-1.

The PDP-1 + Zn2+ complex showeduorescence bandmaximaat 412 and 470 nm.During the addition of pyrophosphate ions tothe solution of thePDP-1 +Zn2+ complex inHEPESbuffer (10mMHEPES, pH ¼ 7.4, H2O–CH3CN ¼ 30 : 70) the emission maxima

Fig. 4 Fluorescence response (I/I0) of PDP-1 (10 mM) in CH3CN–H2O(30 : 70 v/v) to 10 mMof various testedmetal ions (green bar) and to themixture of 10 mM of tested metal ions with 10 mM Zn2+ ion (red bar).

This journal is © The Royal Society of Chemistry 2014

at 470 nm completely disappeared, and a new emission band at515 nm emerged.

Addition of pyrophosphate resulted in the change of uo-rescence from blue to green. Regarding the selectivity of thePDP-1 + Zn2+ complex among anions such as PO4

3�, F�, Cl�,Br�, I�, NO3

�, NO2�, OAc�, PF6

�, ClO4�, HPO4

2� and P2O74�,

the pyrophosphate anion alone shows a uorescence change(Fig. 5–7). The uorescence change aer the addition of pyro-phosphate ion with PDP-1 + Zn2+ complex can be accounted forby the strong interaction of pyrophosphate ion with Zn2+. Theexchange of Zn(II) in the PDP-1 + Zn2+ complex for the pyro-phosphate ion leaves the probe PDP-1 in solution. This wasfurther conrmed by ESI-MS analysis aer addition of pyro-phosphate ion with PDP-1 + Zn2+ complex showing the peak atm/z 577.43 (PDP-1 + H+) (Fig. S5 in ESI†).

DFT calculations

We also performed DFT and time-dependent DFT by theGaussian 03 program34 to make clear the changes in the elec-tronic properties. As we know, the highest occupied molecularorbital (HOMO)–lowest unoccupied molecular orbital (LUMO)gap and the electronic distributions may be used to clarify thechanges in the uorescent properties with metal cation coor-dination and detect the ratiometric response to metal cations.35

PDP-1 and PDP-1 + Zn2+ structures were optimised using theB3LYP/6-31G, LANL2DZ methods, respectively. The optimizedgeometries are shown in Fig. 8. The optimised geometries showthat the distance between the two pyrene units in PDP-1 is 4.537A (C31–C12). This shows clearly the possibility of p–p stackingbetween the pyrene rings. In PDP-1 + Zn2+ the distance changesto 10.230 A (C31–C12); there is no possibility of effective p–p

stacking in the pyrene units.In order to assess the practical utility of the probe PDP-1 and

PDP-1 + Zn(II) ensemble for Zn(II) and pyrophosphate ions

Fig. 5 Fluorescence spectra of PDP-1 + Zn2+ (1 � 10�5 mol L�1) in thepresence of PO43�, F�, Cl�, Br�, I�, NO3

�, PF6�, ClO4

�, HPO42� and

P2O74�, (10 mM) in aqueous HEPES buffer (10 mm HEPES pH 7.4)

containing CH3CN–H2O (30 : 70). Excitation was performed at 350nm.

Anal. Methods, 2014, 6, 2343–2348 | 2345

Fig. 6 Fluorescence emission spectrum of PDP-1 + Zn2+ (1 � 10�5

mol L�1) upon addition of pyrophosphate ions (0–100 equiv.) inaqueous HEPES (10 mM HEPES, pH ¼ 7.4, H2O–CH3CN ¼ 30 : 70).

Fig. 7 Bar chart representation of fluorescence response of PDP-1 +Zn2+ (1 � 10�5 mol L�1) in the presence of PO43�, F�, Cl�, Br�, I�,NO3

�, PF6�, ClO4

�, HPO42� and P2O7

4�, (10 mM) in aqueous HEPESbuffer (10 mm HEPES pH 7.4) containing CH3CN–H2O (30 : 70).Excitation was performed at 350 nm.

Fig. 8 Energy optimized geometry of PDP-1 and PDP-Zn2+ by B3LYP/6-31G and LANL2DZ methods, respectively.

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detection, respectively, in drinking water, tap water and riverwater samples further work was carried out. In order to removeinsoluble substances, the water samples were rst ltered. Allthe samples, with or without addition of zinc and pyrophos-phate ions, at different concentration levels of 0, 50 and 100 mgwere analysed by the probes PDP-1 and the PDP-1 + Zn(II)ensemble (Tables S1 and S2†). The experimental results showthat PDP-1 and the PDP-1 + Zn(II) ensemble is able to measurethe concentrations of zinc and pyrophosphate ions, respec-tively. These results indicate the suitability and applicability ofthe probe PDP-1 for the detection of Zn2+ and pyrophosphateions from real samples.

To account for the variation of uorescence intensity uponthe addition of Zn2+ to PDP-1, we investigated the frontiermolecular orbitals of PDP-1 and its Zn2+ complexes. In PDP-1the HOMO shows that most electron density resides in one ofthe pyrene moieties and the LUMO on the imine-incorporatingdisulphide (spacer) unit, whereas in PDP-1 + Zn2+ HOMOresides on the sulfur unit, and the LUMO on both of the pyreneunits (Fig. 9).

The HOMO and LUMO of PDP + Zn2+ show a remarkabledifference from the PDP-1. The excited states of PDP-1 can bestabilized by p–p interactions between the pyrene units,whereas in PDP + Zn2+ this is not possible. This is further evi-denced by the HOMO–LUMO transition in PDP-1. In the case ofPDP + Zn2+, the HOMO � 1–LUMO + 1 transition shows thepresence of p–p interactions. It is clear that the excimer uo-rescence from PDP-1 is due to the existence of T-shaped stackedpyrene moieties. The emission band at 515 nm originates fromthe static excimer of the pyrene units. These results clearlysuggest that the ratiometric uorescence change is due to aconformational change from the T-shaped, stacked pyrene tothe non-stacked form aer the appendage of zinc ions. In orderto prove the conformational change of PDP-1 upon binding toZn2+ but no other cations (Fig. S11 in ESI†), DFT calculationswere carried out with PDP + Cu(II) ions. The optimized geometry

Fig. 9 Frontier molecular orbital diagram of PDP-1 and PDP-1 + Zn2+.

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of PDP + Cu(II) showed the distance between the two pyrenerings was 5.85 A whereas in the case of PDP-Zn2+ the inter-pyrene ring distance was 10.23 A. Hence PDP-1 showed theuorescence change during the addition of zinc ions by thechange in distance between the two pyrene rings.

Conclusions

In summary, we have designed a new pyrene derivative andsynthesised it by a convenient, single-step reaction. It shows aratiometric uorescent behaviour due to excimer/monomerswitching towards Zn2+ ions and pyrophosphate in aqueousacetonitrile solution. This system exhibits a novel detection ofzinc ions and pyrophosphate through interchange of the exci-mer emission and monomer emission of pyrene.

Materials and methodsSynthesis of PDP-1

0.25 g (1.085 mmol) pyrene-1-carboxaldehyde is mixed with0.120 g (0.5425 mmol) of cystamine hydrochloride in ethanol. Itwas reuxed in the presence of triethylamine 0.35 g (1.5 mmol)for 8 hours under nitrogen atmosphere. On cooling to roomtemperature the desired compound was precipitated; aerrecrystallization with dichloromethane PDP-1 was obtained aspale yellow solid. Yield: 79%, (Mp: 196 �C) 1H-NMR (CDCl3, 300MHz): 9.303 (s, 2H), 8.741–8.710 (d, 2H, J ¼ 9 Hz), 8.510–8.401(dd, 2H, J ¼ 3.3 Hz) 8.308–8.287 (m, 6H), 8.119–7.902 (m, 8H),4.269–4.225 (t, 4H, J¼ 6.6 Hz), 3.290–3.263 (t, 4H, J¼ 6.6 Hz).13C-NMR (CDCl3-75 MHz): 161.45, 132.80, 131.07, 130.40,129.76, 128.57, 128.45, 128.19, 127.26, 126.20, 125.942, 125.74,125.63, 124.78, 124.65, 124.43, 122.37, 61.07, 40.04. ESI-MS: m/z577.42 [(M + H)+] calculated: (576.72). C38H28N2S2: calculatedC, 79.13; H, 4.89; N, 4.86. Found: C, 78.96; H, 4.49; N, 4.72%.

Synthesis of PDP-1 + Zn(II) complex

2 mmol of PDP-1 in chloroform solution was added to theethanolic solution of ZnCl2 (2.5 mmol). During the addition ofzinc ions the colour turned to dark yellow; stirring wascontinued for 30 minutes. Evaporation of the solvent yielded adark yellow solid. 1H-NMR (DMSO-d6, 300 MHz): 9.427 (s, 2H),9.271–9.255 (m, 2H), 8.761–8.730, 8.499 (m, 16H), 7.275–7.260(t, 3H), 4.183–4.144 (t, 2H), 3.070–3.051 (t, 2H), 3.306–3.284 (t,2H), 3.481–3.393 (t, 2H). Molecular formula: C38H28Cl2N2S2Zn.

Acknowledgements

G. S. thanks UGC for a senior research fellowship. G. S., T. A.and D. C. acknowledge DST-IRHPA, FIST and PURSE forinstrumental facilities and funding. We thank Professor S.Krishnaswamy, School of Biotechnology for providing access tothe spectrouorimeter facility.

Notes and references

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