intracellular detection of toxic hypochlorite anion by ...rhodamine b (1 g, 2.88 mmol) in dry...

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July-G- p6.5

J. Indian Chem. Soc.,Vol. 94, July 2017, pp. 673-680

Intracellular detection of toxic hypochlorite anion by using a nontoxic rhodaminebased sensor

Dibyendu Saina, Shyamaprosad Goswami*a and Chitrangada Das Mukhopadhyayb

aDepartment of Chemistry, bDepartment of Centre for Healthcare Science & Technology,

Indian Institute of Engineering Science and Technology, Shibpur,

Howrah-711 103, West Bengal, India

E-mail : [email protected]

Manuscript received 21 April 2017, accepted 08 May 2017

Abstract : A simple bio-compatible 2,4-dinitrobenzene and rhodamine coupled probe was designed and synthe-sized for the detection of highly toxic hypochlorite ion with high selectivity and sensitivity. Practical applicabi-lity of the sensor was established through simple and fast naked-eye detection of ClO–. Absorption and emissionspectra proved the detection ability of the sensor towards ClO–. The non toxic behaviour of the sensor in livingcells increased its practical applicability and it was effectively applied in intracellular detection of toxic ClO– inliving cells.

Keywords : Hypochlorite sensor, Rhodamine hydrazide, naked-eye, intracellular detection, DFT computation.

Introduction

Hypochlorite anion (OCl–) is commonly used indaily life as a bleaching agent, disinfectant for drink-ing water, cooling-water treatment and cyanide treat-ment in the millimolar to micromolar concentrationrange1. However, high level of hypochlorite accumu-lation causes a lot of human diseases, such as cardio-vascular diseases, atherosclerosis, lung injury, neurondegeneration, kidney disease, arthritis, and cancer2,3.Therefore, the detection and precise determination ofHOCl in the biological systems is the new challengefor the researchers. Among different sensitive and se-lective methods, fluorescent probes are superior in thisrespect due to their high sensitivity and fine resolutioncapability4. Moreover, colorimetric chemodosimetersare widely used for the detection of target analytesquantitatively by the naked-eye as well as in vivo cellimaging technique5. Previously, some useful fluores-cent sensors have been reported for the detection ofHOCl6,7. But, most of the reported probes for hy-pochlorite suffer from poor selectivity and sensitivity,

very high response time, and practically unsuitable for

biological application8. Therefore, the construction of

a novel fluorescent chemodosimeters for the easy and

accurate detection of hypochlorite in the living cells

pays great attention.

Rhodamine based sensors are widely used for bio-

logical application due to their well-established photo-

chemical properties such as longer excitation and emis-

sion wavelengths, high fluorescence quantum yield

etc.9. The spirolactam ring closed form of rhodamine

moiety is normally non-fluorescent and by the influ-

ence of the guests (mostly metal ions) ring-opening

state exists to show strong fluorescent character10. Till

now, there are only few reports of using rhodamine

moiety as an effective structural scaffold for designing

fluorescent chemodosimeters of hypochlorite11.

Herein, a rhodamine based sensor coupled with 2,4-

dinitrofluorobenzene (S1) was designed and synthesised

(Scheme 1). The molecule was characterised by NMR

and Mass spectroscopy.

J. Indian Chem. Soc., Vol. 94, July 2017

674

Results and discussion

The sensing ability of the sensor (S1) was studiedwith various anions like F–, Cl–, Br–, I–, CN–, AcO–,HSO4

–, H2PO4–, ClO– in 40% ethanol in water me-

dia. S1 displayed visual colour change from colourlessto pink instantly after the addition of 1.0 equivalent ofClO– (Fig. 1). In contrast, other anions did not exhibitany colour change with S1. UV-Vis absorbance spec-trum showed the peaks at 319 nm for only S1 (1.4×10–5

length UV light (Fig. 3). The solution of S1 remainedunchanged with the other anions. The visual fluores-cence change could be easily observed from the emis-sion spectra of S1 (1.7×10–6 M) in 40% ethanol in

Scheme 1. Synthesis of S1. Reagent and condition : (i) hydrazine hydrate, dry ethanol, reflux, 12 h, (ii) 2,4-dinitrofluorobenzene,dry DMF, r.t., 12 h.

Fig. 1. Visual colour change of S1 with different anions.

M) but after gradual addition of ClO–, a new peak wasgenerated at colour region (559 nm) with increasingpeak intensity (Fig. 2). The bathochromic shift (max319 to 559 nm) in UV-Vis electronic spectra stronglysupports the interaction of S1 with ClO–. This newpeak was the indication of spirolactam ring openingform of rhodamine part by the reaction with hypochlo-rite. No such spectral change was observed in the pres-ence of other anions.

The non-fluorescent solution of S1 turned to redfluorescent in the presence of ClO– under long wave-

Fig. 2. UV-Vis absorption spectra of S1 with 1.0 equivalentClO– in 40% ethanol in water.

Fig. 3. Visual fluorescence change of S1 with different anions.

Colour

S1

Sain et al. : Intracellular detection of toxic hypochlorite anion by using a nontoxic rhodamine based sensor

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water. The emission peak of S1 was visualized at 439nm (exc 350 nm) but after addition of ClO–, a newpeak at 584 nm was generated and its intensity in-creased with the gradual addition of ClO– up to 1equivalent (Fig. 4). No such spectral change was ob-served for other anions. Visually detectable red fluo-

rescence of S1 in the presence of ClO– only alongwith the generation of a new emission peak at 584 nm(exc 350 nm) with ClO– also concluded that S1 is aselective and sensitive fluorosensor of ClO–.

Probable mechanism for sensing of hypochlorite

Based on the previously reported mechanism12, wesupposed that HClO promoted chlorination reactiontook place followed by the opening of the spirolactamring in S1. Here, the hydrazide moiety of S1 wasoxidized by ClO– to form a diimide intermediate(Scheme 2), which experienced further hydrolysis toproduce the strong chromogenic and fluorogenicrhodamine B. The rate of hydrolysis was dependenton solvent because water is highly responsible for thehydrolysis of the diimide intermediate.

Cytotoxicity test

Cytotoxicity test of S1 was performed to knowwhether the compound was toxic or not. The toxicitylevel and practical applicability of the molecule wascarried out using MTT assay (Fig. 5). About 91% ofthese cells remained viable even upon addition of 30

Fig. 4. Fluorescence emission spectra of S1 with 1.0 equiva-lent ClO– in 40% ethanol in water.

Scheme 2. Reaction of S1 with ClO–.

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M concentration of S1 for 24 h which suggested thatthe toxicity level of S1 up to 30 M was low enoughand it could be easily applied for any biological acti-vity including cell imaging study.

Fluorescence cell-imaging study

The long wavelength fluorescence emission prop-erty of the S1 was applied for intracellular hypochlo-rite detection in living RAW 264.7 cells, which provesthe superiority of the sensor. To identify ClO– in liv-ing cell, at first 5 M of S1 was incubated into thecells (5% EtOH in H2O) in Dulbecco’s modified Eagle’smedium (DMEM) for half an hour at 37 ºC. Afterhalf an hour, 5 M solution of ClO– was incorporatedwith the cell and incubated for again half an hour.

Fig. 5. Checking of cell viability using MTT assay for S1 andS1+NaOCl.

Then fluorescence images were captured after process-ing the cells in the proper way. S1 containing cells didnot show any fluorescent image in red channel of fluo-rescence microscope but the cells incubated with bothS1 and ClO– showed a bright red fluorescent in thered channel (Fig. 6). This observation suggested thatS1 could be used as a real time useful molecule fordetection of ClO– in living cells.

Quantum chemical DFT calculation

Quantum chemical DFT calculation was performedusing Gaussian 09 program with the help of Gauss-View 5.0 visualization program13 to investigate thestructural aspects of S1. The structures were optimizedtheoretically using B3LYP/6-311G+(d,p) basis set. Theenergy optimized structure of S1 suggested that 2,4-dinitrobenzene moiety was almost parallel to the xan-thene part of the rhodamine moiety to maintain thestability of the system (Fig. 7a). The optimization en-ergy value of S1 (–2095.6415 a.u.) suggested its veryhigh stability. Molecular electrostatic potential (MEP)diagram displayed two different colour regions, wherered colour signified the electro negative regions andblue colour was the indication of positive regions (Fig.7b). Electron rich red regions were mainly distributedaround the most electronegative centre of the moleculesuch as N and O atoms. These regions were respon-sible for easy deprotonation of the NH group by theinfluence of ClO–.

The highest occupied molecular orbital (HOMO)and lowest unoccupied molecular orbital (LUMO) dia-gram of S1 along with their spatial distributions were

Fig. 6. Confocal microscopic images of living cells incubated with 5 mM S1 with 5 mM ClO– : (a) bright-field image of the cell,(b) red channel fluorescent image, (c) blue channel images of DAPI stained cells and (d) overlay images of b and c in darkfield.

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displayed in Fig. 8. From the diagram of S1, it wascleared that the xanthene moiety contained HOMOorbitals whereas 2,4-dinitrobenzene moiety containedLUMO orbitals (Fig. 8). The orbital diagrams sug-gested the probable energy transfer from HOMO toLUMO i.e. from xanthene part to 2,4-dinitrobenzenepart of S1.

Conclusion

As a conclusion, a simple bio-compatible 2,4-dini-trobenzene and rhodamine coupled probe was devel-oped for the detection of highly toxic hypochlorite ionwith high selectivity and sensitivity. Practical applica-bility of the sensor was established through simple andfast naked-eye detection of ClO–. The detection of

ClO– was carried out using UV-Vis and fluorescencespectral experiments. The non toxic behaviour of thesensor in living cells increased the practical applica-bility of S1 and it was effectively applied in intracellu-lar detection of toxic ClO– in living cells. Quantumchemical DFT calculation displayed the structural as-pects of S1. So, this sensor might be useful in futurefor in vivo detection of ClO– infected cells in the hu-man body.

Experimental

All reagents (AR grade) for synthesis were pur-chased from Sigma-Aldrich chemicals commerciallyand used without further purification. Solvents weredried following standard procedures. 1H NMR spectra

Fig. 8. Energy level HOMO and LUMO diagrams of S1.

Fig. 7. (a) Energy optimized structure of S1, (b) MEP diagram of S1.

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were recorded on a Bruker AV300 instrument usingCDCl3 with TMS as an internal standard. ESI-MS mea-surements were carried out using a microTOF-Q II10337 mass spectrometer instrument. UV-Vis spectraand fluorescence spectra were recorded using JASCOUV spectrometer (1.0 cm quartz cell) and Perkin-ElmerLS 55 fluorescence spectrometer, respectively. Fluo-rescence images of living cells were captured usingepifluorescence microscope (Carl Zeiss).

Synthesis of compound 1 :

Rhodamine B (1 g, 2.88 mmol) in dry ethanol andexcess of hydrazine (2 ml) was taken in a rb flask andthe reaction mixture was refluxed for 12 h. A lightpink precipitate was obtained. The precipitate was fil-tered and washed with ethanol for several times toobtained pure compound 114.

Synthesis of S1 :

Compound 1 was taken in dry DMF and 2,4-dinitrofluorobenzene was added to it. The mixture wasstirred at room temperature for 12 h. The mixture wasthen poured into ice water to get precipitate. The pre-cipitate was then filtered and dried followed by purifi-cation through column chromatography to obtain pureS1.

Characteristic experimental data of S1 :1H NMR (300 MHz, CDCl3) : 8.87 (1H1, s),

7.96 (1H2, d, J=7.5 Hz), 7.75 (1H3, d, J=6.9 Hz),7.63–7.52 (2H4, m), 7.27 (2H5, d, J=7.5 Hz), 6.59(2H6, d, J=9.3 Hz ), 6.40 (1H7, s), 6.24–6.22 (4H8,m), 3.19 (8H9, s), 1.03 (12H10, s);

13C NMR (125MHz, CDCl3) : 165.6, 154.1, 149.0, 148.5, 148.4,137.9, 133.7, 130.9, 129.6, 128.8, 128.2, 124.4,123.5, 122.4, 116.4, 107.9, 97.4, 67.4, 44.1, 12.0;ESI-MS (ES+) (m/z) : 623.41 (M+1).

Procedure for cytotoxicity assay

The cytotoxic effects of the sensor S1 and S1+NaOCl were determined by an MTT assay followingthe manufacturer’s instructions (MTT 2003, Sigma-Aldrich, St. Louis, MO, USA). HCT cells were cul-tured into 96-well plates (approximately 104 cells perwell) for 24 h. On the following day, media wereremoved and various concentrations of S1 and S1+

NaOCl (10, 25, 30, 50, 75, and 100 M) made inDMEM were added to the cells and incubated for 24h. Solvent control samples (cells treated with DMSOin DMEM), no cells and cells in DMEM without anytreatment were also included in the study. Followingincubation, the growth media were removed, and freshDMEM containing MTT solution was added. The platewas incubated for 3–4 h at 37 ºC. Subsequently, thesupernatant was removed, the insoluble coloredformazan product was solubilized in DMSO, and itsabsorbance was measured in a microtiter plate reader(Perkin-Elmer) at 570 nm. The assay was performedin triplicate for each concentration of sensor S1 andS1+NaOCl. The OD value of wells containing onlyDMEM medium was subtracted from all readings toremove background influence. Data analysis and thecalculation of standard deviation were performed withMicrosoft Excel 2007 (Microsoft Corporation).

Fluorescence cell-imaging procedure

Frozen human colorectal carcinoma cell line HCT116 (ATCC : CCL-247) was obtained from the Ameri-can Type Culture Collection (Rockville, MD) andmaintained in Dulbecco’s modified Eagle’s medium(DMEM, Sigma Chemical Co., St. Louis, MO) supple-mented with 10% fetal bovine serum (Invitrogen), peni-cillin (100 g/mL), and streptomycin (100 g/mL).The RAW 264.7 macrophages were obtained fromNCCS, Pune, India, and maintained in DMEM con-taining 10% (v/v) fetal calf serum and antibiotics in aCO2 incubator. Cells were initially propagated in 25cm2 tissue culture flask in an atmosphere of 5% CO2and 95% air at 37 ºC humidified air until 70–80%confluency.

For fluorescence imaging studies, RAW cells,7.5×103 cells in 150 L of media were seeded onsterile 12 mm diameter poly-L-lysine-coated coverslipand kept in a sterile 35 mm covered Petri dish andincubated at 37 ºC in a CO2 incubator for 24–30 h.Next day cells were washed three times with HEPES(pH 7.4) and fixed using 4% paraformaldehyde inHEPES (pH 7.4) for 10 min at rt washed with HEPESfollowed by permeabilization using 0.1% saponin for10 min. Then the cells were incubated with 10 M of


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the sensor (S1) dissolved in 100 L of DMEM at 37ºC for 1 h in a CO2 incubator and observed underepifluorescence microscope (Carl Zeiss). The cells wereagain washed thrice with HEPES (pH 7.4) to removeany excess sensor and incubated in DMEM containingNaOCl (20 M) followed by washing with HEPES(pH 7.4) three times to remove excess free NaOCloutside the cells. Again, images were taken usingepifluorescence microscope. Before fluorescence im-aging, all the solutions were aspirated, and the sampleswere mounted on slides in a mounting medium andstored in dark before microscopic images were ac-quired.

Acknowledgement

Dibyendu Sain acknowledges Science and Engineer-ing Research Board (SERB), Department of Science& Technology, Government of India for awarding Na-tional Post-doctoral fellowship (File No. PDF/2015/000882).

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Electrochemical sensor for simultaneous determination of dextromethorphanhydrobromide and paracetamol at carbon paste electrode modified withsynthesized indium tin oxide nanoparticles and ionic liquid

Mal Phebe Kingsley, Pratima Ashok Sathe and Ashwini K. Srivastava*

Department of Chemistry, University of Mumbai, Vidyanagari, Santacruz (E), Mumbai-400 098, India

E-mail : [email protected], [email protected] Fax : 91-22-26528547

Manuscript received 04 May 2017, accepted 08 May 2017

Abstract : Indium tin oxide nanoparticles were synthesized by solution route using cetyltrimethyl ammoniumbromide as surfactant and used as a modifier of the electrode. An electrochemical study for simultaneous quan-tification of dextromethorphan hydrobromide (DXM) and paracetamol (PA) by adsorptive stripping differentialpulse voltammetry at a carbon paste electrode modified with the composite of indium tin oxide nanoparticlesand 1-butyl-3-methylimidazolium chloride ionic liquid (ITO-IL-CME) is described. Phosphate buffer (0.05 M) atpH 7 was found to be the ideal supporting electrolyte for the electrochemical study. The voltammetric resultsindicate that ITO-IL-CME remarkably enhances the electrocatalytic activity towards the oxidation of DXM andPA in neutral solution. The peak current increased linearly with the concentration for DXM in the range from9.70×10–7 M to 6.20×10–4 M with a correlation coefficient of 0.989 and for PA with the concentration in therange from 3.62×10–8 M to 7.18×10–4 M with a correlation coefficient of 0.995. The detection limits for simul-taneous determination of DXM and PA were 4.66×10–8 M and 8.40×10–9 M for DXM and PA respectively.The proposed procedure was successfully applied for determination of the drugs in pharmaceutical formulations,human serum and urine sample with good apparent recoveries without interference from matrix. Thenanocomposite material modified carbon paste electrode has several advantages such as providing improvedvoltammetric behavior, long time stability, good selectivity, high sensitivity and excellent reproducibility.

Keywords : Dextromethorphan hydrobromide, paracetamol, 1-butyl-3-methylimidazolium chloride, indium tin oxidenanoparticles, modified carbon paste electrode, adsorptive stripping differential pulse voltammetry.

1. Introduction

Dextromethorphan hydrobromide (DXM, (+)-3-methoxy-17-methyl-(9,13,14)-morphinanhydrobromide) is an over the counter nonnarcotic an-titussive drug. It acts through depression of the med-ullary centers of the brain to decrease the involuntaryurge to cough. It affects the signals in the brain thattrigger cough reflex. The drug is beneficial when ad-ministered in recommended doses. However, DXMproduces a range of toxicities when consumed in highdoses and can cause liver damage, heart attack, stroke,and death1. Though DXM is a very effective safe-to-use medicament, abuse of the drug replicates alcohollike effect such as hallucination, paranoia, euphoriaand suicidal tendency2. Number of adolescents in the

United States and Europe intoxicate themselves withmegadoses of DXM viz. 5 to 10 times the dose recom-mended for control of annoying nonproductive coughs.Over dosage of DXM lacks the ability to metabolizethe drug normally, leading to rapid acute toxicity withprofound psychological and physiological effects3. Also,interaction between DXM and other substances (e.g.alcohol, acetaminophen, 3,4-methylenedioxy metham-phetamine and other over the counter cough medicines)produces a synergistic effect that can be very danger-ous4. Several analytical methods have been employedfor the determination of the DXM, like high perfor-mance liquid chromatography5,6, micellar liquid chro-matography7, RP-HPLC8, first-derivative spectropho-tometry9, capillary gas chromatography10 andvoltammetric techniques11,12. With respect to the use


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of voltammetric techniques, very few articles werereported for the quantification of DXM with rare us-age of modified electrodes13.

Paracetamol or N-acetyl-p-aminophenol (PA) is themost widely antipyretic, analgesic drug. It relieves painand fever. Generally, limited use of paracetamol issafe and has no harmful side effects. However, over-dose or long-term use of it may cause adverse effectson health because of the accumulation of toxic me-tabolites, which can lead to severe and sometimes fa-tal hepatotoxicity and nephrotoxicity14. PA is also com-bined with other active ingredients in prescriptionmedicines that treat allergy, cough, colds, flu, andsleeplessness. Varieties of methods have been used forthe determination of PA in pharmaceutical prepara-tions and human plasma like reverse phase high per-formance liquid chromatography15, RP-UPLC16, spec-trophotometry17,18, flow-injection analysis19 andvoltammetric techniques20. PA is present in some phar-maceutical preparations containing DXM in order toachieve synergistic effect as a combinational drug,though with different mechanisms of action21.The pres-ence of analgesic and cough suppressant in a pharma-ceutical formulations work together in the brain todecrease pain and reduce the cough reflex.

Ionic liquids (IL) are salts those are liquid at orabove room temperature. They possess several advan-tages (e.g. high intrinsic conductivity, wide electro-chemical windows, low volatility, high thermal stabil-ity and good solvating ability). Higher currents (bothfaradaic and capacitive) are often observed at ionicliquid-carbon paste composite electrode (IL-CME)compared to traditional bare carbon paste electrodedue to its larger electro-active surface area promotingincrease in electron transfer at the electrode surface22.

Voltammetric methods of analysis have been re-ceiving considerable attention since past few decadesdue to their simplicity, efficiency and low cost. Modi-fication of carbon paste working electrode surface im-parts high sensitivity and selectivity to the analyte res-ponse23. Use of nanoparticles as chemically modifiedelectrodes for electroanalysis of drugs, has enhancedthe scope and analytical applicability of the techniquein the field of drug analysis24.

In view of medical and pharmacological importance,as well as increasing trend of DXM abuse with poten-tial increase in toxicity of combined drug formulationsof DXM and PA on over dosage, there is a compellingneed to develop reliable, selective and sensitive ana-lytical method for simultaneous quantification of DXMand PA in pharmaceutical products and in human bodyfluids. However, to the best of our knowledge, onlyfew reports on voltammetric method have been re-ported for the simultaneous determination of DXMand PA compound25. Therefore, the objective of thepresent study is to develop an electrochemical sensorfor the simultaneous determination of DXM and PA,to understand the electrochemical reaction mechanismsand to determine their diffusion coefficients in solu-tion. Further, it aims to determine simultaneously thelevel of concentration of DXM and PA in pharmaceu-tical and clinical preparations in presence of preserva-tives and excipients with the developed sensor.

2. Material and methods

2.1. Chemicals

All chemicals were of analytical grade and wereused without further purification. Graphite powder(amorphous graphite size, < 50m) was purchasedfrom S.D. Fine Chemicals and mineral oil was fromFluka. Indium chloride (InCl3.4H2O), tin chloridepentahydrate (SnCl4.5H2O), ammonium hydroxide,ethanol, cetyltrimethylammonium bromide (CTAB),DXM, PA and 1-butyl-3-methylimidazolium chlorideionic liquid (IL) were purchased from Sigma Aldrich.Britton-Robinson buffer (BR) solution 0.04 M was usedto investigate pH dependence of DXM and PA. Otherbuffer solutions used were phosphate (K2HPO4/KH2PO4-Phos), tris(hydroxymethyl)amino methane(Tris) and 4-(2-hydroxy ethyl)-1-piperazine ethanesulphonic acid (HEPES). Double distilled water wasused for the preparation of aqueous solutions. Tablets/syrup/lozenges of DXM and tablets/syrup of PA avail-able from local pharmacies were subjected to analysis.

2.2. Instrumentation

Voltammetry measurements and electrochemicalimpedance spectroscopy (EIS) study have been per-

Kingsley et al. : Electrochemical sensor for simultaneous determination of dextromethorphan etc.

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formed on Eco Chemie, Electrochemical Work Sta-tion, model Autolab PGSTAT 30 using GPES soft-ware version 4.9005 and Frequency Response Analyser,software version 2.0 respectively. A three electrodesystem employing an Ag/AgCl (3 M KCl) and plati-num electrode were used as reference and counter elec-trodes respectively. Bare carbon paste electrode (CPE)/synthesized indium tin oxide nanoparticles modifiedcarbon paste electrode (ITO)/1-butyl-3-methyl-imidazolium chloride ionic liquid modified carbon pasteelectrode (IL-CME)/synthesized indium tin oxidenanoparticles with 1-butyl-3-methylimidazolium chlo-ride ionic liquid composite modified carbon paste elec-trode (ITO-IL-CME) were used as working electrodes.The EIS studies were performed in the frequency rangefrom 10–1 to 105 Hz at open circuit potential. The pHmeasurements were performed using an ELICO LI120 pH meter. Conductivity measurement was carriedout using four point probe instrument, Scientific Equip-ment, Roorkee. The energy dispersive spectroscopy(EDAX) and scanning electron microscopy (SEM)measurements were recorded using FEI Quanta-250model, Transmission electron microscopy (TEM) im-ages were registered by FEI TF 30 model, Powder X-ray diffraction (XRD, Rigaku, D/Max-RA,Cu K) andPerkin-Elmer FTIR spectrophotometer version 10.03.07in wave range of 4000–400 cm–1 were used for char-acterization of synthesized indium tin oxidenanoparticles.

2.3. Synthesis of indium tin oxide nanoparticles

A mixture of 1.105 g of InCl3·4H2O and 0.21 g ofSnCl4·5H2O was dissolved in water and 20 mL etha-nol was added followed by adding 0.05 g of CTABsurfactant and drop by drop addition of ammonia solu-tion under constant stirring at ambient condition. Thewhole mixture was stirred for 1 h on magnetic stirrer.It was then transferred to an autoclave and heated at150 ºC for 5 h. The product was centrifuged andwashed with de-ionized water and further by ethanol.Pale blue coloured nanocrystalline indium tin oxidewas obtained after the dried product was subjected toheat treatment at 400 ºC for 3 h in muffle furnace.

2.4. Preparation of CPE, ITO-CME, IL-CME andITO-IL-CME

CPE was prepared by mixing graphite with min-eral oil at composition 70 : 30 (w/w) using a motorand pestle and was homogenized for 30 min. ITO-CME, IL-CME and ITO-ILCME were prepared bymixing graphite powder, indium tin oxide nanoparticlesand/or 1-butyl-3-methylimidazolium chloride, mineraloil with various weight ratios. The pastes were thenpacked into a Teflon micro tip (diameter 0.5 mm) anda copper wire was inserted into the paste to establishan electrical contact. A new surface was regeneratedby pressing out an excess of paste out of the tip andpolishing it against zero grade butter paper.

2.5. Voltammetry procedure – Determination of DXMand PA

Cyclic voltammetry (CV) and differential pulsevoltammetry (DPV) studies were carried out with ap-propriate quantity of the DXM and/or PA with pH 7phosphate buffer (Phos). The solution was then trans-ferred into an electrochemical cell and the measure-ments were carried out at 25±0.2 ºC. N2 gas purgingwas not required as oxygen did not interfere in themeasurements. The cyclic voltammetric experimentswere carried out by sweeping the potential between+0.65 to +1.25 V for DXM and between +0.15 to0.75 V. For simultaneous recording of DPVs for DXMand PA, the potential range were within +0.20 to+1.1 V.

2.6. Sample treatment for scanning electron micro-scope (SEM) and transmission electron microscope(TEM) analysis

Morphology studies of ITO were carried out bySEM and TEM. Samples of ITO for TEM measure-ments were prepared by placing drops of the solution(sample in alcohol) on coated Cu grids and subse-quently drying in air. For SEM analysis, ITOnanoparticles were sonicated in toluene, allowed todry and then employed for analysis.

2.7. Sample preparation – Standard and real sample

The pharmaceutical preparations were Alkox-Dx(Alkem Laboratories Ltd.) and Ascoril-D (Glenmark


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Pharmaceuticals Ltd.) cough syrups each containing10 mg of dextromethrophan hydrobromide per 5 mL,TusQ-D lozenges (Blue Cross Laboratories Ltd.) con-taining 5 mg of dextromethrophan hydro-bromide, P-250 (Apex Laboratories Ltd.) containing 250 mg ofparacetamol, Pyrigesic (East India PharmaceuticalWorks Ltd.) containing 500 mg paracetamol and Alex-P+ (Glenmark Pharmaceuticals Ltd.) containing 5 mgof dextromethrophan hydrobromide and 170 mg ofparacetamol per 5 mL.

Five tablets/lozenges were accurately weighed andfinely powdered with a mortar and pestle. To a corre-sponding weight of the real sample water was added,filtered through a Qualigens (615) filter paper into a100 mL calibrated flask. The residue was washed sev-eral times with water and the solution was diluted tothe mark. Appropriate volume of this solution wasdiluted to 25 mL with phosphate buffer (pH 7.0) andthen transferred to an electrolytic cell for the determi-nation of DXM and/or PA.

To 1 mL of blood serum/urine/syrup, supportingelectrolyte was added and made up to 10 mL and ana-lyzed. Standard addition method was employed forquantification of DXM and PA as individual/combina-tions in pharmaceutical preparations, blood serum andurine samples.

3. Results and discussion

3.1. Characterization of indium tin oxide nanoparticles

3.1.1. X-Ray diffraction studies (XRD) of synthe-sized indium tin oxide nanoparticles

The XRD of indium tin oxide is given in Fig. 1.All diffractions can be indexed as cubic crystal struc-ture of indium oxide. Indium oxide nanoparticles ex-hibits the reflection from the (222) planes as the mostpredominant peak in the X-ray diffraction pattern. Tinatoms are doped into the indium oxide lattice since noevidence of characteristic peaks of Sn, SnO or SnO2were indicated in the spectra. The peaks in the diffrac-tion spectrum are found to be sharp indicating highlycrystalline nature of the nanoparticles. No characteris-tic peaks of impurities were detected.

Table 1 shows that all the peaks can be perfectlyindexed to crystalline indium oxide, not only in theirpeak positions, but also in their relative intensities.

Using Debye-Scherrer’s formula, the crystallite size,Dhkl is given by

Dhkl = K/ cos (1)

where the Scherrer’s constant K is taken to be 0.9, ‘’is the wavelength of X-ray (1.5406 Å), ‘’ is the full

Fig. 1. XRD pattern of indium tin oxide nanoparticles.


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width at half maximum of the peak, ‘’, the Braggangle and ‘D’ is the particle diameter size. The meanparticle diameter of ITO nanoparticles was found tobe 30 nm.

3.1.2. Scanning electron microscopy (SEM) andtransmission electron microscopy (TEM) of synthe-sized indium tin oxide nanoparticles

The morphology and micro-structure of ITO wereinvestigated by SEM and TEM. Fig. 2(A) is the SEMimage of synthesized indium tin oxide nanoparticles,

which is observed to be non-agglomerated with uni-form morphology and of cubic shape. The chemicalconstituent of particles was analyzed by EDAX (Fig.2(B)). The In L1 (3.286 keV) and Sn L1 (3.443keV) peaks show the presence of indium and tin ele-ments with the atomic molar percent determined forindium being 91% and for tin 9% in the lattice of theITO nanoparticles.

The TEM image of synthesized indium tin oxidenanoparticles is given in Fig. 3.

Table 1. XRD data for indium tin oxide nanoparticles

XRD data-d (nm) 4.130 2.924 2.532 1.986 1.789 1.526

Theoretical values-d (nm) 4.130 2.921 2.529 1.984 1.788 1.525

Crystalline plane (hlk) 211 222 400 431 440 622

Fig. 2. SEM image of (A) ITO nanoparticles and (B) Energy-dispersive X-ray spectrum for ITO nanoparticles.


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The TEM examination of synthesized ITO indi-cates that the morphology is homogeneous showing acubic structure.

3.1.3. FTIR spectral analysis of synthesized indiumtin oxide nanoparticles

The peaks at 476, 557, 593 and 849 cm–1 in Fig. 4show the characteristic bands of crystalline In2O3. Theobserved bands at 476 and 557 cm–1 are attributed tothe stretching vibrations of In-O whereas bands at 593and 849 cm–1 are characteristic of In-O bending vibra-tions. The vibrations extending from 2328 and 1293cm–1 indicate the presence of hydrogen bonds involvedin O-H oscillations arising from adsorbed water26.

3.1.4. Conductivity measurements of synthesized in-dium tin oxide nanoparticles

Electrical conductivity measurement of synthesizedindium tin oxide nanoparticles was performed usingthe DC four-point probe technique27 on synthesized

ITO pellets (1.7 cm diameter, 0.42 mm thickness).Three pellets were prepared by pressing about 100 mgof synthesized ITO with an IR pressing tool. Sheetresistance ‘Rs’ across the pellets was determined to be2.41×10–2 /sq, the value of the average electricalconductivity ‘’ calculated from Rs obtained was 98.79S cm–1.

3.2. Effect of pH

The effect of pH on peak current and peak poten-tial for DXM (1.0×10–5 M) and PA (2.5×10–6 M)are shown in Fig. 5(A) and (B) respectively in BRbuffer from pH 2 to 10 at CPE using differential pulsevoltammetry.

It is observed that the peak current of DXM in-creases till pH 7.0 followed by a decrease with furtherincrease in pH. Similarly for PA, the maximum peakcurrent is obtained at pH 7.0. Therefore, pH 7 wasselected for the determination of DXM and PA in theirmixture. The peak potentials for the oxidation of DXMand PA is shifted negatively with increasing pH, indi-cating that protons take part in the electrochemicalreaction processes for both the molecules. A slope of–0.052 V/pH and –0.050 V/pH for DXM and PA wereobserved respectively, suggesting that same numberof electrons and protons were involved for their electro-oxidation. The effects of several supporting electro-lytes (0.1 M) viz. HEPES, phosphate (KH2PO4+K2HPO4), Tris and BR buffer at pH 7 for DXM(1.0×10–5 M) and PA (2.5×10–6 M) on peak currentwere tested as shown in Fig. 6.

Of these, phosphate buffer (Phos) gave the bestresponse in terms of peak height and the peak shape.Optimization of buffer concentration was carried outby varying phosphate concentration in the range from0.01 M to 0.2 M, the best peak response was observedfor 0.05 M of Phos (pH 7) and hence was used forfurther studies.

3.3. Effect of surface modification and optimizationof the amount of the modifier

The cyclic voltammograms for DXM (1.0×10–5

M) and PA (2.5×10–6 M) in 0.05 M of Phos (pH 7) at

Fig. 3. TEM image of ITO nanoparticles.

Fig. 4. FT-IR spectrum of indium tin oxide nanoparticles.


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Fig. 5. DPV plot of (A) Ip vs pH (▲▲▲) and Ep vs pH (❙❙❙❙) for 1.0×10–5 M DXM and (B) Ip vs pH (▲▲▲) and Ep vs pH (❚❚❚❚)for 2.5×10–6 M PA at CPE.

CPE, ITO-CME, IL-CME and ITO-IL-CME is shownin Fig. 7(A) and (B). It is seen that DXM and PAshow irreversible peaks. It was evident from the fig-ures that the modification of the CPE with indium tinoxide and ionic liquid enlarged the peak current con-siderably by about 2.5 and 3 times for DXM and PArespectively, indicating that the surface property ofthe modified electrode has been significantly changeddue to synergistic effect of ITO with IL.

The influence on the amount of ITO nanoparticles

and IL added to CPE was studied for a solution con-taining DXM (1.0×10–5 M) and PA (2.5×10–6 M) inpH 7 by DPV. Table 2 shows the weight ratios for thevarious modified electrode. The peak current increasedwith increase in the percentage weight of nanoparticlestill the ratio of composition (w/w) for graphite : ITOnanoparticles : IL : mineral oil was 45 : 20 : 05 : 30

The DPV peaks are well-resolved for PA and DXMat ITO-IL-CME with the peak potentials at about 0.391V and 0.872 V respectively. The separation of peakpotentials is 0.481 V for PA-DXM which is largeenough to determine PA and DXM simultaneously.

The surface areas for CPE, IL-CME, ITO-CMEand ITO-IL-CME having same bore size using 6 mMmixture of K3Fe(CN)6 and K4Fe(CN)6 system in 0.1N KNO3 by cyclic voltammetric technique was calcu-lated from Randles-Sevcik equation28. It was found tobe 0.00958, 0.0175, 0.0345 and 0.0847 cm2 respec-tively.

3.4. Cyclic voltammetry

The effect of scan rate to investigate electrode re-action mechanism is studied and are shown in Fig.8(A) and (B) for DXM (1.0×10–5 M) and PA (2.5×10–

6 M) at ITO-IL-CME in 0.05 M phosphate (pH 7)

Fig. 6. Effect of supporting electrolytes (pH 7.0) on anodicpeak current of 1.00×10–5 M DXM and 2.5×10–6

M PA at CPE.

JICS-4


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Fig. 7. Cyclic voltammograms for oxidation of (A) of 1.0×10–5 M DXM at; (a) CPE ( ), (b) IL-CME ( ), (c) ITO-CME( ), (d) ITO-IL-CME ( ) vs Ag/AgCl and (B) of 2.5×10–6 M PA at; (a) CPE ( ), (b) IL-CME ( ), (c) ITO-CME ( ), (d) ITO-IL-CME ( ) vs Ag/AgCl in 0.05 M Phos (pH 7); scan rate 100 mV/s at 25 ºC.

respectively. The scan rates were from 50 to 900 mVs–1.

The peak current varied linearly with increase inthe scan rate for DXM (Fig. 9(A)) and PA (Fig. 10(A))with regression equation

Ip = 4.216 + 0.035 [R2 = 0.984; Ip : A, : (mV/s)] (2)

Ip = 3.442 + 0.033 [R2 = 0.995; Ip : A, : (mV/s)] (3)

respectively, validating a typical adsorption-controlled

process. A plot of log Ip against log for 1.0×10–5 MDXM (Fig. 9(B)) and for 2.5×10–6 M PA (Fig. 10(B))was linear with a slope of 0.666 and 0.941 indicatingthat the electrode process is adsorption controlled.

Also, the peak potential shifted to more positivevalues with the increase of scan rate, which confirmedthe irreversibility of the oxidation process for both themolecules (Figs. 9(C) and 10(C)).

For an irreversible reaction the number of elec-trons (n) involved in the oxidation reaction was calcu-

Table 2. Composition of modified electrodes with various weight ratios of graphite : ITO : IL : mineral oil

Electrode Graphite Indium tin oxide 1-Butyl-3-methyl Mineral oil

(mg) nanoparticles imidazolium (mg)

(mg) chloride ionic liquid

ITO-CME 65 5 – 30

IL-CME 65 – 5 30

ITO-IL-CME 1 60 5 5 30

ITO-IL-CME 2 55 10 5 30

ITO-IL-CME 3 50 15 5 30

ITO-IL-CME 4 45 20 5 30

(ITO-IL-CME)

ITO-IL-CME 5 40 25 5 30

ITO-IL-CME 6 40 20 10 30

ITO-IL-CME 7 35 20 5 30

ITO-IL-CME 8 52 8 10 30


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lated from the following equation29 :

Ep – Ep/2 = 47.7/n [mV at 25 ºC] (4)

Ep – Ep/2 value for DXM and PA was found to be 75mV and 55 mV respectively. On substitution in theabove equation and assuming as 0.5, ‘n’ was calcu-lated was found to be 1.2 for DXM i.e. approximatelyone proton is transferred and 1.7 for PA i.e. approxi-mately two proton is transferred in the oxidation pro-cess. Since the oxidation occurred by transfer of thesame number of electrons and protons for both themolecules, the oxidation of DXM is an one electronone proton process and for PA it is two electron two

proton process at the ITO-IL-CME electrode.

Scheme 1(a) and (b) show the probable electro-chemical reaction process for the oxidation of DXMand PA at the ITO-IL-CME.

In order to calculate the electron transfer rate con-stant the following Laviron’s equation was used

RT RTk0 RTEp = E0 + ——— ln ——— + ——— ln (5)

nF nF nF

where, k0 is the electron transfer rate constant, isthe electron transfer coefficient, is the scan rate, n isthe electron transfer number and E0 is the formal po-

Fig. 8. Cyclic voltammograms for the oxidation of (A) 1.0×10–5 M DXM, (B) 2.5×10–6 M PA : for scan rate (a) 50, (b) 100,(c) 200, (d) 300, (e) 500, (f) 600, (g) 700, (h) 800, (i) 900 mV s–1 in 0.05 M Phos (pH 7) at ITO-IL-CME vs Ag/AgCl at25 ºC.

Fig. 9. Plot of (A) Ip vs , (B) log Ip vs log , (C) log Ep vs , for 1.0×10–5 M DXM in 0.05 M Phos (pH 7) at ITO-IL-CME vsAg/AgCl at 25 ºC.


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of k0 were determined to be 330.0 s–1 and 380.3 s–1

for DXM and PA respectively. Large values obtainedfor electron transfer rate constant indicate high abilityof promoting electrons between the molecules and theITO-IL-CME electrode surface.

3.5. Electrochemical impedance spectroscopy (EIS)

Electrochemical impedance spectroscopy (EIS) is asimple and effective way to measure the charge trans-fer resistance (Rct) of the electrochemical reactions29.The EIS measurements were performed for DXM(1.0×10–5 M) and PA (2.5×10–6 M), in a phosphatebuffer solution pH 7 at CPE, IL-CME, ITO-CME and

Fig. 10. Plot of (A) Ip vs , (B) log Ip vs log , (C) Ep vs for 2.5×10–6 M PA in 0.05 M Phos (pH 7) at ITO-IL-CME vs Ag/AgCl at 25 ºC.

Scheme 1(a). Electro-oxidative mechanism for dextromethrophan hydrobromide.

Scheme 1(b). Electro-oxidative mechanism for paracetamol.

Dimer

Dextromethrophan Radical cation Radical

Paracetamol N-Acetyl-p-quinione imine

tential. k0 can be determined from the intercept andslope of the linear plot of Ep vs log . E0 can bededuced from the intercept of Ep vs plot. The values


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ITO-IL-CME. It can be seen from Fig. 11(A) and (B)that each spectrum exhibited a semicircular and a lin-ear portion.

The semicircle corresponds to the charge transferprocess at the electrode at a high frequency range,whereas the linear portion in the spectra is due to thediffusion process in the low frequency range. The di-ameter of the semicircle represents the magnitude ofRct at the electrode surface. The measured EIS datawere fitted with an equivalent circuit known as theRandles equivalent circuit, consisting of ohmic resis-tance (Rs) of the electrolyte solution, the double layercapacitance (Cdl), electron transfer resistance (Rct) andthe Warburg impedance (Zw) resulting from the diffu-sion of ions from the bulk of the electrolyte to theinterface. The values of Rct at CPE, IL-CME, ITO-CME and ITO-IL-CME were 518.9 , 299.3 , 238.5and 208.4 , for DXM and 317.0 , 295.6 ,286.5 and 235.1 , for PA respectively. By com-paring the data obtained, the Rct for CPE is muchhigher than at ITO-IL-CME for both the moleculesimplying that the charge transfer process is relativelyfast at ITO-IL-CME compared to CPE.

3.6. Chronocoulometry (CC)

Chronocoulometry is a controlled-potential techniquein which the measurement of charge as a function of

time is observed. Application of this technique helpsto study of electrode kinetics by, determining the dif-fusion coefficient of the analyte in solution and bydetermining the surface coverage area of the electrodethe extent of the adsorption of the analyte on the elec-trode can be ascertained30. Double-potential stepchronocoulometry permits an “in situ” double layercorrection even in the presence of extensive adsorp-tion. The above technique was applied individually for1.0×10–5 M DXM and 2.5×10–6 M PA in 0.05 Mphosphate (pH 7).

From following equation29 :

Qt = (2nFD1/2 CA t1/2)/

1/2 + nFA 0 + Qdl (6)

the Anson plot of total charge (Qt) in coloumbs vs thesquare root of time (t1/2) in seconds should give alinear relationship. Qdl is the double layer charge inCoulombs, n is the number of electrons, F is the Fara-days constant (96485 C), C is the concentration inmol/cm3, A is the area of the electrode in cm2, 0 issurface coverage in mol cm–2 and D is the diffusioncoefficient in cm2 s–1. The diffusion coefficient andsurface coverage were estimated from the slope andintercept of the Ansons plot at CPE, IL-CME, ITO-CME and ITO-IL-CME as given in Table 3.

Fig. 11. Nyquist plots for the EIS measurements for (A) 1.0×10–5 M DXM at (a) CPE (✧✧✧✧), (b) IL-CME (), (c) ITO-

CME (■■■■), (d) ITO-IL-CME (OOOO); (B) for 2.5 ×10–6 M PA at (a) CPE (▼▼▼▼), (b) IL-CME (XXX),

(c) ITO-CME (), (d) ITO-IL-CME ( ) in the frequency range from 10–1 to 105 Hz, the inset shows an

equivalent circuit was used for data fitting.


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Table 3. Chronocoulometry of 1.0×10–5 M DXM and 2.5×10–6 M PA in 0.05 M Phos (pH 7)

Molecule Electrode Slope Intercept Qads Surface coverage Diffusion coefficient

(C s–½) (C) (10–10 mol cm–2) (cm2 s–1)

DXM CPE 0.367±2.14 0.528±1.79 5.71 1.23×10–3

IL 0.945±3.48 1.280±0.92 7.59 2.45×10–3

ITO 2.510±3.79 3.260±2.13 9.78 4.45×10–3

ITO-IL-CME 7.830±1.81 8.990±4.07 10.99 7.19×10–3

PA CPE 0.113±3.55 7.050±3.77 38.1 4.68×10–4

IL 0.245±2.19 16.10±2.84 47.6 6.58 ×10–4

ITO 0.574±1.03 42.40±1.62 63.7 9.32 ×10–4

ITO-IL-CME 1.53±3.13 160.9±2.20 98.4 10.92×10–4

The values of D and 0 are found to increase fromCPE to ITO-IL-CME indicating that maximum accu-mulation of DXM or PA on ITO-IL-CME from amongall the electrodes studied.

3.7. Adsorptive stripping differential pulse voltam-metry (AdSDPV)

3.7.1. Effect of accumulation parameters – Accu-mulation potential and accumulation time

AdSDPV was employed to study the influence ofdifferential pulse anodic stripping peak current for1.0×10–5 M DXM and 2.5×10–6 M PA on the accu-mulation potential (Eacc) and accumulation time (tacc)at ITO-IL-CME in 0.05 M Phos buffer solution (pH

7). Eacc was determined by employing a potential widowfrom –1.0 V to 0.7 V. The peak current remainedalmost constant when the potential was positive from0 V to 0.7 V for both the molecules. The peak currentgradually increased as Eacc became negative and reachedits maximum at –0.3 V and –0.50 V for DXM and PArespectively (Fig. 12(A)).

When potential shifts still further in the negativedirection, there is drop in peak current with increasein the background current. Thus the optimum accumu-lation potential of –0.50 V was used for subsequentsensitive simultaneous determination of DXM and PA.

At the same accumulation potential, the effect ofaccumulation time on the efficiency of drugs onto the

Fig. 12. Influence of (A) accumulation potential and (B) accumulation time on the oxidation peak current of 1.0×10–5 M DXM and2.5×10–6 M PA in 0.05 M Phos (pH 7.0) at ITO-IL-CME vs Ag/AgCl at 25 ºC.


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working electrode surface was evaluated over a timerange from 0.0 s to 150 s (Fig. 12(B)). Peak currentincreased very rapidly with increasing accumulationtime, which induced rapid adsorption of DXM and PAon the surface of the modified electrode. A steadyenhancement in the peak current was observed overthe range from 0.0 s to 90 s for DXM and from 0.0 sto 110 s for PA and thereafter the peak intensity gradu-ally scales off at longer accumulation time. This indi-cates that, further increase of accumulation time didnot increase the response of DXM and PA on the elec-trode surface due to a complete coverage of electrodesurface. Therefore, the accumulation time was fixedat 110 s with an optimal stirring rate of 1200 rpm forthe analysis.

3.7.2. Effect of potential sweep parameters

To improve the sensitivity of adsorptive strippingdifferential pulse voltammetry for the simultaneous de-termination of 1.0×10–5 M DXM and 2.5×10–6 MPA at ITO-IL-CME, the effect of pulse amplitude,scan rate and pulse width on peak current were inves-tigated. It was found that the peak currents increasedsignificantly with increasing pulse amplitude from 10to 50 mV and then almost remained constant from 60mV to 100 mV. The pulse amplitude chosen for prac-

tical purpose was 50 mV. Faster scan rates resulted inhigher peak currents but background currents also in-creased. Taking peak current and background currentinto consideration the scan rate chosen was 50 mVs–1. The optimum pulse width was 0.05 s.

3.8. Determination of DXM and PA by adsorptivestripping differential pulse voltammetry (AdSDPV)

Based on the optimal operating conditions (Table4), AdSDPV measurements were undertaken in solu-tions containing different concentrations of DXM andPA individually and simultaneously.

Fig. 13(A) and (B) are the voltammograms of DXMand PA respectively carried out individually.

For simultaneous determination, two separate sets

Table 4. Optimal parameters for determination of DXM andPA simultaneously by AdSDPV technique

Parameter AdSDPV

pH 7

Supporting electrolyte 0.05 M Phosphate buffer

Accumulation potential –0.5 V

Accumulation time 90 s

Stirring rate 1200 rpm

Pulse amplitude 50 mV

Pulse width 0.05 s

Scan rate 50 mV s–1

Fig. 13. AdSDPV curves obtained at ITO-IL-CME for (A) DXM at different concentrations (a) 0.6, (b) 0.8, (c) 6.0, (d) 8.0,(e) 60.0, (f) 80.0, (g) 150, (h) 300, (i) 500, (j) 700 M. (B) PA at different concentrations (a) 0.4, (b) 0.8, (c) 2.0,(d) 8.0, (e) 40.0, (f) 60.0, (g) 80.0, (h) 200.0, (i) 400.0, (j) 600.0 M; scan rate 50 mV s–1 in 0.05 M Phos (pH 7.0);pulse amplitude 50 mV. Inset of (A) and (B) shows the linearity graph of DXM and PA respectively.


694

of experiments were carried out. In the first instance,concentration of DXM was increased in the presenceof fixed concentrations of PA and the voltammogramswere recorded (Fig. 14(A)) and vice versa as given inFig. 14(B). For the second instance, both DXM andPA were determined by simultaneously increasing theirconcentrations as given in Fig. 14(C). The linear work-ing range (LWR), empirical limits of detection (LOD)(S/N = 3), linear regression equation (LRE) and cor-relation coefficient (r) were determined (Table 5).

The anodic peak current corresponding to the oxi-dation of DXM and PA correlates linearly with itsconcentration in the entire range of investigation.

From Table 5 it is observed that the LWR andLOD determined for DXM and PA simultaneously arein good agreement with when both were individuallydetermined.

Table 6 shows a comparison of the proposed sen-sor with the reported electrochemical sensors for thedetermination of DXM and PA individually and si-multaneously.

3.9. Validation and interference studies

The precision of the developed method was inves-tigated with respect to both repeatability (single elec-trode), reproducibility (multiple electrodes), rugged-

Fig. 14. AdSDPV curves obtained at ITO-IL-CME for (A) DXM at different concentrations (a) 0.8, (b) 4.0, (c) 20.0, (d) 40.0,(e) 60.0, (f) 80.0, (g) 100, (h) 200 M in the presence of 2.5×10–6 M of PA. (B) PA at different concentrations (a) 0.2,(b) 0.6, (c) 0.8, (d) 40.0, (e) 60.0, (f) 80.0, (g) 200.0, (h) 200.0, (i) 400.0, (j) 600.0, (k) 800.0 M in the presence of1.00×10–6 M DXM. (C) Simultaneous determination of DXM and PA at different concentrations (a) 0.2 and 0.2, (b) 0.4and 0.4, (c) 0.8 and 6.0, (d) 10.0 and 8.0, (e) 20.0 and 20.0, (f) 60.0 and 40.0, (g) 80.0 and 80.0, (h) 100.0 and 200.0,(i) 300.0 and 500.0, (j) 600.0 and 400.0 M respectively in 0.05 M Phos (pH 7.0); scan rate 50 mV s–1; pulse amplitude50 mV.


695

Table 5. Analytical parameters for electrochemical determination of DXM and/or PA in 0.05 M phosphate buffer (pH 7)

Molecule LOD % RSD LWR LRE r

Parameters for individual molecule

DXM 2.61×10–8 2.34 8.00×10–7 Ip (A) = 0.010 (M)+0.049 0.998

to

8.00×10–4

PA 1.58×10–9 3.29 4.00×10–8 Ip (A) = 0.030 (M)+0.349 0.998

to

8.00×10–4

Parameters when DXM concentration increases and PA concentration (2.5×10–6 M) remains constant

DXM 1.58×10–8 1.65 6.00×10–7 Ip (A) = 0.017 (M)+0.788 0.989

to

7.00×10–4

Parameters data when PA concentration increases and DXM concentration (1.00×10–5 M) remains constant

PA 1.45×10–9 2.81 2.00×10–8 Ip (A) = 0.091 (M)+0.350 0.996

to

7.40×10–4

Parameters when DXM and PA increases in concentration simultaneously

DXM 4.66×10–8 3.96 9.70×10–7 Ip (A) = 0.076 (M)+3.505 0.989

to

6.20×10–4

PA 8.40×10–9 2.87 3.62×10–8 Ip (A) = 0.357 (M)+8.137 0.995

to

7.18×10–4

ness and interferents with standard solution of 1.0×10–4 M DXM and 1.0×10–5 M PA in 0.05 M Phosbuffer solution (pH 7) at ITO-IL-CME. Repeatabilitywas assessed by continuous electrooxidative determi-nation of the standard solution with the same ITO-IL-CME electrode for 10 analyses. The anodic currentdecreased by 1.05% and 1.32% with the RSD valueof 1.75% and 2.19% for DXM and PA respectivelyafter completing 10 scans, which showed that the elec-trode responds with good repeatability. Reproducibi-lity was investigated by determination of three repli-cate samples containing standard solution of DXM andPA on five consecutive days using multiple electrodeswhere the mean concentrations were found to be1.09×10–4 M and 0.983×10–5 M with associated RSDvalues of 1.19% and 3.67% respectively. The rugged-ness of the method was assessed by comparison of theintra- and inter-day assay results for DXM and PAthat had been performed by two analysts. The RSD

values for intra- and inter-day assays of analyses per-formed in the same laboratory by two different ana-lysts did not exceed 3.5%, thereby indicating the rug-gedness of the method. The influence of peak currenton DXM and PA in presence of interferents that arecommonly present in biological media and 4-aminophenol (metabolite of paracetamol) were inves-

tigated. Also, DXM and PA are formulated in differ-

ent dosage forms in combinations with drugs like aspi-

rin, caffeine, phenylepherine, ascorbic acid and

cetirizine dihydrochloride, therefore the extent of their

interference along with DXM and PA were tested with

the developed AdSDPV method. Under optimized ex-

perimental conditions, no effect on the determination

of DXM and PA was observed up to 250-fold of Na+,

K+, Mg2+, Ca2+, Cl–, SO42–, 200-fold of glucose

and ascorbic acid, 150-fold of glucose and uric acid,

20-fold of citric acid, 100-fold of aspirin and caffeine,

JICS-5


696

60-fold of phenylepherine, 50-fold of cetirizine

dihydrochloride and 70-fold of 4-aminophenol, indi-

cating that the present modified electrode was highly

selective towards the determination of DXM and PA.

3.10. Application to real matrices

Commercial pharmaceutical samples (tablets) con-taining DXM and/or PA present in tablets were ana-lyzed in order to evaluate the practical applicability of

Table 7. Assay of DXM and/or PA in pharmaceutical preparations (n = 5)

Dextromethrophan hydrobromide Paracetamol

Tablet/Syrup Amount of Amount of drug Tablet/Syrup Amount of Amount of drug

drug in the obtained in the drug in the obtained in the

sample proposed method sample proposed method

(mg) (mg)±RSD (mg) (mg)±RSD

Alex-P+ 5.0 4.8±2.13 Alex-P+ 170.0 172.1±1.17

Tus Q-D 5.0 5.1±2.15 – – –

– – – Pyrigesic 500.0 498.4±2.94

Table 6. Comparison of the performances of electrochemical sensors for DXM and PA

Molecule Modified electrode Detection limit (M) Linear range(M) Refs.

Dextromethrophan Carbon nanotubes-carbon 8.81×10–6 2.50×10–4 11

hydrobromide microparticle-ionic liquid to

composite 3.30×10–3

Reduced graphene oxide 1.50×10–6 5.0×10–6 12

modified screen printed to

electrode after 1.50×10–3

electromembrane

extraction

Hydrophilic carbon nano- 2.90×10–6 8.0×10–6 25

particulates at the surface to

of carbon paste electrode 8.0×10–4

ITO-IL-CME 2.61×10–8 8.0×10–7 This work

to

8.0×10–4

Paracetamol Boron doped diamond 1.70×10–7 5.0×10–7 31

electrode modified with to

Nafion and lead films 2.0×10–4

Nafion/TiO2 – Graphene 2.10×10–7 4.0×10–6 32

modified glassy carbon to

electrode 2.0×10–5

Hydrophilic carbon 1.50×10–8 1.0×10–7 25

nanoparticulates at the to

surface of carbon paste 1.0×10–3

electrode

ITO-IL-CME 2.20×10–9 4.0×10–8 This work

to

6.0×10–4


697

the developed method (Table 7). Recovery experimentson the spiked samples (tablets, blood and urine) werealso carried out to evaluate matrix effects (Table 8).Standard solution additions yielded good average re-coveries which ranged between 97.5% and 102.9%for DXM and 97.6% and 103.2% for PA indicatinghigh reproducibility and reliability of the modified elec-trode.

4. Conclusion

The present work demonstrates a simple and sensi-

tive electrochemical method for the simultaneous de-

termination of dextromethrophan hydrobromide and

paracetamol at carbon paste electrode modified with

indium tin oxide and 1-buty-3-methylimidazolium chlo-

ride ionic liquid. Cyclic voltammetry and electrochemi-

cal impedance spectroscopy study results confirmed

the synergism of ITO nanoparticles and IL with uniquecatalytic activity at the electrode surface. Anodic strip-ping voltammetric technique was used for simultaneousdetermination of DXM and PA at ITO-IL-CME elec-trode and also was used for the first time. On com-parison with unmodified carbon paste electrode andwith some of the previously reported electrodes theITO-IL-CME significantly enhanced the sensitivity, se-lectivity and reliability for individual and simultaneousdetermination of DXM and PA with low detection limitsand large linear working range. The utilization of thedeveloped sensor is an attempt to perform as a goodalternative economically and environmental friendlyfor simultaneous determination of DXM and PA, inquality control of pharmaceutical formulations, testingof blood and urine samples in terms of easy handling,low cost and stability.

Table 8. Recovery test for DXM and/or PA in pharmaceutical, blood serum and urine samples (n = 5) by AdSDPV

Dextromethrophan hydrobromide Paracetamol

Std drug Drug conc. Apparent Std drug Drug conc. Apparent

added 10–6 M recovery (%) added 10–5 M recovery (%)

10–6 M ±RSD 10–5 M ±RSD

Alkox-Dx – 3.54 – – – –

1.95 5.65 102.9±2.14 – – –

5.66 8.97 97.5±1.333 – – –

10.84 14.17 98.5±1.75 – – –

Ascoril-D – 8.24 – – – –

1.67 10.10 101.9±1.48 – – –

3.75 12.17 101.5±3.15 – – –

23.31 31.55 97.7±2.66 – – –

P-250 – – – – 1.12 –

– – – 2.40 3.59 101.9±1.24

– – – 4.62 5.62 97.9±2.91

– – – 9.65 9.59 99.4±2.68

Blood serum – ND – – ND –

7.41 7.43 100.3±1.39 1.18 1.21 102.5±2.46

10.70 10.8 100.8±2.16 2.31 2.35 101.7±2.93

13.8 13.7 99.4±2.94 3.40 3.51 103.2±1.68

21.9 21.7 99.2±1.52 4.44 4.43 99.7±3.35

Urine – ND – – ND –

7.41 7.50 101.2±2.44 3.31 3.35 101.2±2.93

16.70 16.40 98.4±2.86 6.74 6.58 97.6±3.14

26.50 26.30 99.3±1.13 11.90 11.72 98.5±1.87


698

Acknowledgement

One of the authors (MPK) is thankful to the Uni-

versity Grants Commission, New Delhi, India for pro-

viding financial assistance under Faculty Improvement

Programme for this work. We are also grateful to Pro-

fessor Sabir H. Mashraqui, University of Mumbai,

for his valuable inputs.

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699


A novel imidazolyl appended quinoline-hydrazide Schiff base fluorescentchemosensor for precise identification of ZnII

Rakesh Purkait and Chittaranjan Sinha*

Department of Chemistry, Jadavpur University, Kolkata-700 032, India



Abstract : (E)-N-((1H-imidazol-2-yl)methylene)quinoline-2-carbohydrazide (H2L) is synthesized by the reactionof acid hydrazide and carbaldehyde. The probe has been characterized by spectroscopic data (FT-IR, UV-Vis,1H NMR) and is weakly emissive. The H2L selectively binds Zn2+ and upon irradiation at 331 nm in presenceof large number of cations shows high intense emission (em, 575 nm) and serves as a “turn-on” fluorescencechemosensor. The limit of detection (LOD) for Zn2+ is 0.36 M. Formation of the 1 : 1 metal-to-ligand com-plex has been ascertained by Mass spectra and Job’s plot.

Keywords : Quinoline-carbohydrazide, Zn2+-sensor, LOD 0.36 M, 1 : 1 complex, spectral characterization.

Introduction

Elements are essential for the origination, growthand reproduction of living bodies. About 98% of thebody mass of man is made up of nine nonmetallicelements1. Transition metals present in trace and someare in ultratrace level in human body2. Elements suchas iron, zinc, copper etc. are essential components ofenzymes and facilitate their conversion to specific endproducts. Zinc, the second-most abundant (transition)metal following iron in the human body, is an om-nipotent metal and the average body content is 2–3 gin an adult3. The standard daily requirement of zincis 15–20 mg/day. Zinc plays an important role in cellproliferation, immunological and psychological func-tions and in metabolic activity. Plasma zinc levels aredecreased in pregnancy, acute myocardial infarction,infections, and malignancies. It is essential for normalspermatogenesis and maturation, functioning of neu-rotransmitters, development of thymus, epithelializa-tion, taste sensation, and secretion of pancreas andgastric enzymes. The excess “free zinc” may inducepathological diseases such as Alzheimer’s andParkinson’s disease4. Moreover, excess of zinc in theenvironment may reduce soil microbial activity, whichhas phytotoxic effects5–7. Thus detection of Zn2+ ispressing important for monitoring human health. Ex-

ploration of selective and sensitive chemosensor fordetection of ions in solution has been of considerableattention with biological and environmental interest8–

14. In the last two decades, significant number of fluo-rescent probes have been designed and some have beenused successfully in sensing of zinc at ultratrace level.Most of them have been developed based on quino-line15, bipyridyl16, coumarine17, pyrazoline18,tripyrrins19, BINOL20, fluorescein21, rhodamine22,fluorophores. Quinoline based fluorophore15,23 is as-sociated with imine (C=N), amide (-CONH-), sulfo-nyl derivatives etc.; however, quinoline-hydrazide hasnot been used for investigating sensor activity. In thiswork, we have designed and synthesized a hydrazidebased imidazole derivative, (E)-N-((1H-imidazol-2-yl)methylene)quinoline-2-carbohydrazide for the selec-tive and sensitive detection of Zn2+ in presence ofother commonly available metal ions. The DFT com-putation of optimized geometry of H2L and the com-plex has been used to explain the electronic spectralproperties.

ExperimentalMaterials and methods

Quinaldic acid and imidazole-2-carbaldehyde werepurchased from Sigma-Aldrich and quinoline-2-


700

carbohydrazide was synthesized following the publishedprocedure24. All other organic chemicals and inorganicsalts were obtained from commercial suppliers Merckand used without further purification. Aqueous solu-tions were prepared using Milli-Q water (Millipore).Elemental analyses were performed using a Perkin-Elmer 2400 Series-II CHN analyzer, Perkin-Elmer,USA elemental analyzer. UV-Vis spectra were recordedon Perkin-Elmer Lambda 25 spectrophotometer andfluorescence spectra were obtained using a Perkin-Elmer spectrofluorimeter model LS55, FT-IR spectra(KBr disk, 4000–400 cm–1) from a Perkin-Elmer LX-1FTIR spectrophotometer. NMR spectra were obtainedon a Bruker (AC) 300 MHz FT-NMR spectrometerusing TMS as an internal standard. ESI mass spectrawere recorded from a Water HRMS model XEVO-G2QTOF#YCA351 spectrometer. All of the measure-ments were conducted at room temperature. The fluo-rescence quantum yield was determined using fluores-cein as reference with a known quantum yield, R =0.79 in 0.1 M NaOH25. The experimental sample andreference were excited at same wavelength, maintain-ing almost same absorbance and fluorescence weremeasured. Area of the fluorescence spectra were mea-sured using the software available in the instrumentand the quantum yield was calculated by following theformula

AA

2S S SR

2R R S R

(Abs)(Abs)

where, S and R are the fluorescence quantum yieldof the samples and reference; AS and AR are the re-spective areas under emission spectra of the sampleand reference respectively. (Abs)R, (Abs)S are the ab-sorbance of sample and reference at the excitation wavelength and S

2, R2 are the refractive index of the

solvent used for the sample and the reference.

Synthesis of probe, H2L

The condensation of quinoline-2-carbohydrazide(0.187 g, 1.0 mmol) and imidazole-2-carbaldehyde(0.097 g, 1.0 mmol) under stirring condition in dryMeOH (15 ml) for 5 h at room temperature yields agrey precipitate. It was filtered off and washed severaltimes with MeOH and dried in open air. Yield : 88%,

m.p. >200 ºC (Scheme 1). Microanalytical data :C20H13N3O3 Calcd. (Found) : C, 63.39 (63.35); H,4.18 (4.19); N, 26.40 (26.37)%; 1H NMR (300 MHz,DMSO-d6) : 15.29 (1H, s, imidazole-NH), 12.31 (1H,s, NH), 8.63 (1H, s, imine-H), 8.25–7.99 (2H, m),7.99–7.85 (2H, m), 7.72 (1H, d, 18Hz), 7.51 (1H, t,18Hz), 7.26 (1H, d, 15Hz), 7.09 (1H, s) (ESI†, Fig.S1); IR : 3240 cm–1 (hydrazide -NH), 1668 cm–1

(–C=O), 1539 cm–1 (azomethine, -C=N), (ESI†, Fig.S2).

Scheme 1. Synthesis of H2L and the complex, [ZnL(H2O)].

Synthesis of [ZnL(H2O)]

To THF-MeOH (1 : 1, v/v, 10 ml) solution of H2L(1 mmol, 0.265 g), MeOH solution (10 ml) ofZn(NO3)2.6H2O (0.297 g, 1 mmol) was added andrefluxed for 3 h to yield a red precipitate. It was fil-tered off and washed several times with MeOH anddried in open air. Microanalytical data : Calcd. (%)C, 48.37; H, 3.48; N, 20.14; 1H NMR (300 MHz,DMSO-d6) : 8.85 (1H), 8.75 (1H), 8.57 (1H), 8.41(1H), 8.17 (1H), 7.95 (1H), 7.80 (1H), 7.42 (1H),7.28 (1H), 7.09 (1H) (ESI†, Fig. S3); IR : 3434 cm–1

((H2O)), 1529 cm–1 (azomethine, -C=N), (ESI†, Fig.S4).

General method for UV-Vis and fluorescence studies

The probe, H2L (1.32 mg, 0.001 mmol) was dis-solved in THF (5 ml) and 100 L of H2L solutiondiluted using 2 ml THF-MeOH (v/v 1 : 1) containingHEPES buffer (pH 7.2) to make the solution with totalvolume 2.1 ml. Zn(NO3)2.6H2O (2.97 mg, 0.001mmol) was dissolved in water (10 ml). The Zn2+ so-

Purkait et al. : A novel imidazolyl appended quinoline-hydrazide Schiff base fluorescent etc.

701

lution (100 L) were transferred to H2L solution pre-pared above. This procedure for sample solution prepa-ration also maintained for other cations. After mixingspectra were recorded at room temperature. For fluo-rescence study excitation wavelength used was 331nm (excitation slit = 10.0 and emission slit = 10.0).

Theoretical computation

H2L and [ZnL(H2O)] were optimized to generatethe structures by DFT/B3LYP method using Gaussian09 software26,27. 6-311G basis set was used for C, H,N, O, and LanL2DZ basis set was used as effectivepotential (ECP) set for Zn. To ensure the optimizedgeometries represent the local minima, vibrational fre-quency calculations were performed, and these onlyyielded positive eigen values. Theoretical UV-Vis spec-tra were calculated by time-dependent DFT/B3LYPmethod in methanol using conductor-like polarizablecontinuum model (CPCM)28,29. GAUSSSUM was usedto calculate the fractional contributions of variousgroups to each molecular orbital30.


Synthesis and formulation

The condensation of quinoline-2-carbohydrazide andimidazole-2-carbaldehyde synthesised (E)-N-((1H-imidazol-2-yl)methylene)quinoline-2-carbohydrazide(H2L) in good yield (88%). It has been characterizedby spectroscopic data (FTIR, Mass, NMR; ESI†).Molecular ion peak, (H2L+H)+ 265.03 (Mwt., 265.27)supports the molecular identity. The broad band at3240 cm–1 refers to (hydrazide-NH) and strongstretches at 1668 cm–1 and 1609 cm–1 are assigned to(C=O) and (C=N) respectively. The 1H NMR spec-trum of H2L (300 MHz, DMSO-d6) demonstrates sin-glet at 15.29 ppm corresponds to (imidazole-NH);hydrazine-NH appears at 12.31 ppm; imine-H (CH=N)appears at 8.63 ppm; and aromatic protons appear at7.0–8.3 ppm. The reaction of H2L with Zn(NO3)2.6H2O in methanol has isolated mononuclear zinc com-plex, [ZnL.(H2O)]. The complex has shown a broadpeak at 3434 cm–1 corresponds to (H2O) and (C=N)is observed at 1592 cm–1 which is shifted to lowerenergy compared to H2L. Mass spectrum shows mo-

lecular ion peak at 368.05 which may be due to[ZnL(H2O)+Na]+. The absence of (hydrazide-NH)and (imidazolyl-NH) support ionization of probe andits binding with ZnII during synthesis. All other pro-tons quantitatively appear in the spectrum. Thermaltreatment eliminates coordinated H2O at 112 ºC whichis supported by elimination of broad stretch at 3434cm–1 in the infrared spectrum. Thus, the structure pro-posal of probe and the complex (Scheme 1) are estab-lished.

UV-Vis spectroscopic studies

The interaction of H2L with Zn2+ has been exam-ined by spectrophotometric titration of H2L with in-cremental addition of Zn2+ in HEPES buffer (10 mM,pH 7.2) at 25 ºC in the same solvent, and has shownabsorption enhancement at 383 nm and decrement at332 nm, with the isosbestic point at 355 nm (Fig. 1)which suggests that the reaction is clean and straight-forward. The red-shifting of the bands of H2L uponZn2+ addition is attributed to expulsion of intramo-lecular charge transfer (ICT) through the chelation.The change of absorbance is linear until the molarratio [Zn2+] : [H2L] reaches 1 : 1, and no longerchanges with increase in [Zn2+]. It suggests that thestoichiometry between H2L and Zn2+ is 1 : 1. Toestablish the binding stoichiometry of H2L and Zn2+

Fig. 1. Change in absorption spectrum of H2L (50 M) upongradual addition of Zn2+ ions (5 M each) in THF-MeOH (v/v 1 : 1) (pH 7.2).


702

the Job’s plot has been generated by plotting absor-bance against different mole fractions of Zn2+ whilevolume of solution has remained fixed (Fig. 2) and themolar fraction maxima has been obtained at 0.5 molefraction, which indeed supports 1 : 1 complex forma-tion of H2L and Zn2+.

co-ordination of imidazolyl-N, azomethine-N and hy-drazide-O to metal ion (Scheme 1). To get furtherinsight about the complexation reaction the fluorimet-ric titration has been done and [(Fmax – F0)/(F – F0)]vs 1/[Zn2+] has been plotted following Benesi-Hildebrand equation (Fig. 5) and has determined thebinding constant [Kd, 7.6×104]. The limit of detec-tion (LOD) of Zn2+ has been calculated 0.36 M fol-lowing the 3 method (ESI†, Fig. S5). The fluores-

Fig. 2. Job’s plot for the reaction between H2L and Zn2+ inTHF-methanol (1 : 1, v/v).

Fluorescence sensing for Zn2+

Upon excitation the probe (H2L) at 331 nm, a weakemission is observed at 500 nm with fluorescence quan-tum yield (HL) 0.0021. On addition of Zn2+ the emis-sion band is red shifted to 575 nm. The fluorescenceemission of H2L with other cations (Na+, K+, Ca2+,Mg2+, Mn2+, Fe2+, Al3+, Co2+, Ni2+, Pd2+, Cd2+,Hg2+, Cu2+, Ba2+ , Pb2+ and Al3+) in THF-MeOH(v/v 1 : 1) (pH 7.2) is insignificant. Thus, the probe isselectively showing “turn-on” emission to Zn2+ underthe identical experimental condition (Fig. 3)31,32. Onincremental addition of Zn2+ to the solution of H2Lthe fluorescence intensity increases and becomes satu-rated when reached at 1 : 1 molar ratio which resultsenhancement of quantum yield to 0.0819 (39-fold in-crement compared to ligand). The emission intensityof the mixture does not change on excess addition ofZn2+ (Fig. 4). The augmentation in fluorescence in-tensity for H2L+Zn2+ may arise from the eliminationof photoinduced electron transfer (PET) in free H2Land chelation enhancement effect (CHEF) through the

Fig. 3. Change in absorption spectrum of H2L (50 M) upongradual addition of different metal ions (100 M each)in THF-MeOH (v/v 1 : 1) (pH 7.2).

Fig. 4. Change in emission spectrum of H2L (50 M) upongradual addition of Zn2+ ions (5 M each) THF-MeOH(v/v 1 : 1) (pH 7.2).


703

cence enhancement of H2L-Zn2+ complex has beenexamined in presence of other metal ions (Fig. 6).

Quinoline substituted fluorogenic motives15,23 havebeen used in large number in the identification of dif-ferent cations and anions; and the detection of Zn2+

appears in highest account. Some of the literature re-ports (Table 1) are selected considering structural simi-larity of present chemosensor; but quinoline-hydrazideis first time reported herewith.

Effect of pH variation on fluorescence intensity ofH2L and H2L-Zn2+ complex has been studied; it hasobserved that there is no significant fluorescence emis-sion of H2L at the pH range 2 to 12 and in presence ofZn2+ the ligand emits in the pH range between 3.0 to11 (Fig. 7). At high acidic medium (pH 2) ligand may

be protonated or hydrolyse Schiff base while in thebasic medium (pH 12) may precipitate Zn(OH)2 andthus, inhibits the complexation. This indicates that H2Lis useful for detection of Zn2+ in the biological pHthat is at much lower concentration than that of WHOrecommended value (76 M) in drinking water33.

Lifetime data were obtained upon excitation at 450nm, and the fluorescence decay curve was deconvolutedwith respect to the lamp profile. The observed flores-cence decay fits nicely with the bi-exponential decayprofile for both H2L and complex (Fig. 8), which issupported by goodness of fit (2) data in the regres-sion analyses. The average lifetime value of [H2L-Zn2+] (0.5 ns) is longer than that of H2L (0.038 ns).The metal-ligand orbital mixing in [H2L-Zn2+] maybe the reason for the longer lifetime of the excitedstate.

Density functional theory calculation

Geometry optimization of H2L and [ZnL(H2O)] hasbeen performed using DFT calculation with B3LYPmethod. The DFT optimized structure of the complexis a distorted tetrahedral where H2L acts as O,N,Nchelator to Zn2+. The calculated Zn-N (imine), Zn-O(amide carbonyl) and Zn-N (imidazolium-N) distancesare 2.04, 2.08 and 2.02 Å respectively and have beencomparable with similar structure31,32. Upon coordi-nation of metal ion with the H2L, the energy of HOMOincreased relative to those of free H2L while LUMOdecreases its energy relative to those of free H2L. Thedecrease in the LUMO level is more significant indi-cating that the LUMO was more stabilized than the

Fig. 5. Benesi-Hildebrand plot of {(Fmax – Fo)/(F – Fo)} vs1/[Zn2+] by fluorescence spectroscopy.

Fig. 6. Bar chart presenting fluorescence response of H2L in presence of different metal ions.

JICS-6


704

Fig. 7. Effect of pH on fluorescence intensity of receptor H2Land H2L-Zn2+ complex. Fig. 8. Decay profile of H2L and [H2L-Zn2+] complex.

Table 1. Structure of quinolinyl fluorophore, LOD of Zn2+ (and Reference)


705

HOMO. The HOMO-LUMO gap in H2L (3.55 eV)has been decreased in [ZnL(H2O)] (3.31 eV) whichsupports the red shift of absorption band from 332 nmto 483 nm in UV-Vis spectra (Fig. 9).

Conclusion

Quinoline-hydrazide (H2L) has been successfullyused as “turn-on” fluorescence chemosensor to Zn2+

ion in presence of large number of other metal ionsupon irradiation at 331 nm and shows high intenseemission (em, 575 nm). The limit of detection (LOD)for Zn2+ is 0.36 mM which is far below the WHOrecommended limit (76 M). The 1 : 1 metal-to-ligandcomplex has been ascertained by Mass spectra andJob’s plot.

Acknowledgement

Financial support from the Council of Scientificand Industrial Research (CSIR, Sanction no. 01(2894)/09/EMR-II), New Delhi, India is gratefully acknowl-edged. One of the authors (RP) is thankful to Depart-ment of Science and Technology (DST), Govt. of In-dia for providing DST-INSPIRE research fellowship.

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707


Triphenylamine-based oxidized bis(indolyl)methane as fluorogenic andchromogenic chemosensor for anions

Kumaresh Ghosh*a and Indrajit Sahaa,b

aDepartment of Chemistry, University of Kalyani, Kalyani-741 235, Nadia, West Bengal, India


bDepartment of Chemistry, R. K. Mahavidyalaya, Kailashahar, Unakoti-741 235, Tripura, India


Abstract : Triphenylamine coupled oxidised bis(indolyl)methane 1 has been designed and synthesized for opticalsensing of anions in organic (CH3CN) and semi aqueous (CH3CN/H2O, 4 : 1, v/v) medium. The sensory systemexhibited ratiometric change in emission upon addition of fluoride anion in CH3CN. Further, the chemosensor1 displayed selective coloration from yellow to pink in presence of F– anion in CH3CN while in aqueous aceto-nitrile sharp colour change from yellow to pink was observed upon addition of HSO4

– only. The differentialselectivity of 1 in different medium is attributed to the different proton transfer signaling modes.

Keywords : Bis(indolyl)methane, fluoride sensing, hydrogen sulphate sensing, ratiometric sensing, triphenylamine.

Introduction

The significant role that anions play in biological,

environmental and medicinal science has inspired su-

pramolecular chemists to design and synthesize nu-

merous receptors for the recognition and sensing of

anionic substrates1. Owing to their diverse geometry,

low charge to radii ratios, narrow pH window and

high solvation energy, design of anion receptor that

operate with high sensitivity and selectivity towards

desired analytes is particularly difficult. Charge-charge

interactions, hydrogen bonding interactions, coordina-

tion bonds and hydrophobic interactions are the key

tools that have been widely utilized for the recognition

and sensing of anionic species. As a result, numerous

reports that take the advantages of metal ions2,

pyridinium3, imidazolium/benzimidazolium4,

guanidinium5, polyammonium6, urea/thiourea7,

amide8, pyrrole9 and calixpyrrole10 functionalities have

been documented in literature. In this regard, synthetic

receptors that signal the presence of anions through

change in luminescence as well as colour of the solu-

tion have drawn significant attention in recent years11.

Among the various anions, fluoride has received

special attention due to its potential toxicity in various

living systems. Scientific challenges along with soci-

etal issues associated with the smallest anion fluoride

have made the recognition and sensing of this anion

by synthetic hosts is of special interest12. For example,

low concentration of fluoride in human diet has ben-

eficial health effects, particularly in dental care. How-

ever, excess consumption of fluoride has toxic side

effects, related to various human pathologies such as

neurological dysfunction, osteosarcoma etc. Although

significant progress has been made in the field of fluo-

ride recognition and sensing, there are only a few easy-

to-use chemosensors that operate with high sensitivity

owing to its high charge density and high hydration

energy.

The oxidised bis(indolyl)methane is a well estab-

lished chromophoric unit that recognizes anions in both

organic and aqueous organic solvents by exhibiting

color change of the solution. It provides an acidic hy-


708

drogen bond donor moiety and a basic hydrogen-bond

acceptor moiety for anion binding. Shao and co-work-

ers first reported that the oxidised bis(indolyl)methane

is an efficient chromogenic-sensing system for anion13.

This colorimetric anion sensor exhibited selective col-

oration either for F– in aprotic solvent or for HSO4– in

water containing medium based on different proton

transfer signaling modes. The same group modified

the bis(indolyl)methane system to tris(indolyl)methene,

a new kind of unexplored chromogenic molecules14.

These molecules can act as excellent chemosensors

for F– by two stages of proton transfer along with

stepwise drastic colour changes, and could also distin-

guish AcO– and H2PO4– through spectral behaviours.

Kim et al. also designed and synthesized dual colori-

metric sensing system based on this bis-(indolyl)methane

skeleton15.

cellent fluorescent probe and has been extensively used

by us and other groups in molecular recognition stu-

dies16. In the design 1, triphenyl-amine acts as

fluorophore as well as electron donor group and con-

jugated bis(indolyl) skeleton acts as a potential chro-

mophoric unit which contains an acidic hydrogen bond

donor and a basic hydrogen bond acceptor.


The synthesis of the designed compound 1 was

achieved according to the Scheme 1. It starts with 4-

(diphenylamino)benzaldehyde 2 which was synthesized

using literature procedure16d. The condensation reac-

tion of 2 with indole in CH3OH afforded the com-

pound 3 in 90% yields. The subsequent oxidation of 3with DDQ in CH3CN gave the desired compound 1 in

37% yields. All the compounds were characterized

using 1H NMR, 13C NMR, FTIR, and Mass spectro-

scopic tools.

The anion binding properties of 1 were investigated

using fluorescence, UV-Vis, and 1H NMR spectro-

scopic techniques. The receptor 1 exhibited a well de-

fined fluorescence band with maxima at 365 nm and

512 nm upon excitation at 300 nm in CH3CN. The

bands at 365 nm and 512 nm were assigned to

triphenylamine and bis(3-indolyl)methane moieties,

respectively. Upon complexation with the anions, the

emission intensities of both the bands were perturbed

differently depending upon the anionic species (Fig.

1). While the emission intensity at 365 nm was found

to be enhanced greatly upon complexation of H2PO4–,

Scheme 1. Reagents and conditions : (i) KHSO4, dry CH3OH, rt, 4 h, 90%; (ii) DDQ, dry CH3CN, 4 h, 37%.

Colour

1

132

Herein, we intended to study the anion binding

behavior of the oxidised bis(indolyl)methane when it

remains attached to the triphenylamine unit. The pro-

peller-shaped triphenylamine platform serves as an ex-

Ghosh et al. : Triphenylamine-based oxidized bis(indolyl)methane as fluorogenic and chromogenic etc.

709

Fig. 3 demonstrates that the titration of 1 with F–

follows a ratiometric change in emission with an

Fig. 1. Change in emission of receptor 1 (c = 3.55×10–5 M)upon addition of 15 equivalent amounts of putative an-ions in CH3CN.

Fig. 2. Change in emission of receptor 1 (c = 3.55×10–5 M)upon addition of H2PO4

– in CH3CN.

Fig. 3. Change in emission of receptor 1 (c = 3.55×10–5 M)upon addition of F– in CH3CN.

isosbestic point at 468 nm. With the increase in con-

centration of F–, while the intensity of the peak at 365

nm was increased, the emission at 512 nm was de-

creased gradually. Such characteristic spectral change

was observed only with F– and is thus diagnostic to

differentiate F- fluorimetrically from other anions taken

in the study. It is worthy to note that ratiometric fluo-

rescent probes have the important feature in that they

permit signal rationing increase the dynamic range and

provide bait-in correction for environmental effects17.

Other anions such as Cl–, Br–, I–, NO3–, CH3COO–,

ClO4– and HSO4

– interacted very weakly in CH3CN

showing no significant change in emission of 1.

In aqueous CH3CN (CH3CN : H2O, 4 : 1, v/v),

the receptor 1 showed only one emission band at 372

nm upon excitation at 300 nm. However, no signifi-

cant change in emission intensity of this band was

observed upon addition of various anions. Fig. 4, in

this aspect, represents the change in emission of 1upon addition of 15 equivalent amounts of anions in

aqueous CH3CN.

The ground state interaction of 1 with the anions

was investigated through UV-Vis spectroscopic tech-

nique in both CH3CN and aqueous CH3CN (CH3CN :

the peak at ~512 nm was changed merely (Fig. 2).

Among the halides, only F– ion interacted with 1 sig-

nificantly. The change in emission of 1 with the in-

crease in concentration of F– ion is different from the

other anions examined in the present study (Fig. 1).


710

H2O, 4 : 1, v/v) solvents. The receptor 1 showed two

absorption bands at 284 nm and 436 nm along with a

weak shouldering at around 550 nm in CH3CN. The

strong absorption band at 436 nm, responsible for yel-

low colour of the solution, was assigned as the ICT

absorption band of the conjugated bis(indole) skeleton.

The less strong shoulder peak at 550 nm is related to

its intermolecular hydrogen bond interaction, which

disappeared upon addition of polar protic solvent such

as H2O and CH3OH12.

Fig. 5 collectively represents the change in absorp-

tion of 1 upon addition of various anions. Except fluo-

ride, other anions did not show any red or blue shift in

the absorption spectra. Fig. 6 illustrates the change in

absorption of 1 with increase in concentration of F– in

CH3CN. As the concentration of F– ion increases,

absorbance at 436 nm decreases with a blue shift of 35

nm and a new band at 520 nm appeared showing an

isosbestic point at 480 nm. Three other isosbestic points

were noticed at 246 nm, 310 nm and 366 nm. In the

event the colour of the solution changes from yellow

to pink and this is attributed to the formation of

deprotonated receptor (Fig. 7A). Initially, F– ion forms

hydrogen bonding complex and at high concentration,

Fig. 4. Change in emission of receptor 1 (c = 3.55×10–5 M)upon addition of 15 equivalent amounts of putative an-ions in CH3CN/H2O (4 : 1, v/v).

Fig. 5. Change in absorbance of receptor 1 (c = 3.55×10–5 M)upon addition of 15 equivalent amounts of putative an-ions in CH3CN.

Fig. 6. Change in absorbance of receptor 1 (c = 3.55×10–5 M)upon addition of F– in CH3CN.

by virtue of its basic nature, it deprotonates the recep-

tor. The new band at 520 nm was related to the forma-

tion of deprotonated receptor. Such deprotonation was

related to the acidity of the H-bond donor sites and the

particular stability of [HF2]– hydrogen bond complex18.

On the other hand, in aqueous CH3CN (CH3CN :

H2O, 4 : 1, v/v), the compound 1 behaved differently

towards the same anions. In this solvent composition,


711

no characteristic change in UV-Vis spectra of 1, upon

titration with various anions, was observed except for

HSO4– and CH3COO–.

Fig. 8 represents the change in absorbance with

increase in concentration of F–, CH3COO– and

HSO4–. During complexation of AcO–, the band at

455 nm moved to 478 nm with the appearance of a

weak shoulder peak at 540 nm (Fig. 8b). In the case of

HSO4–, the band at 455 nm underwent red shift show-

ing new absorption band at 528 nm, which was gradu-

ally intensified upon complexation (Fig. 8c). This re-

sulted in a visual color change from yellow to pink

(Fig. 7B). Since the HSO4– anion is a relatively strong

acid (pKa = 1.99 in aqueous solution), the HSO4–-

induced change is mainly the consequence of a simple

protonation of receptor 1. The band that develops at

500 nm pertains to the protonated receptor [H21]+,

which was confirmed by the protonation of receptor 1

by using perchloric/acetic acid in CH3CN/H2O or in

CH3CN13.

The interaction of 1 with F– was also probed using1H NMR experiments, carried out in DMSO-d6 (solu-

bility problems in CD3CN in 1H NMR concentration

range prevented the study in CD3CN). The signals for

NH- proton in 1 was difficult to identify. This may be

either due to significant broadening or overlapping with

the other signals appeared in the aromatic region.

However, it was found that the signals for indole ring

protons in 1 underwent upfield shift upon addition of

0–2 equivalent amounts of F– ion (Fig. 9). These ob-

servations reveal that F– first establishes H-bond in-

teraction with the NH subunit of 1, while the second

F– induces the deprotonation of the NH fragment, which

brings electron density onto the -conjugated frame-

work with through-bond propagation, thus causing a

shielding effect and inducing upfield shift. In the full

deprotonated form, the polarization effect is no longer

present since the anion does not remain in proximity

to the receptor.

Fig. 7. Change in colour of 1 (c = 5.00×10–5 M) upon addition of 10 equivalent amounts various anions in (A) CH3CN and (B)CH3CN : H2O (4 : 1, v/v) : (a) 1, (b) ClO4

–, (c) H2PO4–, (d) CH3COO–, (e) HSO4

–, (f) NO3–, (g) F–, (h) Cl–, (i) Br–, (j)

I–.

a b c d e f g h i j

B

a b c d e f g h i j

A

Colour

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Fig. 8. Change in absorbance of receptor 1 (c = 3.55×10–5 M) upon addition : (a) F–, (b) AcO–, (c) HSO4– in CH3CN/H2O (4 : 1,

v/v).

Conclusion

In conclusion, it has been shown that triphenylaminecoupled oxidized bis(indolyl)methane 1 can report theselective recognition of anions through the change inphotophysical properties of the triphenylamine unit.The compound works both in organic and aqueousorganic solvents. The ratiometric change in emission,observed only with F– in CH3CN, is diagnostic todifferentiate F– anion fluorimetrically from other an-ions taken in the study. Also, the sensor exhibitedselective coloration either for F– in aprotic solvent andor for HSO4

– in water containing medium based ondifferent proton transfer signaling modes. The changein color of the receptor solution upon complexation ofF– and HSO4

– is sharp and reproducible. Further work

along this direction is underway in the laboratory.

Experimental

General : Reagents and solvents were purified us-ing standard techniques. Solvents were dried over theappropriate drying agent following standard procedure.Solvents for spectroscopic measurements were of spec-troscopic or HPLC grade. Infrared spectra were re-corded on Perkin-Elmer L120-00A spectrophotometer.1H NMR spectra were recorded at 400 MHz usingBruker instrument. Mass spectra were recorded on API2000 LCMS/MS instrument. Fluorescence measure-ments were done using Perkin-Elmer LS55 and UV-Vis absorption spectra were recorded at room tem-perature using Perkin-Elmer Lambda 25.


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4-(Di(1H-indol-3-yl)methyl)-N,N-diphenylaniline 3

KHSO4 (0.170 g, 1.25 mmol) was added to a mix-ture of indole (0.292 g, 2.5 mmol) and the aldehyde 2(0.342 g, 1.25 mmol) in dry methanol (10 mL), andthe reaction was stirred at room temperature for 4 h.Then water (10 mL) was added to quench the reac-tion, and the aqueous phase was extracted with CH2Cl2(3×10 mL). The organic phase was dried with anhy-drous Na2SO4, and was removed under reduced pres-sure to give the crude product. Repeated crystalliza-tion of the crude product using the mixture solvent(ethyl acetate : petroleum ether = 1 : 3, v/v) affordedthe product 3 as white solid (0.445 g, yield 90%),m.p. 100 ºC (decomposed); 1H NMR (400 MHz,CDCl3) : 7.93 (2H, s), 7.43 (2H, d, J=8 Hz), 7.36(2H, d, J=8 Hz), 7.23–7.15 (8H, m), 7.08–6.94 (10H,m), 6.72 (2H, s), 5.84 (1H, s); 13C NMR (100 MHz,CDCl3) : 147.9, 145.7, 138.5, 136.7, 129.4, 129.1,127.1, 125.6, 124.1, 123.9, 123.5, 122.4, 121.9,119.9, 119.8, 119.2, 111.0; FTIR (KBr, cm–1) : 3417,3057, 3035, 1587, 1505, 1493.

Receptor 1

To a stirred solution of 3 (0.244 g, 0.5 mmol) inCH3CN (10 mL), DDQ (0.068 g, 0.3 mmol) dissolvedin CH3CN (5 mL) was slowly added. The reaction

mixture was further stirred for 4 h, gave a dark redprecipitate, which was filtered, washed with CH3CN,and recrystallized from ethanol to afford pure com-pound 1 (0.100 g, yield 37%), m.p. 140 ºC (decom-posed); 1H NMR (400 MHz, DMSO-d6) : 7.96(2H, s), 7.54 (2H, d, J=8 Hz), 7.41 (4H, t, J=8 Hz),7.33 (2H, d, J=8 Hz), 7.22–7.15 (8H, m), 7.00 (4H,d, J=8 Hz), 6.85 (2H, d, J=8 Hz); 13C NMR (100MHz, DMSO-d6) : 151.8, 149.7, 146.2, 133.4,131.9, 129.8, 129.3, 128.1, 125.4, 124.5, 124.3,123.2, 122.2, 120.8, 119.9, 116.1 (one carbon in aro-matic region is unresolved); FTIR (KBr, cm–1) : 3392,3063, 1608, 1588, 1556, 1529, 1503, 1489; MS(LCMS) C35H25N3 requires 487.20; found 488.4(M+H)+.

General procedures of UV-Vis and fluorescence ti-tration

Stock solutions of the hosts and guests were pre-pared in CH3CN or CH3CN-H2O and 2.5 ml of theindividual host solution was taken in the cuvette. Thesolution was irradiated at the excitation wavelengthmaintaining the excitation and emission slits. Uponaddition of guest anions, the change in fluorescenceemission of the host was noticed. The correspondingemission values during titration were noted. For UV-Vis titration the receptors were dissolved in dry UV

Fig. 9. Partial 1H NMR (DMSO-d6, 400 MHz) spectra of 1 (8.20×10–3 M) in the presence and absence of F– ion.


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grade CH3CN or CH3CN-H2O and 2.5 ml of the indi-vidual host solution was taken in the cuvette. Then,anions, dissolved in dry CH3CN were individuallyadded in different amounts to the receptor solution andthe corresponding absorbance values during titrationwere noted.

Acknowledgement

KG thanks DST [755 (Sanc.)/ST/P/S & T/4G-3/2014, dated 27.11.2014], West Bengal, for providingfinancial assistance. Research grant from UGC, NewDelhi under minor research project [No. F. 5-344/2015-16/MRP/NERO/1083 dated 29.03.2015] is alsogratefully acknowledged.

References

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11. (a) M. Cametti and K. Rissanen, Chem. Commun.,2009, 2809; (b) M. Cametti and K. Rissanen,Chem. Soc. Rev., 2013, 42, 2016.

12. B. N. G. Giepmans, S. R. Adams, M. H. Ellismanand R. Y. Tsien, Science, 2006, 312, 217; (b) D.W. Domaille, E. L. Que and C. Chang, J. Nat.Chem. Biol., 2008, 4, 168; (c) L. E. Santos-Figueroa, M. E. Moragues, E. Climent, A. Agostini,R. Martinez-Manez and F. Sancenon, Chem. Soc.Rev., 2013, 42, 3489.

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Chemosensors based on diazole derivatives

Manjira Mukherjee and Pabitra Chattopadhyay*

Department of Chemistry, The University of Burdwan , Golapbag, Burdwan-713 104, West Bengal, India



Abstract : The heterocyclic diazole compounds and their derivatives not only exhibit a wide range of pharma-cological activity but also can act as bioagent and revolve around its ability to bond to various guests as host.Hence it is useful in designing chemosensors. This review summarizes the most recent and relevant advances inthis area.

Keywords : Diazole, bioagent, chemosensors.

Introduction

Azoles are five-membered heterocyclic compoundshaving a nitrogen atom and at least one other non-carbon atom (i.e. nitrogen, sulfur or oxygen) as partof the ring, diazoles refer to a special class of azolesas these are also five-membered aromatic heterocycliccompounds consisting of three carbon atoms and twonitrogen heteroatoms. Their names have been derivedfrom the Hantzsch-Widman nomenclature (also calledthe extended Hantzsch-Widman system), where thenumbering of the ring atoms in azoles starts with theheteroatom that is not part of a double bond, and thenproceeds towards the other heteroatom. These com-pounds having two double bonds are aromatic in na-ture due to the participation of only one lone pair ofelectrons of each heteroatom in the ring as a part ofthe aromatic bonding in an azole. The two double bondsof the parent compound could be successively reducedto produce reduced analog with fewer like azolinesand azolidines and upon reduction the names of azolesmaintain the prefix e.g. pyrazoline, pyrazolidine.

Such heterocyclic units have many uses. They ex-hibit a wide range of pharmacological activity, ubiqui-tous in nature and play a critical role in many struc-tures within the human body, notably histamine andhystidine which contain imidazole unit. A special in-terest of researchers towards benzimidazole deriva-tives was stimulated by the fact that 5,6-dimethyl-1-

(-dribofuranosyl)benzimidazole is an basic part of thestructure of vitamine B121, and benzimidazole is astructural unit of naturally occurring nucleotide, dueto which it can easily interact with the biopolymers ofliving system, which is responsible for its numerousbiological aspects. Diazoles like benzimidazole andpyrazole ring become an important pharmacophore inmodern drug discovery2–4. Moreover dazoles can beused as bioagent and its ability to bond to variousguests as ligand make it useful in designingchemosensors for various ions. This review coversrecent important development of diazole (here benzi-midazole and pyrazole) as receptors for the recogni-tion of various anions, cations and neutral molecules.

Imidazole/benzimidazole based compounds aschemosensor

Benzimidazole ring behaves as an excellent hydro-gen bond donor moiety in synthetic anion receptorsystems, and the acidity of the NH proton of the imi-dazole can be tuned by changing the electronic proper-ties of the imidazole substituents. On the other hand,the presence of a donor pyridine-like nitrogen atomwithin the ring, capable of selectively binding cationicspecies also converts the imidazole derivatives intoexcellent metal ion sensors. Due to the emissive prop-erties of the aromatic ring, benzimidazole has also beencommonly used in molecular recognition of all cat-ions, anions and neutral molecules. As a result, this


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moiety has been used not only as a binding unit forcations and anions, as the imidazole derivatives do,but also as a fluorogenic antenna. Another fact withthe imidazole ring is the two NH protons of differentacidity caused by the electronic effect of the benzenering. Here we cover important development of benzi-midazole derivative receptors for the recognition ofvarious anions, cations and neutral molecules. We havegrouped the receptors to their ion sensing which in-clude cations, anions, cations-anions both and neutralmolecules which behaving as sensors for different ions.

Chemosensors for cations :

In the field of molecular recognition of cationsGarcía and co-workers had mentioned the macrocyclicligand, 1 (Table 1). The N and S atoms present in thissystem act as recognition sites while the two benzimi-dazole units act as fluorescent antenna for the detec-tion of transition metal cations in water5. Abathochromic shift in the UV-Vis bands upon additionof the corresponding cations is due to the metal coor-dination. Titration experiments carried out by usingthis technique were used to calculate the associationconstant of the different complexes, showing a higheraffinity towards Cr3+, followed by the Fe2+ cation,while other metal cations as Hg2+ or Cd2+ have muchsmaller association constants. The selectivity found inthis receptor has been attributed to the relative Lewisacidity of the cations that dominates the coordinationcapabilities of the receptor. The small selectivity inthe process indicates that fluorescence quenching hasa diffusive behaviour.

Fahrni’s group developed a Zn2+-selectiveratiometric biologically suitable probe (2) showed thecation-induced inhibition of excited-state intramolecu-lar proton transfer (ESIPT) with a series of 2-(2-benzenesulfonamidophenyl)benzimidazole derivativesin emission study6. In the absence of Zn2+ at neutralpH, the fluorophores undergo ESIPT to yield a highlyStokes’ shifted emission from the proton-transfer tau-tomer. Coordination of Zn2+ inhibits the ESIPT pro-cess and yields a significant hypsochromic shift of thefluorescence emission maximum. Due to the modulararchitecture of the fluorophore, the Zn2+ binding af-finity can be readily tuned by implementing simple

structural modifications. The synthesized probes aresuitable to gauge free Zn2+ concentrations in the mi-cromolar to picomolar range under physiological con-ditions.

Guo and co-workers devised a fluorescent 2-(2-pyridinyl)benzimidazole based Zn2+ sensor (3)7. Thesensor demonstrates a Zn2+-specific emission shift andenhancement with a 1 : 1 binding ratio. Due to theZn2+-induced coplanation via reversion/rotation, thesensor behaves as a ratiometric sensor. The intracellu-lar Zn2+ imaging ability of the sensor has been testedin HeLa cells using a confocal microscope.

Rao and co-workers has reported the benzimida-zole-substituted calix[4]arene (4) for the selective rec-ognition of Hg2+ versus a large number of divalentcations (Table 1)8 in MeOH, anhydrous CH3CN andalso in different aqueous mixtures of this organic sol-vent. In 50% aqueous solution, a 10-fold quenching inthe fluorescence was observed only in the case of Hg2+,whereas all other divalent metal cations tested exhi-bited almost no quenching in fluorescent intensity. How-ever, when the amount of water decreases, the selec-tivity for this cation also decreases, due to the effectof a less competitive media. Binding of Hg2+ has beenattributed, by 1H NMR experiments, to the nitrogenatoms in the benzimidazole ring and the oxygen atomsof the amide bridge, as the theoretical calculations alsopredicted. The Hg2+-complex formed has been iso-lated, characterized and studied on surface by TEM,SEM and AFM microscopies.

Minkin and co-workers synthesized a number of 1-(9-anthrylmethyl)-2-aryl-1H-benzimidazoles (5)9. Studyon their luminescent properties and complexing abilityshowed that the sensitivity of these systems to H+ andHg2+ ions is related to fluorescence quenching by theaction of these cations and effectively selective towardsH+ and Hg2+ ions.

Jang’s group synthesized a benzimidazole-basedfluorescent receptor (6) bearing imine linkages withtwo sets of sp2 nitrogens, and investigated its bindingproperties toward various metal ions10. The receptorexhibited a shift in emission band upon binding withFe3+ ions, and no such significant response was no-ticed in other metal ions. The receptor shows a prop-

Mukherjee et al. : Chemosensors based on diazole derivatives

717

erty of selective ratiometric fluorescent probe of Fe3+

ions without interferences of the background metal ions.

Singh and co-workers have synthesized a novel fluo-rescent receptor (7) based upon a benzimidazole moi-ety in a dipodal framework11. The receptor exhibiteda dual fluorescence emission which is quenched uponaddition of Cu2+ or Fe3+. Interestingly, the receptor

offers a ratiometric property and an ‘OR’ logic gateproperty to Cu2+ and Fe3+.

A different approach has been followed by Tangand Nandhakumar who designed a Rhodamine B colo-rimetric chemosensor (8) based on the chemical changesinduced by CuII anion (Table 1)12. UV-Vis experi-ments demonstrated that upon addition of CuII the

Table 1. Benzimidazole based chemosensors recognizing cations


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Table-1 (contd.)


719

Table-1 (contd.)

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change of colour in the solution from colourless topink with appearance of a low energy band. This colourchange has been attributed to a ring-opening processof the spirolactam form of chemosensor (8). Additionof other metal cations does not produce such changesin the absorption spectrum and does not disturb theeffect of CuII in competitivity experiments. Thereversibility of the process has confirmed by the addi-tion of EDTA to the medium that cause the disappear-ance of colour, returning the solution to the initialcolourless appearance.

A rhodamine-benzimidazole conjugate (9) was de-signed and synthesized by Li and co-workers whichdetect selectivity Fe3+ in a 1 : 1 stoichiometry overother metal ions in acetonitrile13. A thousand time fluo-rescence intensity enhancement was observed at themaximum emission wavelength of 582 nm upon theaddition of 10 equiv. of Fe3+ the detection limit was1.5×10–8 M.

Wang and co-workers synthesized a dehydroabietylmolecule (10) with a benzimidazole unit for the mo-lecular recognition of metal cations14. Receptor (10)decreases its fluorescent emission at em = 540 nmupon addition of CuII whilst the rest of the cations donot modify or increase the fluorescent emission. More-over, addition of up to 2 equiv. of CuII also modifiesthe absorption spectrum. The Job’s plot of the UV-Visdata yielded a 1 : 1 stoichiometry for its CuII com-plex.

Ghosh et al. reported a simple tripodal shapedchemosensor (11) for metal ions15. In CH3CN con-taining 0.04% DMSO, upon excitation at 370 nm, thechemosensor exhibited structured emission centred at

418 nm, which increased to a significant extent uponcomplexation of CuII. The other metal ions except Zn2+

and Hg2+ examined in this study did not exhibit anymarked change in emission under a similar condition.Although Cu2+ ion showed strong interaction with (11),Zn2+ and Hg2+ ions exhibited moderate binding.

Jang’s group synthesized a novel benzimidazole-based, anthracene-coupled fluorescent receptor (12)capable of recognizing and estimating the concentra-tions of Fe3+ in semi-aqueous solution by ratiometricestimation16. The sensor can be made highly selectivefor Fe3+ over other metal ions by changing the sol-vent composition.

Ghosh et al. reported a benzimidazole basedchemosensor (13), derived from L-valine17. Thechemosensor effectively recognises Hg2+ ion in theopen cleft in CH3CN containing 0.2% DMSO by ex-hibiting significant enhancement in fluorescence emis-sion. The steric isopropyl groups in (13) play the keyrole in the selectivity. The ensemble of (13).Hg2+

also showed the fluorescence sensing of L-cysteine,homocysteine, and glutathione over the other aminoacids with no thiol group in aq. DMSO (DMSO :H2O, 4 : 1, v/v).

Luxami et al. reported a probe (14) selectively bindwith Zn2+ ions at pH 7.0 (10 mM, HEPES) in CH3CN-H2O (4 : 1) and give a new emission band at 490nm18. These can estimate 20 nM Zn2+ ions as thelowest detection limit. The determination of Zn2+ ionsby probe (14) is not interfered by the presence of othermetal ions expects in the case of Cu2+ causes only thefluorescence quenching. In acetonitrile, the additionof different concentrations of Cu2+ (2 mM, 5 mM, 10

Table-1 (contd.)


721

mM) and a fixed amount of F– (25 mM) to a solutionof (14) (0.25 mM, CH3CN) elaborates Cu2+ ion con-centration dependent NOR, INH and AND Booleanoperators with three distinct emission channels at 585,515 and 400 nm, respectively.

Dessingou’s group showed the benzimidazole con-taining moiety (15) act as a selective “turn-on” fluo-rescence response toward Ag+19. The selectivity of(15) has been explored in aqueous methanol, resultingin selective 8-fold switch-on fluorescence response to-ward Ag+ among 14 different transition, alkali, andalkaline earth metal ions studied. The complexation ofAg+ by (15) has been addressed by ESI-MS, 1H NMR,and UV-Vis spectra. Microstructural features of (15)and its Ag+ complex have been measured by AFMand TEM. The morphological features of (15) aloneand (15) in the presence of Ag+ differ dramaticallyboth in shape and size, and the ion induces the forma-tion of chains owing to its coordinating ability towardbenzimidazole. Further, the in situ [Ag+-(15) ] com-plex was titrated against 20 naturally occurring aminoacids and found that this complex acts as a secondaryrecognition ensemble toward Cys, Asp, and Glu byswitch-off fluorescence.

Singh et al. developed a new benzimidazole-basedreceptor (16) with potential functional groups for ex-cited state proton transfer (ESPT) through ketoenoltautomerism20. The enol form of the receptor selec-tively recognizes Zn2+, allowing it to be used as aratiometric fluorescence sensor in DMSO/CH3CN(1 : 9, v/v). The binding event triggers a blue-shiftedband through the modulation of charge transfer transi-tions. The sensor is applicable for recognizing Zn2+

in the cytoplasm of Saccharomyces cerevisiae.

An imine-linked, benzimidazole-based sensor (17),reported by Jang et al.21, was used for chromogenicrecognition of Mg2+ and fluorescent recognition ofCr3+. Addition of Mg2+ to a solution of (17) inCH3CN-HEPES buffer (8 : 2, v/v, pH 7.0) led to astepwise decrease in absorbance at 400 nm and anincrease at 350 nm with a clear isosbestic point at 385nm. Cr3+ binding with sensor (17) caused a change inthe fluorescence spectra of (17) with quenching at 415

nm and enhancement at 475 nm. In the absence ofCr3+, the enol form of (17) was in equilibrium withits keto tautomer in the excited state. The modulationof the fluorescence spectrum of (17) with the additionof Cr3+ ions was due to the formation of a stableCr3+ complex with the keto form of (17). In addition,(17) was applicable for staining the cytoplasm of mi-crobial cells enriched with Cr3+.

Two Zn2+ ion selective fluorescent probes (18a)and (18b) based on a coumarin Schiff base were de-signed and synthesized by Guo et al. on the basis ofC=N isomerization mechanism22. Both the X-ray crys-tal structure of the zinc complex and the Job’s plotsshowed a 1 : 1 probe-Zn2+ ion identification withhigh selectivity and sensitivity. The probe (18a) wasalso used to image intracellular Zn2+ ions in MCF-7cells with a good performance.

Interestingly, imidazo-benzocrown ether-based iono-phores (19-21) bearing arylthienyl and bithienyl -con-jugated bridges have been evaluated as fluorimetricsensors for CuII and PdII ions (Table 1). Moreover,systems (20 and 21) have also proved to be efficientsensors for basic anions such as F–, due to changes inemission after deprotonation of the imidazole NH bythe F– ion23.

Molina et al. reported a redox, chromogenic, andfluorescent chemosensor molecule (22) based on adeazapurine ring which could selectively detect aque-ous Pb2+ in acetonitrile over other metal ions. Probe(22) showed a redox shift (E1/2 = 0.15 V of the FeII/FeIII redox couple) with a color change from the col-orless to orange, and an emission change of 620-fold,with an unprecedented detection limit of 2.7 gL–1 24. The signal transduction occurs via a reversibleCHEF with this inherent quenching metal ion.

A mercury non-sulfur and simple fluorescentchemosensor (23) based on the benzimidazole groupand quinoline group as the fluorescence signal grouphas been designed and synthesized by Chen’s group25.The receptor could instantly detect the Hg2+ cationover other cations by fluorescence spectroscopy in H2O/DMSO (1 : 9, v/v) solution with specific selectivityand high sensitivity with the fluorescence color change


722

of the solution from blue to colorless only after theaddition of Hg2+ in aqueous media. Moreover, fur-ther study demonstrates that the detection limit on thefluorescence response of the sensor to Hg2+ is 9.56×10–9 M. Test strips based on (23) were fabricated,which could act as convenient and efficient Hg2+ testkits. Thus, the probe should have potential applica-tions in an aqueous environment for the monitoring ofmercury.

Zhang et al. reported a fluorescent sensor (24) basedon benzimidazo[2,1-a]benz[de]isoquinoline-7-onewhich can be used as chemosensor for various metalions26. By the introduction of N,N-bis(pyridin-2-ylmethyl)-ethane-1,2-diamine as an electron-donatingreceptor, chemosensor (24) exhibited weak fluores-cence due to fluorescence quenching by the PET (pho-toinduced electron transfer) process from the lone pairelectrons on the nitrogen atom to the fluorophore. Onthe other hand, the receptor may have a strong bindingability toward metal ions due to its multiple coordinat-ing groups. After binding with metal ions, a large che-lation-enhanced fluorescence (CHEF) effect would beobserved because the chelation abrogated the PET pro-cess giving it possible to be used as a fluorescent sen-sor in pure water.

Mashraqui et al. synthesized a chemosensor (25) intwo steps from readily accessible 2,6-bis(2-benzi-midazole)pyridine. Photophysical studies revealed thatof the several metal ions examined, biologically andenvironmentally significant Zn2+ exhibited highly se-lective emission wavelength shifts under the buffercondition27. In contrast to Zn2+, the coordinativelycompeting and toxic Cd2+ elicited less remarkableoptical responses as evidenced by its two order ofmagnitude lower stability constant compared to that ofZn2+. Moreover, metal ions, viz. Li+, Na+, K+,Mg2+, Ca2+, Ba2+, Co2+, Ni2+, Cu2+, Hg2+ andPb2+ exhibited insignificant optical perturbations evenin concentrations far exceeding Zn2+. Clearly, theprobe has the attributes to selectively target Zn2+ byratiometric analysis under buffer conditions.

A rhodamine-benzimidazole based chemosensor (26)was designed and prepared by Li’s group for Fe3+ via

opening of the spiro-ring to give fluorescent and col-ored species. (26) exhibited high selectivity and excel-lent sensitivity in both absorbance and fluorescencedetection of Fe3+ in aqueous solution with compara-tively wide pH range (5.8–7.4)28. The detection limitof this newly developed probe was shown to be up to2.74 M. The reversibility establishes the potential ofboth probes as chemosensors for Fe3+ detection. Fluo-rescence imaging experiments of Fe3+ in livingMGC803 cells demonstrated its value of practical ap-plications in biological systems. It also displayed en-hanced fluorescence intensities and clear color changesupon recognition. Moreover, fast response (in 10 s)and neutral aqueous medium made it possible to bepractical application for Fe3+ ions. The probe couldbe applied in biological systems for the detection ofFe3+ through confocal laser scanning microscopy ex-periments.

On the basis of fluorescence resonance energy trans-fer (FRET) from benzimidazole to a rhodamine moi-ety, another rhodamine-benzimidazole conjugate (27)ratiometric fluorescent probe has been designed andsynthesized by Goswami et al.29. (27) selectively bindsto Cu2+, showing visually observable changes in ab-sorption and emission behavior, and demonstrates aneffective intracellular Cu2+ imaging ability, allowingit to function as a cytoplasm marker.

Tang’s group reported a phenylbenzimidazolederivatized sensor (28) that behaves as a ratiometricfluorescent receptor for Zn2+ in water has been de-scribed30. In HEPES buffer at pH 7.4, sensor (28)displays a weak fluorescence emission band at 367nm. Upon addition of Zn2+, the emission intensity at367 nm is decreased, concomitantly, a new emissionband centered at 426 nm is developed, thus facilitatesa ratiometric Zn2+ sensing behavior. Sensor (28) bindsZn2+ through a 1 : 1 binding stoichiometry with highselectivity over other metal cations. Sensor displays alinear response to Zn2+ concentration from 0 to6.0×10–5 M, sensor (28) also exhibits high sensitivityto Zn2+ with a detection limit of 3.31×10–7 M.

Li and co-workers reported an optical and fluoro-metric pH sensor (29) by attaching pH sensitive group


723

benzimidazole at the 2-position of borondipyrro-methene31. Due to the electron-donor effect, benzimi-dazole group caused obvious bathochromic shift in theabsorption and fluorescence spectra. Upon protona-tion, the solution exhibited drastic blue-shifted absorp-tion and enhanced fluorescence, and the color changedfrom yellow to green simultaneously. The sensingmechanism was elucidated by quantum chemistry cal-culation approach. Cell experiments were carried outto verify the compound could selectively locate in theacidic lysosome. These results indicated the benzimi-dazole-BODIPY could be used as an optical and “off-on” fluorescent pH indicator.

A highly sensitive and selective benzimidazole basedcolourimetric chemosensor (30) for the efficient de-tection of Ni2+ has been reported by Mondal et al.32.The synthesized chemosensor (30) is highly efficientin detecting Ni2+ over other metal ions that commonlycoexist with Ni2+ in physiological and environmentalsamples. Probe (30) also shows distinct color changefrom orange yellow to blue visible under the “naked-eye” due to specific binding with Ni2+. This colorchange corresponds to a large red shift of the UV-Visspectrum from 403 nm to 600 nm with a distinctisosbestic point at around 500 nm. The cation sensingproperty of the receptor (30) has been examined byUV-Vis spectroscopy. Electronic structure of its Ni2+

complex and sensing mechanism has been interpretedby DFT and TDDFT calculations.

Naphthalene benzimidazole conjugate (31) bearinga hydroxyl group was synthesized by Nandhakumar etal. Its binding properties towards various metal ionswere examined and it showed a high selectivity andsensitivity towards Al3+ ions in CH3CN-H2O solution(1 : 1, v/v, HEPES 50 mM, pH 7.0)33. The sensorwas designed in such a way that the naphthalene actsas fluorophore which is covalently attached to the ben-zimidazole scaffold which contains the heterocyclic ni-trogen atoms. The recognition processes follows a photoinduced electron transfer (PET) mechanism assistedwith the restricted intramolecular C-C single bond ro-tation and are scarcely influenced by other coexistingmetal ions. In addition, determination of Al3+ in a

variety of sewage water samples was also determined.These authors synthesized a quinoline benzimidazole-conjugate (32) for the highly selective detection of ZnII

both by colorimetry and fluorimetry34. Probe (32)senses Zn2+ over other cations as fluorescence “off-on” behaviour in HEPES-buffered CH3CN/H2O(1 : 1, v/v, pH 7.0) solution. A possible mechanism isproposed based on the inhibition of PET and intramo-lecular restricted torsional rotation through the C-Csingle bond between the quinoline benzimidazole-con-jugate. This probe has been reported as a usefulchemosensor to detect Zn2+ ions in much real sampleanalysis. Singh’s group synthesized a novel receptorand multianalyte fluorescent probe with benzimidazolemoieties in a tripodal framework (33)35. The receptordisplays rarely observed metal specific fluorescenceenhancement at two different wavelengths. The recep-tor was investigated for the simultaneous analysis ofCu2+ and Fe3+ and successfully quantified the ionswithout interference over a wide concentration range.

Recently reported some benzimidazole derivativeshaving quinazoline moiety (34-42) (viz. Table 1)showed metal sensing property either by formation ofchelating environment for incoming metal ions. Inter-estingly, in some cases this type of compounds ligatethe metal ions selectively through solvent/metal ionassisted 1,5- tropic shift (34-42). The first examplewhere reported a probe 2,6-bis(5,6-dihydrobenzo-[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol(34) as a selective and sensitive fluorescent probe forZn2+ in a HEPES buffer (50 mM, DMSO : water =1 : 9 (v/v), pH 7.2) at 25 ºC36, where formation ofthe zinc complex needs a [1,5] sigmatropic-type shiftof the secondary N-H proton of the probe (34) prior tobinding with Zn2+ to form a Schiff base structureevidenced by the solid state crystal structure. The se-lective enhancement of fluorescence intensity in pres-ence of Zn2+ is due to the formation of dinuclearZn2+ complex [Zn2(C35H25N6O)(OH)(NO3)2(H2O)]with increase of fluorescence quantum yield of thechemosensor by more than 12-fold in the presence of2 equiv. of the zinc ion. Here, upon complexation, the


724

lone pair of electrons on the N atom of the organicmoiety is no longer available for PET, leading to fluo-rescence enhancement. This probe is also potent toidentify the distribution of Zn2+ ions in the livingcells (A375 and HT-29) using fluorescence micros-copy. Similar type compound (35) have also reportedas a highly selective ratiometric fluorescent sensor forFe2+ at pH 4.0–5.0 and Fe3+ at pH 6.5–8.0 in aceto-nitrile-HEPES buffer (1/4) (v/v) medium37. The probeis a “naked-eye” chemosensor for two states of ironand is efficient for detecting in vitro Fe3+ ions in thebiological organelles by developing a fluorescenceimages. According to the same group, probe (36) wasrationally illustrated as a highly selective cell-perme-able ratiometric fluorescent chemosensor for ReOV ionin acetonitrile : water = 9 : 1 (v/v) at 25 ºC38. Thefluorescence quantum yield of the chemosensor (36)was only 0.198 at 410 nm, and it increased more than3-fold in the presence of 2 equiv. of the ReOV ion at478 nm with almost no interferences of other metalions and relevant anions. In another example a benz-imidazole based fluorescent Cr3+ receptor, (37) wasreported39. The sensor is efficient for detection of Cr3+

in vitro, developing a good image of the biologicalorganelles. Recently there is example of two structur-ally modified quinazoline derivatives (38 and 39) actas highly selective chemosensor for Al3+ ions inDMSO-H2O (1 : 9, v/v) over the other competitivemetal ions40. Both the probes show a red shifted fluo-rescence after addition of Al3+ ions and later the fur-ther fluorescence enhancement is due to chelation en-hanced fluorescence (CHEF) through inhibition ofphotoinduced electron transfer (PET). (38) detects Al3+

ions as low as 9 nM whereas (39) can detect Al3+ ionsas low as 1.48 nM in 100 mM HEPES buffer (water/DMSO, 9/1, v/v) at biological pH within a very shortresponsive time (15–20 s). This two non cytotoxic probecan efficiently detect the intercellular distribution ofAl3+ ions in living cells under fluorescence micro-scope to exhibit its sensible applications in the biologi-cal systems. In this context Chellappa et al. reported aratiometric chemosensor, 6-imidazo-2-yl-5,6-dihydro-benzo[4,5]imidazo[1,2-c]quinazoline (40) selective for

Al3+ ions as low as 5.3×10–7 M in aqueous-DMSO41.A highly fluorescent and efficient ratiometric “turn-on” probe for Hg2+ and Al3+ based on quinazoline(41) has been designed and synthesized by Pandey andco-authors42. Limit of detection (LOD) and associa-tion constants revealed its superior binding affinity andsensitivity toward Hg2+ over Al3+. Paul et al. re-ported structurally characterised quinazolinefunctionalized benzimidazole-based fluorogenicchemosensor (42) as a highly selective colorimetricand fluorescence sensor for Cu2+ ions in DMF/0.02M HEPES (1 : 1, v/v, pH 7.4) medium43. Again, thiscopper(II) complex acts as a metal based highly selec-tive and sensitive chemosensor for S2– ions even in thepresence of other commonly coexisting anions such inDMF/0.02 M HEPES (1 : 1, v/v, pH 7.4) mediumdue to the liberation of probe (42). Quantification analy-sis indicates that these receptors, (42) and its CuII com-plex, can detect Cu2+ ions and S2– ions upto 1.6×10–9

M and 5.2×10–6 M, respectively.

Chemosensors for anions :

The benzimidazole based organic moieties of judi-cial designs are also effective to detect the anions se-lectively. Jang and co-workers reported a compoundcomprise of three bezimidazole units connected by atripodal 2,2,2-trioxiethylamine linker (43) which be-haves as a good I– ion selective chemosensor in CH3CN/water (9 : 1) buffered with HEPES at neutral pH basedon quenching mechanism44. The 1H NMR titrationwith iodide showed that this anion is fundamentallybound by the NH protons of the benzimidazole moi-ety.

The receptors (44 and 45), reported by Jang andco-workers, in which one or two 2-aminobenzimidazolegroups are connected to an anthracene ring are signifi-cant in the field of molecular recognition of anions45.Receptor (44) displayed strong fluorescence emissionin CH3CN, which is quenched with the addition ofhalide, AcO– and H2PO4

– anions. The quenchingmechanism has been attributed to a photoinduced elec-tron transfer (PET) process. Compound (45), bearingtwo benzimidazole units, presents similar results andselectivities, though higher association constants were


725

found and a preference for AcO– over F– was alsoobserved.

Jang’s group synthesized a fluorescent receptor (46)bearing benzimidazole moiety as recognition sites (viz.Table 2)46. The recognition behaviour of the receptortowards various anions has been evaluated in CH3CN.The receptor showed ratiometric fluorescent changesonly with CH3COO–, and it showed no significant re-sponse to any of other anions such as Cl–, Br–, I–,HSO4

–, NO3–, C6H5COO– and H2PO4

–.

Lin and co-workers published a chromogenic “na-ked-eye” anions receptor (47) having a dinitrobenz-imidazole unit connected to a phenol donating groupin an aqueous media47. The presence of the nitro groupsincreases the polarity of the molecule, making it moresuitable for chromogenic detection and increases theacidity of the NH group of benzimidazole. Addition ofF– to a solution of compound (47) induces a clearcolor change that is not observable with other halideanions. The fluoride ion binds strongly with (47) be-cause of its highest electro-negativity and smallest sizeamong all of the anions being investigated here. In theUV-Vis spectrum of the complex formed, a new charge-transfer band appears which is responsible for thiscolour change. The 1H NMR study carried out to in-vestigate the coordination modes illustrates that theNH group of the benzimidazole and the phenolic OHare responsible for the F– ion coordination.

Within the concept of two-arms receptors, Jang andco-workers synthesized two isophthalamide receptorscontaining benzimidazole (48a) and nitrobenzimidazole(45b) units48 and a 1,3-bisurea bearing two benzimi-dazole moieties (49)49. Compound (45b) experiencesa bathochromic shift of the UV-Vis bands with theaddition of F– and AcO– anions in CH3CN/DMSO(99 : 1) observable by “naked-eye”, while the rest of theanions tested do not cause any important variations.When the same titrations are carried out on the relatedreceptor (48a), it exhibited no change in the absorp-tion spectrum upon addition of any of the anions as-sayed. In the case of (49), after addition of 5 equiv. ofthe sodium salt of a large variety of anions, only the

phosphate anion quenches the fluorescence of the re-ceptor in a DMSO/water mixture while the other an-ions do not modify importantly the fluorescence emis-sion. A PET quenching process has been addressed toexplain the fluorescence changes. The 1H NMR titra-tion of the receptor with phosphate exhibits an upfieldshifting of the thiourea and benzimidazole NH protonsinvolved in the recognition process that points to thepresence of hydrogen bonds in the neat receptor thatare stronger than in the complex. The popularity ofthe tweezer-like structures for anion recognition is againdemonstrated with the preparation of two moleculesbearing a phenanthroline, two imide and two benzimi-dazole moieties as receptor units50. Lin’s group re-ported two novel and neutral benzimidazole deriva-tives bearing a 1,10-phenanthroline fluorophore (50)as anion receptors based on “turn-on” and “turn-off”fluorescence responses to various anions in DMSOsolution. In the process of anions binding, there weretwo different fluorescent responses in presence of an-ions : a quenching of the fluorescence emission for F–

and AcO– ions and an enhancement of the fluores-cence emission for Cl–, Br– and I– ions. Two differentluminescent mechanisms of the receptors resulting fromvarious anions were exploited to rationalize quenchingand enhancement of the fluorescence emission : a photo-induced electronic transfer mechanism (PET) and theincrease of the rigidity of the host molecules, respec-tively. In particular, chloride could be recognized se-lectively from the anions tested according to changesof fluorescence spectrum.

Tomapatanaget and co-workers synthesized anionreceptors (51 and 52) fabricated from the imidazoleunit and anthraquinone moieties. 1H NMR spectros-copy and UV-Vis titrations in DMSO-d6 and DMSO,respectively, showed that both receptors underwentdeprotonation at the NH moiety of the amide-an-thraquinone unit in the presence of basic anions suchas F– and AcO– 51. These phenomena gave a dramaticcolor change due to charge transfer transition corre-sponding to the shift of max from 371 nm to 489 nm.Redox chemistry of (51) and (52) in the presence ofanions (F–, Cl–, AcO–, BzO–, and H2PO4

–) using cy-


726

clic voltammetry showed the different cyclicvoltammogram (CV) responses upon addition of vari-ous anions. In the case of (51) with various anions, theCV changes are dependent on the basic strength ofanions in order of F– > AcO–, BzO– > H2PO4

– >Cl–, Br–. Interestingly, the CV responses of (52) withH2PO4

– exhibited the most significant changes. Thus,(52) has an excellent electrochemical selectivity to-ward H2PO4

– ion.

Ghosh et al. designed and synthesized an anthracene-appended benzimidazolium-based receptor (53). Thereceptor shows selective recognition of iodide in theexcited state by exhibiting quenching of emission ofanthracene52. In the ground state, receptor shows dif-ferent selectivity and prefers to bind bromide withhigher binding constant value as established from NMRtitration experiment. Other anions in the study indi-cated weak or no interaction. Hydrogen bonding andcharge-charge interactions are the forces responsiblefor strong binding. The interaction properties of thenew receptor were evaluated by 1H NMR, UV-Vis

and fluorescence spectroscopic methods. The an-thracene-linked bis(benzimidazole)diamide (54) has alsobeen described as a simple receptor for the selectivedetection of metal ions and organic sulfonic acids53.Differences in the monomer/excimer ratio of fluores-cent emission of the anthracene moieties were used todetect the presence of diverse organic acids in CHCl3and CH3CN/water (4 : 1) solutions. The change inemission was considerable in the presence ofmethanesulfonic acid and p-toluenesulfonic acid whilethe addition of acetic, mandelic or trifluoacetic acidsless perturbed the emission of receptor (54) due to theprotonation of host by the most acidic sulfonic acidsand the formation of different ion pairs. Changes inthe absorption spectrum correlate well with the fluo-rescence results. Moreover, metal complexation wasstudied in CH3CN and addition of increasing amountsof transition metal cations decreases both the mono-mer and the excimer emission, although the effect onthe excimer band is more pronounced. Cu2+, Co2+

and Ni2+ ions cause the most marked changes in com-parison to the other transition metals.

Table 2. Benzimidazole based chemosensors selective for anions


727

Table-2 (contd.)

((

))

JICS-9


728

Table-2 (contd.)


729

Ghosh et al. designed and synthesized a new an-thracene-coupled benzimidazolium-based tripodal,tricationic fluorescent chemosensor (55)54. Receptorexhibits high degree of selectivity towards H2PO4

– inCH3CN through anion-induced quenching of emissionalong with the formation of a weak excimer complexin the excited states. Furthermore, receptor shows se-lective sensing of ATP over ADP and AMP by exhi-biting an increase in emission in aqueous CH3CN(CH3CN : H2O, 3 : 2, v/v). The electrostatic charge-charge interaction along with both conventional(NH···X; X=O, halides) and unconventional(C+H···X; X=O, halides) hydrogen bonding betweenthe host and the guest molecule synergistically inter-plays in the complexation. The anion-binding proper-ties of receptor were understood by 1H NMR, UV-Visand fluorescence spectroscopic methods.

Eichen et al. reported cyclo[2]benzimidazole (56)as a new host for anions that turns on its luminescenceup to 150-fold upon binding55. Photoexcitedcyclo[2]benzimidazole undergoes an efficient non-ra-

diative deactivation through an excited-state intramo-lecular protontransfer (ESIPT) mechanism. Upon bind-ing an anion, the ESIPT pathway is blocked, resultingin an increase in the luminescence efficiency.

Lee and co-workers synthesized a novel 1,3,5-sub-stituted triethylbenzene derivative (57) with a 2-aminobenzimidazole moiety as a binding and signal-ing subunit56. The sensor was tested in a bufferedCH3CN/H2O (99 : 1, v/v) solution and found to beselective for iodide as demonstrated by thephotophysical properties obtained through UV-Visabsorption and fluorescence spectroscopy analyses.

A simple CN– sensor (58) bearing a naphthol andan imine group was designed and synthesized byZhang’s group, which showed both a colorimetric anda fluorescence “turn-on” response for cyanide ions inaqueous solution57. The probe shows an immediatevisible change in color from yellow to orange whichthen becomes colorless followed by a final change topink, when cyanide is added; these color changes canbe readily observed visually. The mechanism for the

Table-2 (contd.)


730

cyanide ion sensing was established by using 1H NMRand FT-IR spectroscopy and mass spectrometry. More-over, test strips based on the sensor were fabricated,which could act as a convenient and efficient CN– testkit.

Zhang et al. reported a series of novel acridinederived bis-benzimidazolium macrocyclic fluorescentsensors (59 and 60)58. X-Ray crystal structures de-monstrated the self-assembly behavior of thesecyclophanes in the solid state driven by hydrogen bondand – interactions. Anion binding studies of thesesensors revealed a significant effect of the macrocy-clic size and rigidity for H2PO4

– sensing via the obvi-ous “turn-on” as well as bathochromic-shift in fluo-rescence emission. Different cavity size or rigidity ofthe sensors showed different bathochromic-shifts of36 to 126 nm in fluorescence emission induced byH2PO4

–, which resulted in significant color changesof fluorescence from blue to orange red, orange, greenand blue-green respectively. The unique fluorescenceresponse toward H2PO4

– may be attributed to H2PO4–-

induced assembly of sensors forming the excimer be-tween two acridine rings to a different extent.

A series of pyridine-coupled benzimidazolium-basedreceptors (61, 62 and 63) have been designed and syn-thesized by Ghosh et al. In the series, only receptor(61) is structurally appealing in the selective recogni-tion of H2PO4

– in CHCl3 as well as in CH3CN over aseries of other anions59. The ratiometric change inemission with a triplet band at 420 nm is the distinc-tive feature of selective recognition of H2PO4

– inCHCl3. In CH3CN, a “turn-on” response is selectivelyobserved. Binding studies have been carried out usingfluorescence, UV-Vis, 1H NMR and 31P NMR spec-troscopic techniques. Experimental results have beencorrelated with the theoretical findings.

Molina et al. reported a benimidazole based recep-tor (64) which senses aqueous H2PO4

– and HP2O73–

anions through different channels in DMSO. A red-shift of the emission band with an important chelationenhanced fluorescence effect and a large cathodic shift

of the ferrocene. The recognition event has also beenstudied by 1H NMR spectroscopy. It is worth men-tioning that a series of ruthenium complexes contain-ing bis-benzimidazole derivatives have been identifiedas able to target mitochondria and induce caspase-de-pendent apoptosis in cancer cells through superoxideoverproduction60. Following with the family of ruthe-nium complexes, Ye and co-workers prepared mono-nuclear complexes with 2,2-bisbenzimidazole and thecorresponding 4,4-bismethylated ligand61. In a simi-lar way as in the aforementioned complexes, com-pounds (65) and (66) experienced successivedeprotonations in CH3CN with F– and AcO– that werefollowed by UV-Vis, emission spectroscopy, 1H NMRand electrochemical methods. Additionally, a stronghydrogen bond coordination of the less basic anions isobserved in emission spectroscopy and 1H NMR ex-periments. A theoretical paper published by Zhang andco-workers studies the coordination/deprotonation pro-cess in this sort of ruthenium bis(benzimidazol-2-yl)imidazole complexes62. Computational results, ob-tained with (TD)-DFT methods in the Gaussian09 pro-gram, support a proton transfer process for the spec-troscopic and electrochemical changes experienced inthese compounds.

Liu and co-workers reported a fluorescence sensor(67) bearing NH and OH subunits displayed a highlyselective and sensitive recognition property for fluo-ride over other anions63. Fluoride-driven ESPT, poorlyused in anion recognition and sensing, was suggestedto be responsible for the fluorescence enhancementwith a blue shift of 35 nm in the emission spectrum.Singh et al. reported a Co3+ complex of similar typeof benzimidazole-based probe (68) and evaluated as asensor for I– and HSO4

– in aqueous media64. The com-plex showed electrochemical changes with I– over otheranions, whereas it had a ratiometric response towardsHSO4

– ions in UV-Vis spectroscopic study even in thepresence of other anions.

A simple, tailor made receptor (69) based on azodye featuring with benzimidazole unit with hybrid


731

-OH and -NH binding sites was synthesized and charac-terized by Singh’s group. The addition of CN–, AcO–,F–, and H2PO4

– in acetonitrile solution of (69) re-sulted in a large bathochromic shift from 315 nm to470 nm allowing “naked-eye” color change from col-orless to orange65. Anion sensing characteristics weredetermined using visual inspection, UV-Vis and 1HNMR spectroscopy pointing toward the improved chro-mogenic ability after introduction of -N=N- group inthe azo dye (69). The mechanism of anion bindingwith receptor (69) showed 1 : 2 (69/Anion) and theassociation constants were found in the order of AcO–

> CN– > F– > H2PO4–.

1,3-Bis(5,6-dimethyl-1H-benzo[d]imidazol-2-yl)benzene (70) a neutral tridentate ligand, is synthe-sized and employed as a chemosensor by Iyer et al.for the detection of different anions with highest affi-nity for fluoride66. The detection ability of this ligand(70) is confirmed using UV-Vis spectroscopy, fluo-rescence spectroscopy and 1H NMR techniques. An-ion binding studies using 1H NMR and fluorescencespectroscopy revealed that (70) exhibits high selecti-vity for fluoride over other anions.

A benzimidazole-based compound (71) built on thepiperazine motif has been designed and synthesized byGhosh et al.67. The chemosensor (71) is highly selec-tive and sensitive towards ATP over ADP and AMPin CH3CN/H2O (1 : 1, v/v, using 10 mM HEPESbuffer, pH 6.4) as evidenced by the significant changein emission. Furthermore, sensor (71) is cell perme-able and can detect the presence of ATP in Humancervical cancer cells (HeLa).

Samanta’s group synthesized a new dibenzimidazolediimine sensor (72) for selective detection of acetateion68. Significant “naked-eye” recognized color changeof (72) solution from light yellow to pink upon addi-tion of only acetate ion is accompanied with near infrared (NIR) emission exploiting excited state intramo-lecular proton transfer (ESIPT).

Sen Gupta et al. synthesized ESIPT based benzimi-dazole derivative and investigated their photophysical

behaviour towards various anions69. The probe (73)has been used for selective estimation of F– ions ascompared to other anions and signaled the binding eventthrough formation of new absorption band at 360 nmand emission band at 420 nm. The probe (73) showedfluorescence behaviour towards fluoride ions throughhydrogen bonding interactions and restricted the ESIPTemission at 540 nm from OH to nitrogen of benzimi-dazole moiety to release its enol emission at 420 nm.

In another work Sen Gupta et al. synthesized ex-cited state intramolecular proton transfer based benz-imidazole derivative having push-pull effect has beensynthesized and investigated their photophysical be-havior towards various anions70. The probe (74) hasbeen used for selective estimation of F– and CN– an-ions and signaled the binding event through formationof new absorption band at 465 nm. The probe (74)opens different emission channels at 425 nm in thepresence of CN– ions and two new emission bands at435 nm and 365 nm in case of F– ions. The probe (74)behaved as chemodosimeter for CN– ions which havebeen proved by 1H NMR and whereas fluoride causedhydrogen bonding interactions with probe (74) andrestricted the ESIPT emission at 505 nm from OH tonitrogen of benzimidazole moiety to release its enolemission. The differential behaviour of F– ions andCN– has been confirmed through DFT calculations.

A sensitive fluorescent probe, 2,2-bisbenzimidazole(75), for CN– has been developed by Liu and co-work-ers. This structurally simple receptor displays greatselectivity for the cyanide anion over other commoninorganic anions in an aqueous environment71. In ad-dition further study demonstrates the lower detectionof the fluorescence response of the sensor to CN– is in10–9 mol/L range. Thus, the present probe should beapplicable as a practical system for the monitoring ofcyanide concentrations in aqueous samples.

In recent study two new 2-(2-aminophenyl)benz-imidazole-based HSO4

– ion selective receptors, (76)and (77) have synthesized72a. Both receptors (76 and77) behave as highly selective chemosensor for HSO4

–


732

ions at biological pH in ethanol-water HEPES buffer(1/5) (v/v) medium over other anions. Theoretical andexperimental studies showed that the emission effi-ciency of the receptors was tuned successfully throughsingle point to ratiometric detection by employing thesubstituent effects. Using 3s method the LOD for HSO4

–

ions were found to be very low. The structure prop-erty relationship of the bisulphate ensemble was estab-lished in another work reported by the same authors72b.Here quinazoline based receptor (78) selectively sensesHSO4

– ions of nanomolar region in 0.1 M HEPESbuffer (ethanol-water : 1/5, v/v) at biological pH overother competitive ions through the proton transfer fol-lowed by hydrogen bond formation and subsequentanion coordination.

Benzimidazole based for both cations and anions :

In the field of both recognition of cations and an-ions the Cu2+ and cyanide successive recognition fea-ture was examined by Tang and co-workers. A benz-imidazole-based imine linked sensor (79) (viz. Table3) exhibits highly selective and sensitive recognitionproperties to Cu2+ in CH3OH/H2O (1/1, v/v, HEPES10 mM, pH 7.0) solution with a 1 : 1 binding stoichi-ometry73. The in situ prepared Cu2+ complex solu-tion displays high selectivity to cyanide through Cu2+

displacement approach and possesses excellent toler-ance to other common interference anions. The detec-tion limits of sensor (79) to Cu2+ and Cu2+ complexto cyanide were estimated to be 1.82×10–8 M and1.62×10–6 M, respectively. This Cu2+ and cyanidesuccessive recognition feature of sensor (79) makes ita potential utility for Cu2+ and cyanide detection inaqueous media.

Saluja’s group synthesized a benzimidazole-basedimine linked receptor (80) for fluorescent recognitionof Cu2+ in a CH3CN/H2O (8 : 2, v/v) solvent sys-tem74. The sensor was highly sensitive and selectivefor Cu2+ detection at low detection limit. The Cu2+

complex with the receptor acted as a sensor for a phos-phate anion and the phosphate recognition event re-stored the fluorescence intensity of the receptor.

Das et al. synthesized and characterized a benzimi-dazole-based tripodal fluorescence derivative (81).Combining (81) with Cu2+ shows excellent selectivitytoward sulfide anions75. The binding stoichiometry of(81) with Cu2+ is established by Jobs’ plot analysisand mass spectral evidence. The initial fluorescenceof (81) is lost on complexation with Cu2+ and is re-gained in presence of sulfide. The “off-on” behaviorof (81) is studied by fluorescence, UV/Vis and massspectroscopic methods.

To realize highly selective relay recognition of Cu2+

and sulfide ions, a simple benzimidazole-based fluo-rescent chemosensor (82) was designed and synthe-sized by Tang’s group76. Sensor (82) displays rapid,highly selective and sensitive recognition to Cu2+ in100% water solution at pH 6.0. The in situ generatedCu2+ complex solution exhibit a fast response andhigh selectivity toward sulfide anion via Cu2+ dis-placement approach. The detection limits of sensor(82) to Cu2+ and Cu2+ complex to sulfide anion wereestimated to be 3.5×10–7 M and 1.35×10–6 M, re-spectively. Proof-of-concept experiment results dem-onstrate that sensor (82) has potential utilities for Cu2+

and sulfide ion concentration evaluation in real watersamples. This successive recognition features of sen-sor (82) makes it has a potential utility for Cu2+ andsulfide anion detection in water.

The 2-(2-aminophenyl)benzimidazole (2-APBI)derivatized fluorescent sensor (83) that also behavesrelay recognition of Cu2+ and S2– in water solution(pH 7.4) has been developed by the same authors77.Sensor (83) displays excited-state intramolecular pro-ton transfer (ESIPT) featured two emission bands andperforms highly selective and sensitive recognition toCu2+ through two emissions simultaneous quenching.The on-site formed Cu2+ complex exhibits excellentselectivity to S2– with fluorescence “off-on” responsevia Cu2+ displacement approach, which exerts ESIPTrecovery. Thus, through modulation the ESIPT stateof sensor (83), relay recognition of Cu2+ and S2– inwater has been achieved.


733

A fluorescent probe 6-(2,4-dihydroxyphenyl)-5,6-dihydrobenzoimidazo[1,2-c]quinazoline (84) was de-signed and synthesized by Wang’s group78. Its fluo-rescence can be quenched upon the addition of Cu2+

in aqueous media. The binding constant for Cu2+ and(84) was determined to be 9.56×103 M–2 in DMSO/H2O (1 : 1, v/v). The crystal structure of the CuII

complex, revealed that the Schiff base complex formedin the response system of probe (84) to Cu2+ via Cu2+-assisted C-N bond breakage of the quinazoline ring in(84) to form a Schiff base. Consequently, complexwas used as a “turn-on” fluorescence chemosensor fordirect detection of CN–. It showed high selectivity toCN– over a number of anions in the aqueous media.CN– replaced the probe in the complex to form[Cu(CN)x]

2–x and fluorescence recovered. The detec-tion limit of CN– was 4.0×10–6 M. The cell imagesshowed that the complex could be used to detect intra-cellular CN–.

Benzimidazole-based derivative (85) has been de-signed and synthesized by Nandhakumar and co-au-thor as a fluorescent probe highly selective to Cu2+ inHEPES-buffered CH3OH/H2O (1 : 1, v/v, pH 7.0)solution79. The in situ formed Cu complex is furtherutilized to sense the cyanide ions with high selectivityand fluorescence enhancement performance. In addi-tion, the detection limit of CuII complex for CN– wasalso evaluated following the above mentioned methodand was calculated to be 1.86×10–5 M, which is greaterthan the upper limit (1.9 M) for cyanide in drinkingwater set by the World Health Organization. This re-sult indicates that Cu complex has a limitation in de-tection of cyanide in drinking water, but it still has thepotential utility to detect cyanide concentration in highlycyanide polluted water. This result indicates that Cucomplex has a limitation in detection of cyanide indrinking water. But it still has the potential utility todetect cyanide concentration in highly cyanide pollutedwater. Tang’s group reports another example of se-quential recognition of Cu2+ and CN– by a new ben-zimidazole-containing quinazoline based probe (86)80.

Probe (86) exhibits highly selective and sensitive fluo-rescence “off-on” recognition properties to Cu2+ ionin EtOH/H2O (1/1, v/v, HEPES 20 mM, pH 7.0)solution with a 2 : 1 binding stoichiometry. The in situprepared CuII complex solution displays high selecti-vity to CN– through fluorescence relay enhancement.This Cu2+ and CN– sequential recognition via fluo-rescence relay enhancement make probe (86) has apotential utility for Cu2+ and CN– detection in aque-ous media. In this case the detection limit of CuII com-plex for CN– was also evaluated and was calculated tobe 1.74×10–6 M, which is smaller than the upper limit1.9 M for cyanide in drinking water defined by theWorld Health Organization, thus Cu complex probehas a potential utility in detection of cyanide in drink-ing water as well as cyanide polluted water. Subse-quently, the reversibility of the cyanide recognitionprocess was also evaluated. Upon further addition ofexcess Cu2+ to the Cu complex + CN– solution, thefluorescence spectrum of the resulted solution is al-most identical with that of Cu complex solution, whichdemonstrates that the cyanide recognition process isreversible.

In this field a water soluble non-fluorescentcopper(II) complex (87)81 behaves as a highly selec-tive and sensitive for HSO4

– ions through the enhance-ment of fluorescence of the system based on intermo-lecular hydrogen bonding assisted chelation enhancedfluorescence (CHEF) process in “turn-on” style, whichhas been confirmed by systematic optical techniquesand electrochemical studies. This mode of sensingpathway and binding of HSO4

– ions with the receptor(87) has also been validated by optimizing the struc-tures of Cu complex and HSO4

– adduct with the helpof theoretical calculations. This non-cytotoxic probesenses HSO4

– ions as low as 3.18×10–7 M in water :DMSO (9 : 1, v/v) at biological pH (using 1 mMHEPES buffer) and it is also useful for the detectionof intracellular HSO4

– ions under a fluorescence mi-croscope.


734

Table 3. Benzimidazole based chemosensors for both cations and anions

For neutral molecules :

Amongst various reports where compounds behaveas sensor for neutral molecules an azo dye-coupledbenzimidazole-based receptor (88) was synthesized andinvestigated as a receptor for metal ions in semi-aque-ous medium by Singh et al.82. The receptor recog-nizes Cu2+ with high selectivity over other metal ions.The resultant complex was found to selectively bindoxalic acid via counter ion displacement.

L-Valine derived benzimidazole based bis-urea (89)

has been designed and synthesized by Ghosh et al.The bis-urea (89) (viz. Table 4) is found to act asenantioselective chemosensor for tartrate. The sensorshows greater fluorescence response for L-tartrate inDMSO83. In comparison, less steric receptor (90) ex-hibits a marginal preference for D-tartrate over the L-tartrate in DMSO and validates the steric role of thesubstituents around the benzimidazole motif in (89)toward enantioselective recognition of tartrate.

Wang’s group designed and synthesized 1,3,5-


735

tri(1H-benzo[d]imidazol-2-yl)benzene derivatives, as afluorescent chemosensor (91) (which has structural simi-larities with compound (57)) for the detection ofnitroaromatic explosives, by simple N-hydrocarbylation84. Among 16 obtained compounds,compound (91) has the best capability for detection of

picric acid (PA), having good selectivity and high sen-sitivity. The detection of PA with (91) solution-coatedpaper strips at the picogram level is developed. Asimple, portable, and low cost method is provided fordetecting PA in solution and contact mode. Benzimi-dazole receptors have also been used for the recogni-

Table 4. Benzimidazole based chemosensors for neutral molecules

JICS-10


736

tion of neutral receptors as carboxylic acids, hydrogenbond donors, as urea or barbital, and O-methylatedaminoacids. Neutral molecules are in general weaklybound and a better control of the complementarity be-tween host and guest is then required.

Moran and co-workers showed a receptor (92) forcarboxylic acids that connects two benzimidazole unitsthrough a xanthene molecule in a tweezer-like struc-ture85. The cavity created between the two benzimida-zole arms effectively binds methanol, DMSO or ace-tone in chloroform and the structure of the complexeswas determined by single X-ray crystal diffraction.When carboxylic acids were studied, it was observedthat the acid group protonates one of the nitrogen at-oms present in the benzimidazole moieties while theother remains neutral and participates in the coordina-tion of the resulting anion. When the acid used wasanthracene carboxylic acid, a strong decrease of theemission intensity was observed compared to the freeacid through a PET mechanism responsible for thequenching of fluorescence. The single crystal struc-ture for the complex shows how the benzimidazole onthe amide arm protonates while this NH and the otherthree NH groups coordinate the carboxylate moiety.

Gale and co-workers prepared a simple tweezer-like receptor (93) connecting two benzimidazole unitsthrough a 2,6-pyridinedicarboxylic amide86. Receptor(93) binds preferentially with barbital over the struc-turally related urea, thiourea or imidazolinone inDMSO/MeNO2. In the crystal structure of the com-plex a good complementarity is observed that leads tothe formation of four hydrogen bonds.

Other examples of the use of benzimidazole inmolecular recognition are the two related macrocycleswith the general structure (94) in which these hetero-cycles are connected through polyether alkyl chains87.These compounds have been used for the recognitionof O-methylated -aminoacids in chloroform. In thestudy it has been observed how the length of the mac-rocyclic arms influences the selectivity due to thecomplementarity of hydrogen bonds and – stack-ing.

Das et al. developed a benzimidazole-derived ICT-based probe, (95) for trace level determination of wa-ter88. In presence of water, the “naked-eye” color of(95) changes from red to yellow, while it turns togreen from red under UV light. Upon addition of wa-ter, (115) shows a ratiometric absorbance change inmethanol.

Recently a newly designed rhodamine-benzimidazolehybrid molecule (96) has been developed89 as a FRET-based chemosensor for the selective detection of tracelevel water in both polar protic and aprotic organicsolvents.

Pyrazole based compounds as chemosensor

Pyrazoles are important nitrogen containing 5-mem-bered heterocyclic compounds with stronger fluores-cence, have higher hole-transport efficiency and ex-cellent emitting blueness property. Therefore,pyrazoline derivatives have widely been used as whit-ening or brightening reagents for synthetic fibers, fluo-rescent chemosensors for recognition of transition metalions, hole-transport materials in the electrophotogra-phy and electroluminescence fields90. Moreover,pyrazoline with membrane permeability, low toxicity,and high quantum yield render the fluorophore attrac-tive for biological applications91,92. However, a fewpyrazoline derivatives are as effective “turn-on” fluo-rescent sensors in literature.

Li and co-authors have developed a chromogenicand fluorescent probe (97), (Table 5) based on an an-ion catalyzed intramolecular hydrogen transfer, whichdisplayed drastic changes in UV-Vis absorption as wellas fluorescence emission intensities showing selectivelyfor F– over other anions93.

According to Li’s group two simple fluorescentanion receptors based on 1-phenyl-3-methylpyrozole-5-one-4-one-phenylhydrazone (98) and 1-phenyl-3-methylpyrozole-5-one-4-one p-nitrophenyl-hydrazone(99) with similar configuration exhibited different an-ion binding behaviors in DMSO solution94. The ex-perimental results indicate that (98) bind anions suchas F–, AcO–, H2PO4

– ions in 2 : 1 host-guest com-plexation fashion, while (99) bind anions in 1 : 1 host-


737

guest complexation in the solution. Here, the anionbinding ability of the receptors both (98 and 99) wasevaluated through UV-Vis titrations. Upon the addi-tion of fluoride ions in case of (98) a significant de-crease in the absorption of 394 nm and appearance oftwo new absorption peaks at 306 nm and 431 nm withtwo isosbestic points at 346 nm and 410 nm. But, inthe case of (99), out of two absorption bands at 407nm and 537 nm, a significant decrease in the absorp-tion band at 407 nm and an increase in the absorptionof 537 nm upon addition of F– ions accompanied by a“naked-eye” color changes from red to purple havebeen recorded. Here, (98 and 99) displayed a tauto-meric equilibrium between the hydrazone form andthe azophenol form. Where the hydrazone form domi-nated in the solution. Upon addition of the strong ba-sic anions, the hydrazone form would be transferredto the azophenol form, which interacts with anionicanalytes and is responsible for the spectral changesand “naked-eye” color changes, established also bythe 1H NMR titration experiment. Upon excitation at397 nm, (98) showed a weak emission at 415 nm andthe visible fluorescent enhancement was observed withaddition of the strong basic anions such as F–. How-ever, (99) exhibited a fluorescent emission band cen-tered at 667 nm where the ex is 450 nm and suchemission would be gradually quenched upon additionof fluoride ions. The possible explanation for this re-sult might be that the receptors (98 and 99) employedtwo different signaling transduction mechanisms foranion binding. Upon complexation with anions, theconfiguration of (98) was rigidified, resulting in inhibi-ting vibrational and rotational relaxation modes of non-radiative decay, and thus the fluorescent enhancementof (99) takes place. In the case of (99), introduction ofan electron-withdrawing substituent (-NO2) would bea photoinduced electronic transfer (PET) from thepyrazole unit (a fluorophore) to the electron-withdraw-ing substituent (-NO2) and as a result the fluorescentemission would be gradually quenched upon additionof fluoride ions. Merkoc et al. used N-alkylaminopyrazole ligands (100 and 101) in a proof-of-concept fluorescent sensor that can operate as a

simple ‘paper test’ for rapid and highly sensitive mer-cury detection in water95. A significant enhancementof fluorescence emission in aqueous media for the Hg2+

detection at concentrations lower than 0.1 ppb isachieved. A linear range from 10 to 100 ppb [Hg2+]was obtained in the paper-based system. Two respec-tive contradictory “turn-on” and “turn-off” fluores-cence mechanisms in the ‘paper test’ and water solu-tion (pH 1) systems can be explained by the concept ofPET, CHEF and H-bonded interaction.

Zhang’s group develop a pyrazoline-based fluores-cent sensor (102) for biological Zn2+ detection96. Thesensor shows good binding selectivity for Zn2+ overcompeting metal with 40-fold fluorescence enhance-ment in response to Zn2+ presumably due to the block-ing of the photoinduced electron transfer (PET) pro-cess upon complexation with Zn2+. The probe is cell-permeable and can be used to detect intracellular zincions in living neuron cells.

The synthesis, characterization, binding anddeprotonation studies with anions for four 5-(1H-indol-3-yl)-pyrazolyl derivatives (103-106) have been de-scribed by Ahmad et al.97. It is worthy to mention thatsensor (103) shows a drastic change in absorption spec-trum (ca. 335 nm) and colour (colourless to blue) uponaddition of F– in DMSO solution due to thedeprotonation of indole -NH proton, as confirmed by1H NMR titration. Sensor (105) recognizes F– andCN– ions by deprotonation mechanism with visiblecolour change of the solution in a similar manner tothat of (103). However, in contrary to (103 and 105),sensor (104) binds with F–, CN–, H2PO4

–, AcO– andPhCOO– ions exploiting hydrogen-bonding interactionwith the shifting of absorption band to longer wave-length and subsequent colour change of the solution.Compound (106) recognizes F– without any visualcolour change and its binding is studied by 1H NMRtitration to acquire the important information about thenature of binding between F– and (106).

Zhao and co-authors present the preparation of apyrazoline compound and the properties of its UV-Visabsorption and fluorescence emission98. The structureof the compound was characterized by IR, 1H NMR


738

and HRMS. Moreover, this compound can be used todetermine Hg2+ ion with selectivity and sensitivity inthe EtOH : H2O = 9 : 1 (v/v) solution. This sensorforms a 1 : 1 complex with Hg2+ and shows a fluores-cent enhancement with good tolerance of other metalions. The association constant Ka measured for coor-dination of the sensor with Hg2+ was 3.03×104 M–1,and the detection limit of the sensor toward Hg2+ was3.85×10–10 M. Detailed information on the interac-tions between (107) and Hg2+ ion from 1H NMR spec-troscopic studies suggested that the nitrogen atoms inthe benzimidazole, thioamide and pyrazoline of thesensor (107) participated to the complex with Hg2+.In addition, the fluorescent probe has practical appli-cation in cells imaging.

Wong and co-workers told about a luminescentcyclometalated iridium(III) complex-based chemosensor(108) bearing a zinc-specific receptor, tris(2-pyridylmethyl)amine, and the 3-phenyl-1H-pyrazoleligand showed a pronounced luminescence color changefrom blue to green upon addition of Zn2+ ions to asolution of iridium(III) complex99. It is attributed tothe suppression of photoinduced electron transfer uponcomplexation of iridium(III) complex with Zn2+ ions,which interaction has been established by UV-Vis ab-sorption titration, emission titration, and 1H NMR ti-tration. Selectivity of this iridium(III) complex (108)for Zn2+ over other common metal ions and the appli-cation of this probe in visualizing intracellular Zn2+

distribution in live zebrafish have been demonstrated.

Table 5. Pyrazole based chemosensors recognising ions


739

The design, synthesis, and photophysical proper-ties of a new fluorene-based fluorescent chemosensor,4-((E)-2-(2-(benzo[d]thiazol-2-yl)-9,9-diethyl-9H-fluoren-7-yl)vinyl)-N,N-bis((3,5-dimethyl-1H-pyrazol-1-yl)methyl)benzenamine (109), is described by Frazerand co-workers for the detection of Al3+100. Fluores-cent probe (109) exhibited absorption at 382 nm andstrong fluorescence emission at 542 nm (fluorescencequantum yield, F, of 0.80). The capture of Al3+ bythe pyrazolyl aniline receptor resulted in nominal change

in the linear absorption (372 nm) but a large hypsoch-romic shift of 161 nm in the fluorescence spectrum(542 to 433 nm, F=0.88), from which Al3+ wasdetected both ratiometrically and colorimetrically. Thelarge hypsochromic shift could be attributed to a re-duction in intermolecular charge transfer (ICT). Thisdramatic result found with Al3+ and the probe may bea consequence of the size and polarizability of the Al3+

ion. The Al3+ is believed to interact strongly with thepyrazolyl nitrogens and, even though the resulting

Table-5 (contd.)


740

molecular assemblies are unknown, it is believed torestrict the excited state vibrations of the chromophore,resulting in an increase in fluorescence properties. Asingle set of isobestic points was observed in both theabsorption and emission spectra, suggesting a singleequilibrium was reached in complexation. The addi-tion of other metal ions, namely Mg2+, Ca2+, Mn2+,Fe2+, Co2+, Ni2+, Cu2+, Zn2+, Cd2+, Hg2+ andPb2+ produced only minimal changes in the opticalproperties of this probe. The emission band of thisprobe was also accessed by two-photon excitation inthe near-IR, as two-photon absorption (2PA) is impor-tant for potential applications in two-photon fluores-cence microscopy (2PFM) imaging. The 2PA crosssection of the free fluorenyl ligand (109) was 220 GMat 810 nm and 235 GM at 810 nm for the Al-ligandcomplex, practically useful properties for 2PFM. Theprobe exhibited a peak absorption at 382 nm and agradually blue-shift upon the sequential addition ofAl3+. The emission spectra of the free ligand titratedwith Al3+ resulted in the appearance of a newfluorecence band at 432 nm, shifted from 532 nm forthe pure ligand.

Two novel rhodamine-pyrazolone-based colorimetric“off-on” fluorescent chemosensors for Fe3+ ions weredesigned and synthesized by Jadeja et al. usingpyrazolone as the recognition moiety and rhodamine6G as the signalling moiety101. The photophysical prop-erties and Fe3+-binding properties of sensors (110)and (111) in acetonitrile-aqueous solution were alsoinvestigated. Both sensors successfully exhibit a re-markably “turn-on” response, toward Fe3+, which wasattributed to 1 : 2 complex formation between Fe3+

and the two receptors. After the addition of metal ions,UV/Vis absorption spectra showed a distinct changeand the appearance of a new spectral band with a maxi-mum at 530 nm for Fe3+ ions. The absorption band at530 nm in the case of Fe3+ is due to the formation ofa delocalized xanthane moiety of rhodamine by selec-tive Fe3+-induced ring opening of spirolactam, whichalso explains the change in colour from colourless topink in the presence of this Fe3+ ion. Without metalions, probe (110) and (111) show almost no fluores-cence upon excitation at 530 nm, suggesting that re-ceptors (110) and (111) exist in a ring-closed non-

fluorescent spirolactam conformation.

Das et al. synthesized a new chemosensor (112)and its spectral response was screened with most al-kali, alkaline earth, transition and lanthanide metal ionsin THF-water (2 : 3, v/v, pH 7.2, 20 mM HEPESbuffer) medium102. This is found to bind four differ-ent transition metal ions such as Hg2+, Cu2+, Ag+

and Ni2+ in mixed aqueous solution at pH 7.2; whilebinding affinity towards the Hg2+ is found to be anorder of magnitude higher as compared to other threecations mentioned. The fluorescence quenching of thesemetal ions could be explained based on the spin-orbitcoupling phenomena. Observed luminescence changefor Hg2+ was more appreciable than the other threemetal ions for a comparable concentration of the re-spective metal ion. Chemosensor (112) has ananthracenyl unit as the signalling moiety, covalentlycoupled to an N-N donor receptor fragment. Fluores-cence based emission read-out signal of the anthracenylunit is governed by different photoinduced electrontransfer (PET) processes in the absence and presenceof the coordinated metal ion. Experimental studies re-vealed that pyridyl pyrazole, used as the receptor frag-ment for Hg2+ showed a higher affinity for this ion inaqueous/organic media. Experimental results reveal thatthe (112) could be used for colorimetric detection ofHg2+, present in the lower organism like Pseudomo-nas putida.

Yang’s group reported a new pyrazole-based fluo-rescent sensor, 5-amino-3-(5-phenyl-1H-pyrrol-2-yl)-1H-pyrazole-4-carboxamide (113), and studied for fluo-ride anion (F–) detection in organic or water-contain-ing solution103. This compound displayed both changesin UV-Vis absorption and fluorescence emission spec-tra upon addition of F–. With increasing of F–, blueemission intensity increases drastically and reaches satu-ration with 607-fold enhancement at 424 nm. The re-sults indicate that (113) has highly selectivity for fluo-ride detection over other anions, such as Cl–, Br–, I–,HSO4

–, H2PO4– and AcO– in DMSO or aqueous

DMSO solutions. 1H NMR titration and other experi-ments confirm that the sensing process is mainly from


741

the deprotonation of the pyrazole-NH in (113). Be-cause of the high charge density and small size, fluo-ride as strong base can deprotonate the compound (113)to afford deeply the heterocyclic conjugated anion whichgives strong emission when excited at 365 nm.

Qin’s group reported an original Schiff base typefluorescent chemosensor 1-phenyl-3-methyl-5-hydroxypyrazole-4-carbaldehyde(benzoyl)hydrazone(114) for Cu2+104. An obvious fluorescence quench-ing only for Cu2+ demonstrates that ligand (114) ex-hibits high selectivity and efficient signaling behaviortoward micromolar concentration of Cu2+ comparedwith other metal ions. At the same time, the coordina-tion form between ligand and Cu2+ is elucidated viacrystal structure. The paramagnetic nature of Cu2+

and the Cu2+-F interaction was the cause of fluores-cence quenching.

A structurally characterized naphthelene-pyrazolconjugate (115) behaves as an AlIII ion-selectivechemosensor through internal charge transfer (ICT)-chelation-enhanced fluorescence (CHEF) processes in100 mM HEPES buffer (water-DMSO 5 : 1, v/v) atbiological pH with almost no interference of othercompetitive ions105. The probe can detect Al3+ ionsas low as 31.78 nM within a very short response time(15–20 s).

Conclusions

The important progress achieved in the develop-ment of diazole based receptors can be exemplified bythe number of systems that have recently been de-scribed in this field. In most cases, such receptors areable to recognize either cations or anions. Althoughtoo many diazole derivatives have been devised asprobes for the detection of several types of cationic/anionic/neutral species with help of differentphotophysical processes, comparatively more imida-zole/benzimidazole based compounds including theirmetal chelates have showed imperative photophysicalproperties in favour of developing the fluorescentchemosensors highly selective for the target species.Among the explored chemosensors for cations, sev-eral Zn2+ sensors have been reported. Here, the ben-

zimidazole based compound, 2,6-bis(5,6-dihydrobenzo[4,5]imidazo[1,2-c]quinazolin-6-yl)-4-methylphenol36 is the most superior one as it is highlysensitive and can detect Zn2+ ions as low as 0.53 nMin almost aqueous medium at biological pH, and thisprobe is also efficient to detect the distribution of Zn2+

ions in living cells like other diazole-Zn2+ sensors106.Interestingly it is noteworthy to mention that the diazolederivative is capable to detect Al3+ ions as low as 9.0nM in green solvent system comparable with the low-est LOD available in the literature107. In case of aniondetection, this derivatives are not good enough com-pared to the detection cations in terms of LOD, sol-vent systems. The diazole derivatives are also impor-tant to detect the neutral molecules like picric acid,tartarate, etc.; some toxic anions and also cations ofdifferent oxidation states. And, this review indicatesthat more imidazole/benzimidazole based compoundshave been explored in developing the chemosensorsfor different species compared to the pyrazole basedchemosensors. As a consequence of this background,we thus predict that this topic constitutes an area ofsupramolecular chemistry that is ripe for future growth.

Acknowledgement

Financial support from Council of Scientific andIndustrial Research (CSIR), New Delhi, India is grate-fully acknowledged.

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A novel thiourea-based colorimetric chemosensor for sensitive and selectivedetection of sulphate ion in semi-aqueous medium

Aman Bhasina, Simanpreet Kaurb, Mayankc, Narinder Singh*c and Navneet Kaur*a

aDepartment of Chemistry, Punjab University, Chandigarh-160 014, India


bCentre for Nanoscience and Nanotechnology (UIEAST), Punjab University, Chandigarh-160 014, India

cDepartment of Chemistry, Indian Institute of Technology, Ropar (IIT Ropar), Rupnagar-140 001,Punjab, India



Abstract : Within the pool of naturally available anions, the sulphate anion has gained a certain level of notori-ety by playing a critical role in contaminating the animal food chain, apart from disturbing marine life via pa-ralysis of certain ecological cycles. Under such circumstances, it becomes imperative to regulate the influx ofthis potentially hazardous anion into the ecosystem. An important section of the remediation strategies in thisarea would involve an efficient sensing and quantification of this toxic anion, particularly in the natural watersystems. In continuation of our efforts to exploit the unique photophysical properties of ligand-tagged nanoparticlesto achieve selective ion-recognition potential, this report highlights the chemical synthesis and chemosensing ef-ficacy of a new and selective sulphate-recognising organic receptor OR in aqueous medium. Upon decoration ofOR onto ZnO nanoparticles, the nanohybrid OR@ZnO demonstrated a rapid response and selectivity towardsthe hydrogen sulphate anion, as marked by a significant alteration in the absorbance and fluorescence profileof OR@ZnO in aqueous system with a significantly low limit of detection (100 nM).

Keywords : ZnO, surface decoration, sulphate, thiourea, fluorescence, sensor.

Introduction

Inorganic sulphates are naturally available in sev-eral minerals, including barite (BaSO4), epsomite(MgSO4·7H2O) and gypsum (CaSO4·2H2O)1. Amongstthe several non-transition metal sulphates, sodium, po-tassium and magnesium sulphates are all highly solublein water whereas many heavy metal sulphates (likebarium sulfate) are less soluble. In addition to a plethoraof industrial applications, inorganic sulphates also har-bor potential biological relevance at physiological con-centrations2. For instance, the sulphate moiety consti-tutes an integral part of several polysaccharides (foundin animal tissues) like heparin sulphate, which uponbinding to a variety of protein ligands, regulates bio-logical processes like cell signaling , angiogenesis andregulation of enzyme activity3. In the recent decades

and as a consequence of the industrial revolution, ameteoric rise in the concentration of the inorganic sul-phate ion in natural aquatic systems has been broadlyattributed to acid rain, non-regulated use of sulphate-containing fertilizers and oxidation of pyrite depositsin the subsoil4. A serious consequence of an overloadof this otherwise harmless sulphate ion is the aberrantincrease in phosphorus levels in the aquatic system,leading to an uncontrolled growth of certain algae andlower oxygen levels in the water systems. Such a phe-nomenon, known as eutrophication, poses a seriousecological hazard to marine life5. In addition, anotherphytotoxic effect of sulphate-overdose has been increas-ingly realised in the form of an excess generation andaccumulation of methyl mercury in the deep waters6–8.Therefore, in due recognition of the ubiquitous nature


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of the sulphate anion and a unique sulphatemethyl mer-cury relationship, it is imperative to recognise andquantify sulphate concentrations in the natural waterbodies in order to promote potential remediation strat-egies for specifically limiting the production and accu-mulation of MeHg in the sediment pore-water.

In an attempt to do so, there has been a growinginterest in developing highly selective chemical recep-tors to facilitate a facile and selective recognition andquantification of the sulphate ion in water systems9. Incontrast to a broad spectrum of metal ion-sensors, sev-eral reports in the past have harboured a certain biastowards positively-charged/metal-containing chemicalreceptors as a favourite class for anion detection inaqueous medium10,11. Interestingly, certain naturalanion-binding systems exhibit high affinity and selec-tivity for certain substrates under physiological condi-tions, apparently upon shielding of the latter fromneighbouring solvent molecules via recognition into acleft or cavity of the folded polypeptide chains. Forexample, sulphate binding proteins (SBP) responsiblefor anion transport in bacteria bind sulphate excep-tionally well in water via hydrogen bonding12. How-ever, some critical factors that may impede the bind-ing of sulphate anion to neutral receptors in aqueoussystems include the former’s relatively high hydrationenergy, thereby making the anion strongly hydrophilicand difficult to detect in aqueous phase13. Such chal-lenges encountered in the detection of sulphate-anionin aqueous systems have been often cited in scientificliterature. For example, despite a high selectivity forsulphate anion in organic solvent medium, Shao et al.demonstrated a limited environmental applicability oftheir novel indole-based chemosensor in aqueous me-dium14. Molina et al. have also postulated a similartrend by demonstrating a measurable sulphate-recog-nizing potential of a ferrocene-incorporated chemore-ceptor in acidic medium, whose sensitivity, however,diminishes drastically in a neutral medium15. In theabove and certain other related studies, the sulphate-sensing ability of potential chemoreceptors has beenproposed to heavily rely on protonation of the host

receptors, followed by a subsequent anion coordina-tion within the receptor cleft via H-bonding and elec-trostatic interactions16. Therefore, a rational incorpo-ration of specific sulphate-binding chemical moietiesinto a suitable neutral host would be ideal for selectiverecognition of a sulphate anion in aqueous medium,thereby circumventing the need for protonation of thereceptor ligand, prior to sulphate detection.

In this regard, several urea/thiourea-based hostligands are amongst the most successful group of neu-tral chemical receptors for recognising sulphate ion ina highly competitive aqueous medium10,17. This maybe attributed to a facile hydrogen-bonding interactionbetween the acidic H-bond donor moiety (NH of hostligand) and sulphate anion (guest), thereby ensuring asensitive chemical affinity between the two species18.Upon an evaluation of previously reported sulphate-sensing chemosensors, a major concern regarding theirenvironmental relevance lies in their efficacy in physio-logical medium and their limit of sensitivity.

In due recognition of the same and our long-stand-ing interest in finely tunable ion-recognition proper-ties of biocompatible ZnO nanoparticles, we have ex-ploited this unique “amine-sulphate chemistry” in thechemical synthesis of a novel OR@ZnO nanohybrid19.The subsequent modulatory effects of this nanosensor,resulting in a sensitive and selective recognition of aphysiologically relevant sulphate-ion, has been dis-cussed henceforth.


Synthesis and characterization of organic receptor(OR) : The rationale of this investigation is to obtainthe selectivity of a chemical receptor towards a spe-cific anionic guest; which is being governed throughmultiple interactions between host and guest in acomplementary fashion20,21. The work is themed onthe fact that synthetic receptors with flexible arms pos-sess a shape complementary to tetrahedral anions be-cause the geometry and orientation of the host mol-ecule favour the formation of stable host-guest com-plexes22. This concept has been corroborated with the

Bhasin et al. : A novel thiourea-based colorimetric chemosensor for sensitive and selective detection etc.

747

excellent work of Wu et al.; which highlighted theexceptional sulphate-binding properties of a novel tri-podal hexaurea through the encapsulation of the tetra-hedral anion with six urea groups involving multiplehydrogen bonds23. The reported chemical sensors in-volve multiple steps and tedious purification techniquesfor the synthesis of such functional organic receptors18.However, the organic receptor projected in our workOR was synthesized by a relatively shorter and facileprotocol. The OR was furnished as a yellow solid inquantitative yield (86% overall) through the nucleo-philic addition of a commercially available derivativeof isothiocyanate to a secondary amine (Fig. 1A). Thelatter, in turn, was easily synthesized by sodium-boro-hydride-mediated reduction of a Schiff base which wassynthesized by a one-step condensation reaction be-tween commercially available salicylaldehyde and n-propyl amine in water-free methanol18. Purity of thetitle ligand L was confirmed by its detailed physico-chemical characterization using IR, 1H/13C NMR andelectrospray ionisation (ESI) mass spectroscopy in ad-dition to elemental analysis. In accordance with theresults obtained from the IR spectrum, an observationof a distinct stretching frequency band at 1621 cm–1,which is characteristic of an imine-linked (-CH=N)functional group, provided evidence of the formationof the final product L. As per the result obtained fromNMR spectroscopy, the peak corresponding to the

iminic functionality (-CH=N) at 7.27 ppm and at 165.9ppm in the 1H NMR and 13C NMR, respectively, con-firmed the complete consumption of the reactant, 2-hydroxy benzaldehyde24. In addition, an upfield shiftin the NMR signal (3.7 ppm) corresponding to theCH2-N protons of L, relative to a rather deshieldedenvironment around its precursor proton in benzalde-hyde (10.64 ppm) can be related to the lower elec-tronegativity of a nitrogen atom (as in L).

In order to identify the mass spectrum of the recep-tor ligand L on the basis of its mass/charge (m/z) ra-tio, a solution of the same was subjected to ionisationvia electrospray technique25. The appearance of anintense molecular ion peak (at 346.1) confirmed thepresence of a protonated adduct of OR. A close dif-ference in the observed and calculated percentage ofthe individual elements in OR further confirmed itspurity in bulk.

1H NMR (400 MHz, DMSO-d6) : 9.93 (1H, br,-OH), 9.72 (1H, s, -NH), 8.1 (2H, d, Ar-H), 7.6(2H, d, Ar-H), 7.13 (2H, m, -CH2), 6.86 (1H, d, Ar-H), 6.81 (1H, t, Ar-H), 4.9 (2H, s, Ar-CH2), 3.7(2H, t, -N-CH2) and 0.86 (3H, t, -CH3);

13C NMR(400 MHz, DMSO-d6) : 178.34, 154.90, 152.28,149.82, 147.80, 142.22, 128.35, 123.75, 123.25,119.12, 115.08, 39.70, 39.28, 39.08 and 11.01; ESI-MS m/z = 346.1 [M+H]+; CHN analysis Calcd. for

Fig. 1. (A) Chemical structure of organic receptor (OR) depicting the signal subunit; potential binding sites for anion recognitionand moieties to anchor the receptor (OR) over the ZnO; (B) Possible binding mode of organic receptor (OR) over thesurface of ZnO and projecting the H-bonding sites for anion recognition.

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C21H22N2OS : C, 59.11; H, 5.54; N, 12.16. Found :C, 59.77; H, 5.88; N, 12.51.

Surface decoration of ZnO with organic receptor (OR)and characterization of OR@ZnO

ZnO is a wide band gap semiconductor, possessinghigh exciton binding energy with a stable Wurtzitestructure. It has attracted exhaustive efforts into anelucidation of its unique applications in antireflectioncoatings, solar cells, diode lasers and antibacterial prop-erties26. Nowadays, ZnO nanoparticles, in conjuga-tion with a plethora of organic and bio-chemical ligandsare being popularly utilized as a potential ion-sensingcandidate due to their immense selectivity and sensi-tivity. A significant biocompatibility and highly ad-justable photophysical properties in the presence ofthe ion-of-interest renders ZnO nanoparticles as a de-sirable analytical probe27. In an attempt to explore apotential ion-sensing capability of a new thiourea-basedaromatic derivative OR the new organic receptor wasfurther decorated onto ZnO nanoaggregates in orderto achieve a stable analytical sensor system in aqueousmedium28,29. Details of the procedure for synthesis ofOR-linked ZnO nanoparticles include mixing an alco-holic solution of Zn(ClO4)2.6H2O (595 mg, 2.0 mmol)and an alcoholic solution of NaOH (120 mg, 3.0 mmol).A white product was separated out and the productwas washed and dried at 150 ºC. Compound OR (804mg, 3 mmol) was mixed with freshly prepared ZnOnanoparticles (100 mg) under constant stirring at 25 ºCin dry CHCl3 and the solution was refluxed for 15 h.The progress of the reaction was monitored with IRand UV-Vis absorption spectroscopy. Upon comple-tion of reaction, the final product was isolated by cen-trifugation and washed successively with ethanol andwater. The procured product OR@ZnO was dried for24 h at 50 ºC. The size, morphology, chemical com-position and spectroscopic features of the receptor-capped Zn nanoparticles were elucidated by XRD,SEM, EDAX, solid state UV-Vis and fluorescencestudies, respectively. The particle size distribution ofthe receptor-decorated ZnO nanoparticles was estimated

by dynamic light scattering (DLS) studies using a probebased Metrohm microtrac Ultra nanotrac particle sizeanalyzer. The SEM image depicts that modified ZnOnanoparticles form supramolecular self-assembledmorphology with growth limited in particular direc-tions and thereby generating tripodal geometry (Fig.2A). The EDAX analysis of OR@ZnO points towardsthe simultaneous presence of zinc, oxygen and car-bon, thereby distinctly confirming a coating of the or-ganic receptor OR onto the surface of ZnO (Fig. 2B).The thickness of OR-capped ZnO nanoparticles(OR@ZnO) was characterized by dynamic light scat-tering (DLS), which estimates the hydrodynamic dia-meter of OR@ZnO to be 560 nm (Fig. 2C). Herein,variation in the particle size measured by SEM (200nm, in this case) and DLS was observed. This can beattributed to the fact that SEM only estimates the sizeof the particle core neglecting the surrounding solventlayer. In contrast, DLS studies measures the size ofthe solvent-adhering particles under the influence ofbrownian motion, thereby making it a more relaibletechnique in comprehending and optimizing thenanoparticles’ performance in analytical and biologi-cal assays. In contrast, SEM estimates the size of theparticles without the hydration layer and is a morereliable technique to measure the size of coatednanoparticles26. The solid state UV-Vis absorption spec-tra of pure ZnO exhibit a peak at 360 nm correspond-ing to the exciton state of ZnO (Fig. 2D, SupportingInformation)29. The max of the absorption spectra ofOR@ZnO was blue-shifted as compared to that ofpure ZnO (Fig. 2D). The solid-state photolumines-cence spectrum of OR@ZnO, which was recorded atan excitation wavelength of 325 nm, displayed an in-tense peak at 330 nm and 456 nm (as of pure ZnO)(Fig. 2E); however, the intensity of a characteristicgreen band at 570 nm (corresponding to pure ZnO)quenched in case of OR@ZnO. According to Dijken’spostulate, the visible emission band can be attributedto the formation of a recombination center (Vo**),where the valence band hole is trapped onto the sur-face, which then tunnels back into the oxygen holes


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containing one electron (Vo*)27. This recombinationof a shallow trapped electron with a deeply trappedhole in a Vo** center is responsible for visible emis-sion, thereby explaining the role of multiple surfacedefects in pure ZnO as a probable cause leading tointense emissions in the visible region. Such defects atthe grain boundaries of ZnO may enable a probableconjugation with -OH and -CH=N groups of OR,thereby resulting in a decrease in interfacial defects inOR-capped ZnO and a consequent lowering in greenregion emission (Fig. 2E). Finally, X-ray powder dif-fraction pattern of the prepared OR@ZnO nanopowder,calcined at 500 ºC was also recorded (Fig. 2F), de-picting scattering angles (2) of 31.79, 34.40, 36.25,47.56, 56.56, 62.82 and 67.91, corresponding to re-flections from 100, 002, 101, 102, 110, 103 and 112planes of the crystal, respectively. All diffraction peaksare in good agreement with literature reports, which

can be indexed as the hexagonal Wurtzite structure ofZnO with lattice constants of a = 3.253 Å and c =5.207 Å. No other peak (except assigned for ZnO)was visualized, thereby confirming the purity of thesample. In accordance with Debye-Scherrer’s equa-tion (D = 0.89/ cos ), where is the wavelengthof incident X-ray (0.15418 nm), is the full width athalf maximum (FWHM) of diffraction peak and isthe diffraction angle, the average crystalline size ofthe ZnO nanocrystals was estimated to be 30.55 nm.

Anion recognition properties of OR@ZnO : Thetitle ligand OR belongs to a class of thiourea-basedderivatives (Fig. 1A). As discussed earlier, such typeof compounds possess multiple NH (as hydrogen bonddonor) functionalities, therefore being able to demon-strate ion-sensing potential receptors by facilitating an-ion coordination through multiple hydrogen bond-ing13,14. However, due to the rather high hydrophili-

Fig. 2. (A) SEM image of OR@ZnO, displaying a self-assembled morphology due to its structure directing nature; (B) EDXA ofOR@ZnO depicting the presence of zinc and oxygen along with carbon, thereby confirming the capping of organic ligandOR onto ZnO nanoparticles; (C) Size distribution analysis of OR@ZnO (representing a hydrodynamic diameter of 560nm); (D) Solid-state UV-Vis absorbance profile of OR@ZnO; (E) Solid-state fluorescence emission spectra of OR@ZnO(upon excitation at 320 nm); (F) X-Ray powder diffractogram of OR@ZnO (average crystallite size of OR@ZnO esti-mated to be 30 nm).

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city of biologically relevant anions, a chemical recog-nition and quantification of such ionic species has poseda major challenge in aqueous phase12. This, in turn,has been attributed to the water-mediated disruption ofH-bonding and/or electrostatic interactions between theion of interest and host species30. In order to explore aprobable chemical affinity of OR@ZnO towards a poolof tetrabutyl ammonium salts of physiologicallyrelevant anions (F–, Cl–, Br–, I–, CN–, CH3COO–,HSO4

–, PO43–, NO3

– and ClO4–), its photophysical

properties were inspected by UV-Vis and fluorescencespectroscopy in HEPES buffered acetonitrile/H2O(98 : 2) system. Accordingly, due to the presence ofan arene-conjugated thiocarbonyl moiety, the nativeabsorption spectrum of OR@ZnO exhibited an intenseabsorption band (at 450 nm) in the near UV region(Fig. 3A). This characteristic peak is primarily attrib-uted to -* transitions resulting from an electron di-pole movement, commonly observed in aromatic car-bonyl compounds31. Further, upon an addition of smallaliquots of a solution of each tetrabutyl ammoniumanion salt (5 equivalents) to a solution of OR@ZnO,the absorption profile of OR@ZnO experienced a dis-tinct hypochromic shift and a slight bathochromic shiftin the receptor’s charge transfer band (460 nm) onlyin the vicinity of hydrogen sulphate anion. This couldapparently be attributed to certain alterations in theelectronic levels (HOMO/LUMO) of OR@ZnO, uponcoordination to the electron-rich hydrogen sulphateanion32. In addition, the absorption spectrum of nativeOR@ZnO displayed an uphill slope from 380–465nm, in contrast to a downhill slope observed in thesame region in the vicinity of hydrogen sulphate ion.A similar trend was, however, not observed in thepresence of other anions which indicated towards acertain physico-chemical sensitivity of OR@ZnO to-wards the hydrogen sulphate anion.

On the other hand, OR@ZnO nanoaggregates didnot experience any marked alterations in its UV-Visabsorption spectrum in the presence of any of the se-lect metal ions, thereby pointing towards an inherentinability of the metal cations in altering the electronic

environment of the chemical sensor in its ground state.In order to corroborate the selective binding ofOR@ZnO with HSO4

– in aqueous medium, titrationstudies were carried out. Accordingly, upon a succes-sive addition of HSO4

– (0–80 M) to a solution of thechemoreceptor OR@ZnO in aqueous medium, a dis-tinct drop in the intensity of its native absorption peakat 455 nm was observed with a concomitant formationof a new peak at 360 nm (Fig. 3B). In consequence, aclear isobestic point was observed at 390 nm, indicat-ing the formation of a well-defined OR@ZnO-HSO4

–

scaffold (Fig. 3B). The titration experiment indicateda considerable linearity (98%) within a concentrationrange of 0 M to 50 M (Fig. 3C). Using these titra-tion data, the nanoaggregates OR@ZnO were foundto detect HSO4

– ions in aqueous phase upto 100 nM(3 method)33. Upon an introspection of the library ofpotentially novel HSO4

–-sensing chemosensors pro-posed in the past, the ultra-sensitivity of OR@ZnOmatches closely to one of our recently reported andhighly sensitive HSO4

–-detecting chemosensor10,33.Such a significantly low detection limit is encouragingenough to introduce this newly synthesized receptorinto the elite class of environmentally relevant andhighly selective HSO4

–-sensing chemosensors. To sum-marize, this titration experiment confirmed a selectiveand sensitive affinity of OR@ZnO towards HSO4

–

ion in aqueous medium. In order to further validatethe on-field applicability and selective chemical affin-ity of OR@ZnO towards HSO4

– in a competitive aque-ous medium, an interference experiment was carriedout by subjecting a solution of OR@ZnO to HSO4

–

and a pool of select interfering anions (F–, Cl–, Br–,I–, CN–, CH3COO–, PO4

–, NO3– and ClO4

–), simul-taneously. In accordance with the results obtained, therewas an insignificant variation in the absorbance pro-file of HSO4

– -tagged OR@ZnO in the presence ofthe competing anions (Fig. 3D). Therefore, it couldbe deduced that OR@ZnO selectively coordinates withHSO4

– irrespective of the presence of other competinganions.

The physico-chemical stability of ligand-capped


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nanoparticles are influenced by their local environ-ment. Presence of electrolyte ions in the immediatevicinity may influence the charge on the nanoparticlesurface (basically in first order by the Debye-Hückeleffect), thereby resulting in electrochemical instabilityand consequent agglomeration34. A similar degradativeeffect on the aggregation state of nanoparticles mayalso be exerted by a pH-mediated protonation/deprotonation of acidic groups on surface of ligand-coated nanoparticles. As a consequence, such envi-ronmental factors serve as critical parameters in gov-erning the stability and ion-sensing relevance of

fluorophore-labelled NPs under water. In order to iden-tify an appropriate pH range in which nanoaggregatescan selectively probe HSO4

–, acid/base titrations werecarried out. The absorbance of the title ligand-coatedZnO nanoaggregates OR@ZnO was observed to varynegligibly within a pH range of 2.7 to 11.2 (Fig. 4A).To conclude, a change in pH over a broad range ap-parently exerted a minimal effect on the physico-chemi-cal stability of the nanoaggregates of R1. The effect ofionic strength on the ion-sensing potential of thenanoaggregates OR@ZnO was investigated by com-paring the absorbance spectrum of native OR@ZnO

Fig. 3. Variation in UV-Vis absorption profile of OR@ZnO in HEPES buffered acetonitrile/water (98 : 2 v/v) solvent system : (A)In presence of test anions; (B) Upon successive addition of sulphate anion (0–80 M); (C) Linear regression plot betweenconcentration of sulphate ion added (0–50 M) and change in absorbance of OR@ZnO with a linear regression coefficientof 0.9779; (D) Competitive binding studies of OR@ZnO nanoaggregates (1) towards sulphate anion (2) in the presence ofother interfering anions : F– (3), Cl– (4), Br– (5), I– (6), CN– (7), CH3COO– (8), PO4

– (9), NO3– (10) and ClO4

– (11).

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(0.5 M) with the absorbance spectrum obtained uponaddition of 0–100 equiv. of the tetrabutyl ammonium(TBA) salt of perchlorate to OR@ZnO (Fig. 4B)33.In accordance with the procured data, there was a neg-ligible variation in the absorbance curves, thereby in-dicating that even an excessive addition of salts yieldsa marginal effect on the ion-sensing ability of thenanoaggregates OR@ZnO. In order to investigate theresponse time of OR@ZnO towards HSO4

–, the ab-sorbance spectrum of the same was recorded in theabsence and presence of different concentrations ofHSO4

– (15, 30, 50 M) and analyzed as a function oftime. In accordance with the data obtained (Fig. 4C),the absorbance of OR@ZnO corresponding to the se-lect concentrations of HSO4

– did not vary after 35seconds of start of the experiment. In other words, the

response period of OR@ZnO towards HSO4– was

found to be less than 35 s.

Proposed mechanistic pathway for selective bindingof receptor N1 to sulphate anion

There is ample scientific evidence which confirmsthe critical reliance of certain biological sulphate-iontransporters on hydrogen bonding for the selective bind-ing and transport of this hydrophilic anion across thecellular membrane11. For instance, an elucidation ofthe X-ray structure of the sulphate-binding site in aspecific sulphate-binding protein reveals the encapsu-lation of the anion in a peptide pocket via H-bondingwith the N-H and O-H groups of the protein. In addi-tion, ion-sensing studies and structural data of the sul-phate-engulfed single crystals of a plethora of syn-

Fig. 4. (A) Effect of pH on stability of OR@ZnO nanoaggregates in aqueous medium; (B) Salt perturbation studies of ion-sensingOR@ZnO nanoaggregates in aqueous medium upon addition of (Bu)4N

+ClO4– (0–100 equiv.); (C) Time-dependent

response of OR@ZnO towards varying concentrations of HSO4– (15, 30, 50 M).

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thetic urea-based receptors further validate the signifi-cant role of H-bonding interactions in the sulphate-recognizing ability of chemical receptors with a com-mon amide/thioamide containing structural motif10,11.In due consideration of the same, a probable mode ofsensing of sulphate ion by the title thiourea-based ligand1B, via a potential H-bonding interaction with the acidicH-bond donor moiety (NH and OH) of the receptorligand has been postulated.

Conclusion

A diverse library of potential chemical sensorsagainst the physiologically relevant sulphate anion havebeen proposed in the past34–37. However, a limitedwater solubility and relatively low sensitivity continueto restrict their applicability in the immediate environ-ment. In due consideration of the same, we propose anew thiourea-based ligand OR as a potentially novelsulphate ion-sensing ligand. The new thiourea-basedreceptor OR was quantitatively synthesized by thenucleophilic addition of a secondary amine to a phenylisothiocyanate derivative under ambient reaction con-ditions. Upon capping onto environmentally compa-tible ZnO nanoparticles, the novel nanohybridOR@ZnO demonstrated a distinct chemical sensiti-vity and selectivity towards sulphate anion over otherbiologically relevant ions in aqueous medium, with asignificantly low detection limit of 100 nM. This po-tentially novel sulphate sensor may find industrial ap-plicability in the global fight against sulphate-inducedwater pollution.

Acknowledgement

AB and NK are grateful to CSIR (Project No.0(0216)/14/EMR-II) for the financial support in pur-suing this research work.

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2. J. W. Pflugrath and F. A. Quiocho, Nature, 1985, 314,257.

3. J. Habicher, T. Haitina, I. Eriksson, K. Holmborn, T.

Dierker, P. E. Ahlberg and J. Ledin, PloS One, 2015,10, e0121957.

4. E. C. H. E. T. Lucassen, A. J. P. Smolders, G. Boedeltje,P. J. J. Van den Munckhof and J. G. M. Roelofs, J.Veg. Sci., 2006, 17, 425.

5. M. Chen, X. H. Li, Y. H. He, N. Song, H. Y. Cai, C.Wang, Y. T. Li, H. Y. Chu, L. R. Krumholz and H. L.Jiang, Water Res., 2016, 96, 94.

6. C. C. Gilmour, D. A. Elias, A. M. Kucken, S. D. Brown,A. V. Palumbo, C. W. Schadt and J. D. Wall, Appl.Environ. Microbiol., 2011, 77, 3938.

7. M. C. Gabriel, N. Howard and T. Z. Osborne, Environ.Manag., 2014, 53, 583.

8. P. M. L. Leon, B. M. T. Hilde and G. M. R. Jan,Environ. Sci. Technol., 1998, 32, 199.

9. R. Kumar and P. Ghosh, Chem. Soc. Rev., 2012, 41,3077.

10. (a) K. Tayade, B. Bondhopadhyay, K. Keshav, S.K. Sahoo, A. Basu, J. Singh, N. Singh, D. T.Nehete and A. Kuwar, Analyst, 2016, 141, 1814;(b) K. Kaur, V. K. Bhardwaj, N. Kaur and N.Singh, Inorg. Chem. Comm., 2012, 18, 79.

11. S. Kubik, Chem. Soc. Rev., 2010, 39, 3648.

12. J. W. Pflugrath and F. A. Quiocho, J. Mol. Biol.,1988, 200, 163.

13. (a) K. H. Stern and E. S. Amis, Chem. Rev.,1959, 59, 1; (b) Y. Marcus, "Ion Properties",Marcel Dekker, New York, 1997; (c) Y. Marcus,H. D. B. Jenkins and L. Glasser, Dalton Trans.,2002, 20, 3795.

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17. C. J. Fowler, T. J. Haverlock, B. A. Moyer, J. A.Shriver, D. E. Gross, M. Marquez, J. L. Sessler,M. A. Hossain and K. B. James, J. Am. Chem.Soc., 2008, 130, 14386.

18. R. Kumar and P. Ghosh, Chem. Commun., 2010,46, 1082.

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20. R. Prohens, G. Martorell, P. Ballester and A.Costa, Chem. Commun., 2001, 16, 1456.

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A new highly selective and ratiometric chromogenic sensor for Cu2+ detection

Puspendu Roy, Sangita Das, Krishnendu Aich, Saswati Gharami, Lakshman Patra andTapan Kumar Mondal*

Department of Chemistry, Jadavpur University, Kolkata-700 032, India



Abstract : A new azo-phenol based chemosensor has been designed, synthesized and its structure is confirmedthrough different spectroscopic techniques. The synthesized chemosensor (HL) has shown a marked colorimetricresponse from light yellow to purple upon complexation with Cu2+ which is easily detectable through nakedeyes, makes the sensor efficient one to detect Cu2+ and remain nonresponsive in presence of other competitivemetal ions. The 1 : 1 complex formation of HL with Cu2+ is determined from Job’s plot analysis and massspectroscopic studies. Association constant (Ka, 8.94×104 M–1) of HL-Cu2+ complex suggests HL has high af-finity towards Cu2+.

Keywords : Azo dye, chemosensor, colorimetric detection, ratiometric, Cu2+ sensor, DFT calculations.

1. Introduction

Cu2+ is the third most abundant essential trace el-ement after Fe3+ and Zn2+ in human body and playsa crucial role in many important physiological pro-cesses1,2. Copper is being used as catalyst in a varietyof biological processes and it is the cause of its re-quirement in every living organism3. Oxygen trans-portation, hormone maturation, and signal transduc-tion etc. are the important biochemical activities ofCu2+ 4. For the formation of hemoglobin, red bloodcells and bones of the human system, copper is treatedas an essential element5. Besides its usefulness, cop-per ion at excessive concentration is very toxic. Dueto its adverse effect on living beings, Cu2+ is alsotreated as a notable metal pollutant6. Enormous use ofCu2+ in our daily life makes it easily accumulate inenvironment which exposes to human body throughcontamination of food and water. The U.S. Environ-mental Protection Agency (EPA) has set limits forcopper in drinking water and food to 1.3 ppm7. Manysevere neurodegenerative diseases, such as Alzheimer’sand Wilson’s diseases, amyotrophic lateral sclerosis,Menke’s syndrome and hematological manifestations

are the effect of disorder in CuII metabolism8–12. Thus,it is important to monitor the concentration of copperion in environmental sample.

In this regard, an optical sensor for Cu2+ ion, whichleads a prominent change in color is in high demand.Colorimetric sensors have been studied actively overthe recent decades due to their simple, inexpensive,and rapid implementation13,14. Especially, colorimet-ric sensors are quite promising because the change ofcolor can easily be noticed by the naked-eye. Thus,the colorimetric sensor requires much less labor andneed no equipments15,16. Therefore, the developmentof new chemosensor for Cu2+ detection, especiallythose which exhibit selective ratiometric Cu2+ detec-tion is in high demand which not only contributes tothe accurate measurement of the intensities of the twoabsorbance peaks, but also results in a huge ratiometricvalue.

Herein, we have reported the synthesis and photo-physical characterizations of ONS donor thioether con-taining azo phenol based derivative showing a promi-nent ratiometric sensing towards Cu2+. Experimentalstudies reveal that the sensor exhibits a remarkable


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affinity for Cu2+ in acetonitrile media which is asso-ciated with a color change from light yellow to pinkdue to the enhancement of ICT (internal charge trans-fer) effect.

2. Experimental

2.1. Materials and methods

CuCl2.H2O was purchased from Arrora Matthey,Kolkata, India. 2-(Ethylthio)benzenamine was synthe-sized following the published procedure17. All otherchemicals and solvents were of reagent grade and wereused without further purification.

Microanalyses (C, H, N) were performed using aPerkin-Elmer CHN-2400 elemental analyzer. HRMSmass spectra were obtained on a Waters (Xevo G2 Q-TOF) mass spectrometer. The electronic spectra wereobtained on a Lambda 750 Perkin-Elmer spectropho-tometer in acetonitrile solution. IR spectra were re-corded on RX-1 Perkin-Elmer spectrophotometer inthe spectral range 4000–400 cm–1 with the samples inthe form of KBr pellets. 1H NMR spectra were re-corded in CDCl3 on a Bruker (AC) 300 MHz FT-NMR spectrometer in the presence of TMS as internalstandard.

2.2. Synthesis

2.2.1. Synthesis of 4-chloro-2-((2-(ethylthio)phenyl)-diazenyl)phenol (HL)

A solution of 2-(ethylthio)benzenamine (3.06 g, 0.02mol) in 1 : 1 HCl (10 mL) was cooled in an ice bath.An ice cold solution of NaNO2 (2.0 g in 10 mL water)was added under stirring. This diazotized solution wasadded to an ice cold solution of Na2CO3 (6 g in 25mL) and 4-chlorophenol (2.56 g, 0.02 mol) with vig-orous stirring. An orange-red precipitate was observed.The precipitate was filtered and washed with cold water,then dried over CaCl2. The product was purified fur-ther by column chromatography using silica gel (60–120 mesh) and an orange-red band was eluted by 30%(v/v) ethyl acetate-petroleum ether mixture. Yield 3.7g (72%). The receptor HL has been characterized byelemental and mass spectral analysis along with sev-eral other spectroscopic techniques (IR, UV-Vis, NMRetc.).

Anal. Calcd. for C14H13ClN2OS (HL) : C, 65.34;H, 5.09; N, 10.89%. Found : C, 65.13; H, 5.01; N,10.77%; IR data (KBr, cm–1) : 3434 (O-H), 1421(N=N); 1H NMR (CDCl3, 300 MHz) (ppm) :12.67 (1H, s), 7.95 (1H, s), 7.85 (1H, d, J=8.0 Hz),7.28–7.45 (4H, m), 7.02 (1H, d, J=8.8 Hz), 3.04(2H, q, J=7.2 Hz), 1.40 (3H, t, J=7.3). HRMS m/z,280.19 (Calcd. for C14H13N2OS+Na+ : 280.321).

2.3. Synthesis of the HL-Cu2+ complex

CuCl2.H2O was added in acetonitrile light yellowcolored solution of HL and the stirring was continuedfor 4 h. The solvent was then removed under reducedpressure in rotary evaporator. The residue was washedby cold water followed by n-hexane and dried in aCaCl2 desiccator. Yield was, 0.093 g, 78%. Anal.Calcd. for C14H12Cl2CuN2OS : Calcd. (%) : C, 43.03;H, 3.10; N, 7.17. Found (%) : C, 43.04; H, 3.09; N,7.21. HRMS m/z, 413.523.

Scheme 1. Synthesis of chemosensor HL : (i) Na/dry MeOH/ethyl iodide, (ii) NaNO2/HCl/4-chlorophenol inNa2CO3 (0–5 ºC).

Scheme 2. Synthesis of HL-Cu2+ complex.

2.4. General method for UV-Vis titration

2.4.1. UV-Vis method

Stock solution of the receptor HL (10 M) wasprepared in CH3CN at 25 ºC. The aqueous solution ofthe guest cations using their chloride salts in the order

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of 10–4 M were prepared. Solutions of various concen-trations containing host and increasing concentrationsof cations were prepared separately. The changes inUV-Vis spectra of receptor (10 M) upon gradual ad-dition of metal ion solutions were recorded.

2.4.2. Job’s plot by absorbance methodFor Job’s plot experiment, a series of solutions

containing HL (10 M) and Cu2+ (10 M) were pre-pared in such a manner that the sum of the total metalion and HL volume remained constant (4 ml) in MeCN :H2O (1 : 5, v/v) medium. Job’s plots were drawn byplotting A versus mole fraction of Cu2+ (A =change of intensity of the absorption spectrum at 515nm).


3.3. Cation sensing studies of HL

3.3.1. UV-Vis studyAbsorption study of the receptor upon gradual ad-

dition of increasing concentrations of Cu2+ (0–2.0equiv.) is shown in Fig. 1. The probe shows two promi-nent absorbance peaks at 323 nm and 410 nm in ab-sence of any guest analytes. After addition of Cu2+ asignificant changes in the absorbance profile was no-ticed. Upon gradual addition of Cu2+ to the solutionof HL (10 M) in MeCN : H2O (1 : 5, v/v) mediumthe absorption band at 410 nm gradually decreases anda new band appears at 515 nm which gradually in-creases after incremental addition of Cu2+ with a promi-nent isobestic point at 466 nm. The intensity of thenew band (at 515 nm) increases appreciably with pro-gressive addition of Cu2+ (up to 2 equiv.) (Fig. 1).

This photophysical changes leads to a prominentcolor change of the solution from light yellow to pink.This large red shift (105 nm) of the absorption spectraof HL is attributed due to the enhancement of ICTmechanism after complexation with guest Cu2+ ion.The absorbance at 515 nm exhibits a good linearitywith added Cu2+ concentration with a very good R2

value (Fig. 2). The detection limit of the sensor forCu2+ is determined from the absorption spectralchange, upon addition of Cu2+ to be 6.26×10–8 M,using the equation DL = K Sb1/S, where K = 3, Sb1is the standard deviation of the blank solution, and S isthe slope of the calibration curve. Upon incrementaladdition of Cu2+ solution, a complete reduction in theintensity at 410 nm band accompanied by a ratiometricincrease in intensity at 515 nm was observed whichachieved saturation after the addition of a ~1.0 equiva-lent (10 M) solution of Cu2+ ions (Fig. 1). The ratioof two absorption intensities (A410/A515) also main-tained a good linear relationship with [Cu2+] (Fig. 3).

Fig. 1. Changes in UV-Vis spectra of HL upon gradual addi-tion of Cu2+ in MeCN : H2O (1 : 5, v/v).

Fig. 2. The linear response curve of HL at 515 nm dependingon the Cu2+ concentration.

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The selectivity and interference are the two veryimportant parameters to determine the novelty of anyreceptor. Now the sensitivity and selectivity of theprobe HL towards Cu2+ were examined by employ-ing different guest analytes. In addition of other guestions (except Cu2+) no noticeable change was observedin the absorbance profile (Fig. 4). Upon addition ofCu2+, the absorbance of the sensor at 515 nm was


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increased by 27-fold. Competition study with differentmetal ions (30 M) in the solution of HL (10 M) andCu2+ (20 M) suggesting that there is no significantinterferences of other metal ions (Cd2+, Co2+, Cr3+,Fe3+, Hg2+, K+ , Mg2+, Na+ , Mn2+, Ni2+, Al3+,Pb2+, In3+ and Zn2+) for the detection of Cu2+ (Fig.5). This phenomenon indicates that the sensor can beemployed conveniently for Cu2+ detection by simplevisual inspection. So the selectivity of HL towardsCu2+ is thus well proven.

MeCN : H2O (1 : 5, v/v) at 25 ºC. The UV-Vis spec-trum in each case with different host-guest ratio butequal in volume was recorded. Job’s plots were drawnby plotting I.Xhost vs Xhost (I = change of inten-sity of the absorbance spectrum at 515 nm during ti-tration and Xhost is the mole fraction of the host ineach case, respectively). From the Job’s plot titrationit can be concluded that the stoichiometry between HLand Cu2+ is 1 : 1.

The stoichiometry of the probe in the presence ofCu2+ is found to be 1 : 1, which is supported by theJob’s plot (Fig. 6) and confirmed by HRMS experi-ment. The HRMS spectrum of the HL-Cu2+ complexgives a signal at m/z 413.5, which may be for[Cu(L)Cl+Na]+ species. Further to understand thestability of the HL-Cu2+ complex association constantcalculated according to the Benesi-Hildebrand equa-tion. Ka was calculated following the equation statedbelow :

1/(A – Ao) = 1/{Ka(Amax – A0) [Mx+] n}

+ 1/[Amax – A0]

Here, A0 is the absorbance of receptor in the absenceof guest, A is the absorbance recorded in the presenceof added guest, Amax is absorbance in presence ofadded [Mx+]max and Ka is the association constant,where [MX+] is [Cu2+]. The association constant (Ka)could be determined from the slope of the straight lineof the plot of 1/(A – A0) against 1/[Cu2+] and is found

Fig. 3. Absorbance intensity ratio at A515/A410 nm of HL de-pending on the Cu2+ concentration.

Fig 4. UV-Vis spectra of HL (10 M) in MeCN : H2O (1 : 5,v/v) in presence of different metal ions (3 equiv.).

Fig. 5. Competition study using UV-Vis method, after additionof different analytes (30 M) in the solution of HL (10M) in presence of Cu2+ (20 M).

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To understand the interaction of HL and Cu2+ insolution Job’s plot by absorbance method was per-formed. Stock solution of same concentration of HLand Cu2+ was prepared in the order of 10 M in

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Fig. 6. Job’s plot diagram of receptor for Cu2+ (where Xh isthe mole fraction of the host and I indicates the changeof absorbance intensity at 515 nm).

to be 8.94×104 M–1 (Fig. 7). Thus the experimentsstrongly suggest that there is a strong association be-tween the radii of Cu2+ and the cavity space in theprobe HL, which ultimately gives rise to strong inter-action between Cu2+.

of absorption intensity between pH 8–11. Receptor canform stable complex with Cu2+ between pH 5–8. Atvery low pH (<3), there is a tendency to combinewith proton, hence become less effective in detectionof Cu2+. So it is clear that our synthesized receptorcan sense Cu2+ ion between pH 5–8.

Fig. 7. Benesi-Hildebrand plot from absorption titration data ofreceptor HL (10 M) with Cu2+.

For extending the scope of experiment of synthe-sized probe HL, we have studied the sensing at differ-ent pH medium. The absorption intensity at 515 nm ofthe receptor is almost unaffected at different pH con-dition but in presence of Cu2+ ion there is large change

Fig. 8. Absorbance response of HL and HL-Cu2+ at 515 nm(10 M) as a function of pH.

4. Conclusion

A new azo-phenol based chemosensor has beensuccessfully synthesized and characterized by severalspectroscopic techniques. HL showed a marked colo-rimetric response from light yellow to purple in pres-ence of Cu2+ which is easily detectable through nakedeyes. Moreover, the sensor showed a selective colorchange in presence of Cu2+ and remain non respon-sive in presence of other competitive metal ions. The1 : 1 host-guest complex formation of HL with Cu2+

is determined from Job’s plot analysis and mass spec-troscopic studies. Association constant (Ka, 8.94×104

M–1) of HL-Cu2+ complex suggests HL has high af-finity towards Cu2+.

AcknowledgementFinancial supports received from the Council of

Scientific and Industrial Research, New Delhi, India(No. 01(2831)/15/EMR-II) and the Department of Sci-ence and Technology, New Delhi, India (No. SB/EMEQ-242/2013) are gratefully acknowledged. P. Roy,S. Gharami and L. Patra are thankful to UGC, NewDelhi for their fellowships.

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Orbital and molecular design of new naphthyl-salen type transition metalcomplexes toward DSSC dyes

Marin Yamaguchia, Yuki Tsunodaa, Shinnosuke Tanakaa, Tomoyuki Haraguchia, Mutsumi Sugiyamab,Shabana Noorc and Takashiro Akitsu*a

aDepartment of Chemistry, Faculty of Science, Tokyo University of Science, Shinjuku-ku, Tokyo, Japan


bDepartment of Electrical Engineering, Faculty of Science and Technology, Tokyo University of Science,Chiba, Japan

cDepartment of Chemistry, Aligarh Muslim University, Aligarh-202 002, Uttar Pradesh, India


Abstract : Dye sensitized solar cells (DSSC) emerged as a new class of low cost energy conversion devices. Inthis study, we investigated a class of transition metal complexes [ML(CH3OH)]; M = Mn(1), Fe(2), Co(3), Ni(4),Cu(5) and Zn(6) and H2L is naphthyl-salen Schiff base ligand. The COOH group of the complexes leads tobetter ability for adsorption of dyes. These complexes were characterized employing elemental analysis, powderX-ray analysis, IR, UV-Vis-NIR electronic spectroscopy and so on. Light absorption of the all the complexesshows that -conjugated naphthyl ring makes these complexes efficient as dyes in DSSC. However, spatial dis-tribution of excited orbitals associated with absorption properties (UV-Vis absorption spectra) calculated by TD-DFT method revealed one of the important factors about orbitals and molecular design of metal complexes forDSSC dyes.

Keywords : Schiff base complexes, orbital design, TD-DFT calculation, UV-Vis spectra, dye sensitized solar cell(DSSC).

Introduction

In the year 1991, Grätzel and O’Regan firstly re-ported on a low cost organic material type solar cell,namely dye sensitized solar cells (DSSC) based onTiO2 materials. Thus, DSSC is one of the organictype solar cells, producing electrical energy by lightharvesting of dye molecules. An electron is excited bythe photoexcitation of the dye and transferred to con-duction band of TiO2 which transfers to positive elec-trode and finally returns to ground state dye moleculevia oxidation-reduction reaction of electrolyte. Atpresent, DSSC are the most extensively investigatedarea owing to their applications as low cost photovol-taic devices especially suited for building and automo-biles integrated PV and portable or indoor light har-vesting applications. Many researches have been per-formed on DSSC using different type of dyes1a.

For example, Ru2+ polypyridyl complexes,[Ru(dcbpyH2)2(NCS)2], [Ru(4,4-dicarboxy-2,2-bipyridine)2(NCS)2] (commonly called "N3")1b hadbeen widely used as dye component and shows higherconversion efficiency because of long excitation life-time; however, the metal is rare and expensive2–7.

In addition, reports on transition metal complexeshave demonstrated that organic ligands play importantrole in enhancing the luminescent color and intensity.Especially, narrowing HOMO-LUMO gap makes ex-pansion of absorption band toward NIR region8–10.Although some organic molecules and complexes havebeen developed as dyes in DSSCs, design and synthe-sis of organic ligands and their transition metal com-plexes for application on DSSCs is still a great chal-lenge11–17. In the design of complexes, Schiff baseligands have been widely used as ligands due to their


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convenient synthesis.

Herein we have focused on designing of dyes basedon salen type Schiff base complexes. We also considerthe effect of substituents on the absorption conversionperformance theoretically. Our prediction shows thatthe first row transition metal complexes may be effec-tively exploited as dyes. In the present article, wepresent the synthesis and characterization of the newtransition metal complexes [ML(CH3OH)]; M =Mn(1), Fe(2), Co(3), Ni(4), Cu(5) and Zn(6) and H2Lis naphthyl-salen Schiff base ligand and discusses theeffect of central metal on absorption characteristic alongwith computational calculations. The complexes 1-6were employed to prepare DSSC devices, which couldcompensate for the absorption of N3 in the high-en-ergy band region of the visible spectrum. Thus, per-formance of DSSCs based on complexes as sensitizersis investigated in detail.

Experimental

Chemicals and solvents :

All starting materials such as 1-vinyl-napthalen-2-ol and 2,3-diamino-succinic acid and metal saltsM(OAc)2.H2O were obtained from commercial sources.The solvents like methanol and DMF, DMSO werereagent grade and used as supplied.

Instrumentation :

Elemental analyses (C, H, N) were carried out witha Perkin-Elmer 2400II CHNS/O analyzer at the To-kyo University of Science. FT-IR spectra were re-corded as KBr pellets on a JASCO FT-IR 4200 plusspectrophotometer in the range 4000–400 cm–1 at 298K. UV-Vis-NIR absorption spectra and diffuse reflec-tance electronic spectra were measured on a JASCOV-570 UV-Vis-NIR spectrophotometer. Cyclicvoltammetry (CV) was performed on a AIOS/DY2323with a traditional three electrodes. The working elec-trode and auxiliary electrode was Pt disc. Ag/AgCland Pt wire electrode, respectively. Magnetic studieswere performed using a Quantum design MPMS-XLSQUID magnetometer. The X-ray diffraction pat-terns were collected at 298 K on Rigaku Smart lab(CuK radiation) at the University of Tokyo. Powdercrystal structure analysis was carried out with a Rigaku

PDXL2, commercially available program package.

Computational calculations :

All calculation was performed by the Gaussian 09Wsoftware Revision A.02 (Gaussian, Inc.)18. CalculatedUV-Vis spectra was gained by TD-DFT with B3LYPfunctional. Basis sets were Lan12DZ on central metalion to consider the effect core potential and 6-31(d) onother atoms.

Synthesis :

Naphthyl-salen Schiff base ligand (H2L) and 5 weresynthesized according to the reported method by us19.To methanol solution of 1 mmol H2L ligand, 1 mmolM(OAc)2·xH2O (M = Mn, Fe, Co, Ni, Cu and Zn)was added and dissolved. The reaction mixture wasstirred for 2 h, subsequently filtered and collected atroom temperature.

1 : Yield 17.9%; IR (KBr, cm–1) : 457w, 508w,565w, 657w, 725w, 753w, 831w, 979w, 1038w,1097w, 1191w, 1250w, 1293w, 1340w, 1362m,1385m, 1456w, 1478w, 1542s, 1599s, 1618s (C=N),1727w, 2950w, 3075w, 3253w; Anal. Calcd. forC27H22MnN2O7 : C, 52.96; H, 3.62; N, 4.57. Found :C, 51.95; H, 3.32; N, 4.79%.

2 : Yield 75.0%; IR (KBr, cm–1) : 501w, 566w,618w, 654w, 751w, 831w, 863w, 990w, 1040m,1028s, 1128s, 1192m, 1212m, 1243m, 1295w, 1342m,1364m, 1384m, 1423w, 1453w, 1540m, 1579s, 1600s,1617s (C=N), 1728w, 2962w, 3066w, 3200w; Anal.Calcd. for C27H22FeN2O7 : C, 51.96; H, 3.62; N,4.57. Found : C, 51.87; H, 3.39; N, 4.77%.

3 : Yield 61.4%; IR (KBr, cm–1) : 505w, 567w,659w, 753w, 829w, 977w, 1037w, 1099w, 1188w,1251w, 1295w, 1340m, 1361m, 1389m, 1435w,1452w, 1510w, 1541m, 1590s, 1619s (C=N), 1731w,2954w, 3076w, 3252w; Anal. Calcd. forC27H22CoN2O7 : C, 52.28; H, 3.57; N, 4.52. Found :C, 51.98; H, 3.48; N, 4.84%.

4 : Yield 25.0%; IR (KBr, cm–1) : 506w, 571w,655w, 751m, 829w, 982w, 1045w, 1106w, 1199m,1260w, 1294m, 1364m, 1391m, 1485w, 1542s, 1586s,1603s, 1618s (C=N), 1721w, 2941w, 3074w; Anal.Calcd. for C27H22NiN2O7 : C, 52.32; H, 3.58; N,4.52. Found : C, 51.98; H, 3.48; N, 4.05%.

Yamaguchi et al. : Orbital and molecular design of new naphthyl-salen type transition metal complexes etc.

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6 : Yield 50.0%; IR (KBr, cm–1) : 505w, 575w,723w, 752w, 835w, 974w, 1032w, 1101w, 1199m,1282m, 1371m, 1419m, 1483w, 1518w, 1546m, 1575s,1627s (C=N), 1731w, 2938w, 3062w, 3208w; Anal.Calcd. for C27H22ZnN2O7 : C, 51.21; H, 3.50; N,4.42. Found : C, 51.86; H, 3.45; N, 4.78%.

DSSC fabrication :

Negative electrodes were prepared by adding 1.5 gof TiO2 (P25) to 1.5 g polyethylenegycol (PEG) and15% (v/v) of acetic acid. The mixture was stirred for1 h, resulting in a paste. The paste was extended into1 cm2 of Indium Tin Oxide (ITO) glass 2.5 cm2

by squeegee method. Then the ITO was heated up to450 ºC for 30 min. Finally, the glass was immersed indye (1-4, 6) solutions (5×10–4 M) for 24 h. Electro-lyte was composed of mixture of 0.127 g I2, 0.127 gof dimethyl-3-propylimidazolium iodide (DMPImI),0.134 g of LiI and 0.405 g of tertbutylpyridine (TBP)in 10 mL MeCN. 2–3 drops of the resulting solutionwere added before measurement.

We prepared three types of positive electrode (ITOglass). One ITO glass was coated with graphite using10B pencil. Another was ITO glass with rough sur-face, on which graphite was pasted using 10B pencil.The other ITO glass was exposed to burning candleand covered with graphite. We employed the type 3positive electrode for measurements (Fig. 1).


Synthesis :

The Schiff base ligand was synthesized employingthe condensation reaction of 1-vinyl-naphthalen-2-ol

and 2,3-diamino-succinic acid in the ratio 2 : 1. Thereaction of the preformed Schiff base with metal ac-etate, M(OAc)2·xH2O (M = Mn(1), Fe(2), Co(3),Ni(4) and Zn(6)) afforded polycrystalline solid com-pounds (Scheme 1). 2,3-Diamino-succinic acid andCu(5) complex was previously synthesized and char-acterized thoroughly and reported19. The stoichiom-etry of the rest of the complexes 1-4 and 6 obtainedwas established through elemental analysis and FT-IRwhich was confirmed by structural characterization.

IR spectral studies :

FT-IR spectra of the complexes have provided con-clusion for the mode of coordination from the dianionicmoiety (nap)2– to the metal ions. The positions of bandsare summarized in Experimental section. The spectraof all the complexes contain a band in 1617–1627cm–1 range is the characteristic of (C=N) iminic bond.The spectra also contained bands in 1721–1731 cm–1

range, represent the characteristic stretching frequencyfor free uncoordinated -COOH of the unionized car-

Fig. 1. Schematic of the typical DSSC (SLG soda lime glass;ITO indium doped tin oxide).

Scheme 1. Preparation and chemical structures of 1-6.

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boxylic functions of ligand. The appearance of me-dium intensity band in low frequency region charac-teristic of M–N(~570 cm–1) and the bands M–O(~505 cm–1) stretching vibrations were also presentin the spectra of the complexes.

X-Ray crystallographic studies :

The results of the X-ray analysis on crystals of thecomplexes 1-4 and 6 with refinement parameters are

summarized in Table 1. As shown in Fig. 2, the struc-tures of the independent molecules in the unit cells arepresented for respective complexes (significant atomsare labeled). The selected bond lengths are summa-rized in Table 2. The selected bond angles for thecomplexes (1-3) and (4 and 6) are compiled in Table 3and Table 4 respectively. All the complexes are withincommon range for the related ones20. The metal ions

Fig. 2. Crystal structures of complexes (a) [MnL(CH3OH)](1), (b) [FeL(CH3OH)](2), (c) [CoL(CH3OH)](3), (d) [NiL(CH3OH)](4),(e) [CuL(CH3OH)](5) and (f) [ZnL(CH3OH)](6).

(e)

(c) (d)

(a) (b)

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Table 1. Crystal data and structural refinement for 1-4 and 6

1 2 3 4 6

CCDC 1540630 1540629 1540628 1540631 1540627

Empirical formula C27H22MnN2O7 C27H22FeN2O7 C27H22CoN2O7 C27H22NiN2O7 C27H22ZnN2O7

Formula weight 541.41 542.32 513.37 545.17 551.89

Crystal system Triclinic Monoclinic Monoclinic Triclinic Triclinic

Space group P-1 P2 P2 P-1 P-1

a (Å) 11.21(12) 14.10(2) 10.779(7) 9.39(8) 12.568(19)

b (Å) 15.47(6) 12.87(2) 16.177(4) 16.06(3) 17.41(2)

c (Å) 9.63(11) 110.11(2) 7.740(11) 7.977(9) 10.893(18)

(º) 92.46(11) 95.43(15) 109.83(18)

(º) 111.79(10) 106.02(2) 99.04(18) 107.62(7) 102.46(11)

(º) 93.28(15) 99.48(7) 71.10(10)

V (Aº3) 1550(3) 1921.1(6) 1332.8(6) 1118(2) 2096(6)

Z 2 2 2 2 2

Rwp (%) 2.86 3.97 1.92 5.66 3.90

Table 2. Selected bond lengths (Å) for 1-4 and 6

1 2 3 4 6

Mn–O1 1.933(2) Fe–O1 1.9499(3) Co–O1 1.8961(5) Ni–O1 1.878(4) Zn–O1 2.312(3)



Mn–N1 2.144(3) Fe–N1 2.1312(6) Co–N1 1.9104(7) Ni–N1 1.944(3) Zn–N1 2.321(3)

Mn–N2 2.130(14) Fe–N2 2.1490(3) Co–N2 1.9305(5) Ni–N2 2.142(3) Zn–N2 2.268(3)

Table 3. Selected bond angles (º) for 1-3

1 2 3

O1–Mn–O3 101.76(8) O1–Fe–O3 102.533(4) O1–Co–O3 92.085(17)

O2–Mn–O3 97.76(10) O2–Fe–O3 96.518(16) O2–Co–O3 93.355(8)

O1–Mn–O2 97.76(10) O1–Fe–O2 108.420(4) O1–Co–O2 89.902(7)

O1–Mn–N1 80.99(17) O1–Fe–N1 83.554(5) O1–Co–N1 91.488(7)

O1–Mn–N2 147.30(6) O1–Fe–N2 147.158(9) O1–Co–N2 164.861(4)

O2–Mn–N1 162.23(2) O2–Fe–N1 161.950(5) O2–Co–N1 173.835(2)

O2–Mn–N2 83.73(10) O2–Fe–N2 84.626(9) O2–Co–N2 91.471(5)

O3–Mn–N1 92.54(11) O3–Fe–N1 93.845(16) O3–Co–N1 92.596(7)

O3–Mn–N2 106.23(8) O3–Fe–N2 105.795(5) O3–Co–N2 102.880(16)

N1–Mn–N2 79.49(11) N1–Fe–N2 78.341(6) N1–Co–N2 85.632(5)

occupy the inner chamber of the Schiff base ligandand exhibits penta-coordinated geometry whereby thedianonic (nap)2– binds in tetradentate fashion via twooxygen (O1 and O2) and two nitrogen (N1 and N2) ofthe Schiff base ligand which are approximately copla-nar. The tetragonality index, = (–)/60, where and are given by opposing angles in the coordinationpolyhedron. The values of are zero and unity, forperfect square pyramidal and trigonal bipyramidal

respectively. The values of for complexes are foundto be 0.248(1), 0.246(2), 0.149(3), 0.184(4) and0.164(6). According to these values, the coordinationgeometry around the metal ions is best described asdistorted square-based pyramidal with one methanol ataxial position.

Magnetic properties :

Magnetic susceptibility measurements for powder


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samples (1-4) were carried out by the SQUID basedmagnetometer in the temperature 5–300 K. The mag-netic susceptibilities are shown as a function of tem-perature in Fig. 3. The magnetic data were fitted byemploying Curie-Weiss law, = C/(T+). Magneticmoments were obtained from the relation =2.828(T)1/2 (B.M.). The number of unpaired elec-trons (n) was predicted from by the relation ={n(n+2)}1/2 21. The observed effective magnetic mo-ment along with number of unpaired electrons are

shown in Table 5. The n values are in agreement withexpected d-electron configurations of the coordinationgeometries determined.

Table 4. Selected bond angles (º) for 4 and 6.

4 6

O1–Ni–O3 101.23(9) O1–Zn–O3 114.57(13)

O2–Ni–O3 99.35(8) O2–Zn–O3 100.29(13)

O1–Ni–O2 95.50(11) O1–Zn–O2 85.10(15)

O1–Ni–N1 87.98(11) O1–Zn–N1 76.51(12)

O1–Ni–N2 156.43(5) O1–Zn–N2 152.17(3)

O2–Ni–N1 167.51(2) O2–Zn–N1 94.57(12)

O2–Ni–N2 87.88(12) O2–Zn–N2 108.86(11)

O3–Ni–N1 91.70(8) O3–Zn–N1 162.034(13)

O3–Ni–N2 101.21(9) O3–Zn–N2 87.21(16)

Fig. 3. The m vs T and mT vs T plots for complexes 1-4; Inset : The –1 vs T plots.

Table 5. Effective moment () for the paramagnetic com-plexes (1-4)

Complexes (B.M.) No. of unpaired electrons

1 6.03 5

2 4.64 4

3 4.05 3

4 2.91 2

UV-Vis-NIR electronic spectra and TD-DFT calcu-lations :

The electronic spectra of the 3×10–5 M DMF solu-tions of the complexes are shown in Fig. 4(a). Theintense high energy absorption bands appear at ~270nm (Band 1) and a weak band at ~400 nm (Band 2) inall complexes are due to –*. The complex 4 showshighest absorption coefficient whereas in complex 6,band 2 is broad and prominent as compared to other

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complexes. From these observations, it may be con-cluded that structure of the ligand affects the absorp-tion bands. Comparing with the absorption spectra ofRu2+ polypyridyl complexes, [Ru(dcbpyH2)2(NCS)2],N3, apparently all the complexes could better com-pensate for the absorption of N3 in the low wave-length region of the visible spectrum22. Therefore, weapply complexes (1-6) as dyes to apply in the DSSCdevices. The high molar extinction coefficient indi-cates that they possess a better light-harvesting abilityin this low wavelength region compared with N3.Meanwhile the ligand has carboxylic groups, whichcould adsorb on the TiO2 surface.

The solid state adsorption spectra were also ob-tained to gain insight for the d-d transitions in thecomplexes 1-6 shown in Fig. 4(b). It has been foundthat absorption band at ~700 nm appears in all com-plexes is due to d-d transitions.

The electronic transition in UV spectra of com-plexes 1-6 have been carried out employing TD-DFT(B3LYP/6-31=G(d,p)] method and provided in Fig.5. The molecular orbitals (MOs) HOMO and LUMOwere provided in Fig. 6. To support the DFT geom-etry optimizations, the calculated geometries were com-pared with the experimental spectra data. A compari-son of the theoretical data for the complexes allows us

Fig. 4. (a) Electronic absorption spectra for 1-6 (3×10–5 M DMF). (b) Solid state diffuse reflectance spectra for 1-6.

(a)

(b)

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768

to assess the extent of spectra and to correctly describethe electronic spectra.

The broadening of the peak in Fig. 5 is generatedby two peaks in the absorption spectra at ~300 nmand 400 nm. Band 1 and 2 arise due to (–*) charge

transfer of the naphthyl ring. It is clear from Fig. 5 thatthese two bands are comparable, but complex 6 showthe broadest absorption band among all molecules.

It may also be concluded from Fig. 6 that the com-plex 4 shows longest wavelength. It is plausible to

Fig. 5. Calculated UV-Vis-NIR spectra.

Fig. 6. Orbital distribution (around HOMO or LUMO) before and after intraligand transitions for 1-6, transition energies (inwavelengths) are 329.0, 322.6, 325.3, 361.4, 327.3, and 323.7 nm, respectively.

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conclude that visual appearance of electron transitionby band 2 that causes power generation are shown inFig. 6. It may also be concluded that it is only thecomplexes 4 that can be used as dye as this follow thetrend for DSSC power generation effective. In con-trast to 4, other complexes are not suitable for thispurpose due to unsuitable orbital distribution of ex-cited states (far from -COOH groups).

Table 6. Summary of electrochemical parameters of the complexes 1-6

Complexes Epa Ep

c Epa Ep

c HOMO LUMO

(V vs Ag/AgCl) (V vs Ag/AgCl) (V vs NHE) (V vs NHE) (eV) (eV)

1 –1.170 0.307 –0.970 0.507 –4.95 –3.47

2 –1.150 0.476 –0.950 0.676 –5.12 –3.49

3 –1.170 0.103 –0.970 0.303 –4.74 –3.47

4 –1.120 0.187 –0.920 0.387 –4.83 –3.52

5 –1.300 0.313 –1.100 0.513 –4.95 –3.34

6 –1.130 0.545 –0.93 0.745 –5.19 –3.51

Scheme 2.Schematic orbital energy levels of (a) TiO2 and I2 and (b) comparison with complexes (1-6) used as dyes in DSSCs(upper lines are HOMO and lower line are LUMO of the complexes).

(a) (b)

cyclic voltammograms (CV), these sensitizers have lowlying highest occupied molecular orbital (HOMO) lev-els from –4.74 to –5.19 eV. All HOMO are lowerthan the redox potential of I–/I3– (–4.85 eV vs vacuum),indicating the favorable regeneration. The lowest un-occupied molecular orbital (LUMO) energy of the com-plexes are –3.47(1), –3.49(2), –3.47(3), –3.52(4),–3.34(5) and –3.51(6) eV, respectively. All calculated

Electrochemical properties :

We investigated the UV-Vis absorption spectra ofcomplexes 1-6. The HOMO-LUMO energy levels arecrucial in selecting sensitizers for DSSCs, which weredetermined by cyclic voltammetry in a 0.1 Mtetrabutylammonium perchlorate (TBAP). The elec-trochemical parameters are compiled in Table 6. Asestimated from the onset oxidation potentials in the

LUMOs are higher than the conduction band of TiO2(–4.40 eV vs vacuum), leading to effective electrontransfer (Scheme 2).

Characterization parameters for DSSC :

The solar to electricity conversion efficiency () ofthe solar cell is calculated by the photocurrent densitymeasured at short-circuit (Jsc), the open-circuit photo-voltage (Voc), the fill factor of the cell (FF) and the

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intensity of the incident light (Pinc) by the relation,

= FF(VocJsc/Pinc)

FF = (Imp×Vmp)/(Jsc×Voc)

where, Imp is the maximum power point current, Vmpis the maximum point voltage.

Photovoltaic properties of DSSCs :

The complexes (1-6) were assembled into sensi-tized DSSCs devices to study the influence of the elec-tron-donating group and coordination on the DSSCsperformance was tested under irradiation of 100 mWcm–2 by ADCMT 6241 DC voltage/current source/

monitor. The data of Jsc, Voc and FF for these devicesare shown in Fig. 7 and presented in Table 7. Thehigher value of Voc corresponds to higher harvest ofthe light energy. The efficiencies are found in the or-der 4 > 6 > 1 > 5 > 3 > 2. Complex (4) and (6)show higher Voc and Jsc, which indicates the higherefficiency as compared to the other complexes. Thestrongest light harvesting ability of the complex 6 isalso supported by the appearance of band 2 in calcu-lated electronic spectra. However, band 2 of 4 indicat-ing the highest Voc value is ascribed to electron trans-fer towards -COOH group.

Fig.7. The J-V plots for complexes 1-6.


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The metal order of efficiency is Ni > Zn > Mn> Cu > Co > Fe as listed in Table 6. Efficiencies ofDSSC with 4 and 5 showed an order of magnetitelarger value than other cells, this is due to compara-tively higher Voc and Jsc.

The enhanced Jsc values will be discussed in con-junction with the incident photon-to-current electronconversion efficiency (IPCE) spectra shown in Fig. 8and the parameters are provided in Table 6 to knowthe degree of energy conversion from light harvestingtoward electricity at each light wavelength. The com-plex (4) show the highest value (7%) around 350 nmcorresponds to band 2 in calculated UV-Vis spectra.This transfer results in high value of IPCE. Complex6 show the efficiency () next to 4 and also exhibit thehigh IPCE value too. It may be concluded that com-plexes 4 and 6 can be exploited as dyes in DSSC cellsand increase the conversion efficiency of the cell.

Conclusion

The newly synthesized naphthyl-salen type mono-nuclear complexes of Mn, Fe, Co, Ni, Zn have beencharacterized using elemental analysis and FT-IR. Thestructures are ascertained through X-ray diffraction oncrystal analysis. The metal ions in all the complexeswere pentacoordinate, square pyramidal geometry. TheUV-Vis-NIR, TD-DFT calculation, magnetic proper-ties were also measured and all the five complexesalong with the Cu complex previously synthesized andcharacterized by us. Similar to previous systems23,UV-Vis-NIR electronic spectra of these complexes werealso measured and inference of electron transfer ofeach absorption band is estimated by TD-DFT calcu-lations. The complexes (1-6) were assembled into sen-sitized DSSCs devices to study the influence of theelectron-donating group and coordination in the DSSC.The Ni-nap and Zn-nap show comparatively strongerabsorption, high Voc, Jsc and show suitable HOMO-LUMO band of dye for electron cycle in cell as com-pared to other complexes. Thus, these two complexesmay be exploited as dye in DSSC.

CCDC 1540627-1540631contain the supplementarycrystallographic data. These data can be obtained freeof charge via <http://www.ccdc.cam.ac.uk/conts/retrieving.html>, or from the Cambridge Crystallo-graphic Data Centre, 12 Union Road, Cambridge CB21EZ, UK; Fax : (+44) 1223-336-033; or E-mail :[email protected].

Table 7. The parameters of DSSC performance for 1-6

Dye Voc Jsc FF (V) (mA cm–2) (%)

N3 0.6803 6.0247 0.26 1.0824

Mn-nap 0.0861 0.0084 0.28 2.05×10–4

Fe-nap 0.396 0.0050 0.26 0.52×10–4

Co-nap 0.425 0.0103 0.26 1.15×10–4

Ni-nap 0.1668 0.0305 0.29 1.48×10–4

Cu-nap 0.0304 0.0264 0.22 1.75×10–4

Zn-nap 0.0739 0.0660 0.26 1.26×10–4

Fig. 8. IPCE spectra for fabricated DSSC with complexes 1-6.

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Acknowledgement

This XRD work was conducted at Advanced Char-acterization Nanotechnology Platform of the Univer-sity of Tokyo (Professor Kazuhiro Fukawa), supportedby "Nanotechnology Platform" of the Ministry of Edu-cation, Culture, Sports, Science and Technology(MEXT), Japan.

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2. A. Hagfeldt, G. Boschloo, L. Sun, L. Kloo and H.Pettersson, Chem. Rev., 2010, 110, 6595.

3. T. P. Brewster, S. J. Konezny, S. W. Sheehan, L. A.Martini, C. A. Schmuttenmaer, V. S. Batista and R. H.Crabtree, Inorg. Chem., 2013, 52, 6752.

4. M. Komatsu, J. Nakazaki, S. Uchida, T. Kudo and H.Segawa, Phys. Chem. Chem. Phys., 2013, 15, 3227.

5. D. G. Pogozhev, P. A. Schauer, J. B. Garcia, B. R.Fancy and C. P. Berlinguette, J. Am. Chem. Soc., 2013,135, 1692.

6. D. Wang, Y. Wu, H. Dong, Z. Qin, D. Zhao, Y. Yu,G. Zhou, B. Jiao, Z. Wu, M. Gao and G. Wang, Or-ganic Electronics, 2013, 14, 3297.

7. S. Sinn, B. Schulze, C. Friebe, D. G. Brown, M. Jager,J. Kubel, B. Dietzek, C. P. Berlinguette and U. S.Schubert, Inorg. Chem., 2014, 53, 1637.

8. A. El-Shafei, M. Hussain and A. Atiq, J. Mater. Chem.Soc., 2012, 22, 24048.

9. M. Hussain, A. El-Shafei and A. Islam, Phys. Chem.Chem. Phys., 2013, 15, 8401.

10. M. He, Z. Ji and Z. Huang, J. Phys. Chem. (C),2014, 118, 16518.

11. E. Jakubikova and D. N. Bowman, Acc. Chem.Res., 2015, 48, 1441.

12. I. Liu, Y. Hou. C. Li and Y. Lee, J. Mater.Chem. (A), 2017, 5, 240.

13. B. Kilic, S. Turkdogan and A. Astam, Sci. Re-

ports, 2016, 6, 27052.

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16. M. Castillo-Valles, J. M. Castan and J. Garin,RSC Adv., 2015, 5, 90667.

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18. M. J. Frisch, G. W. Trucks, H. B. Schlegel, G.E. Scuseria, M. A. Robb, J. R. Cheeseman, G.Scalmani, V. Barone, B. Mennucci, G. A. Peterson,H. Nakatsuji, M. Caricato, X. Li, H. P. Hratchian,A. F. Izmaylov, J. Bloino, G. Zheng, J. L.Sonnenberg, M. Hada, M. Ehara, K. Toyota, R.Fukuda, J. Hasegawa, M. Ishida, T. Nakajima, Y.Honda, O. Kitao, H. Nakai, T. Vreven, J. A.Montgomery (Jr.), J. E. Peralta, F. Ogliaro, M.Bearpark, J. J. Heyd, E. Brothers, K. N. Kudin,V. N. Staroverov, R. Kobayashi, J. Normand, K.Raghavachari, A. Rendell, J. C. Burant, S. S.Iyengar, J. Tomasi, M. Cossi, N. Rega, J. M.Millam, M. Klene, J. E. Knox, J. B. Cross, V.Bakken, C. Adamo, J. Jaramillo, R. Gomperts, R.E. Stratmann, O. Yazyev, A. J. Austin, R. Cammi,C. Pomelli, J. W. Ochterski, R. L. Martin, K.Morokuma, V. G. Zakrzewski, G. A. Voth, P.Salvador, J. J. Dannenberg, S. Dapprich, A. D.Daniels, O. Farkas, J. B. Foresman, J. V. Ortiz,J. Cioslowski and D. J. Fox, Gaussian, Inc.,Wallingford CT, 2009.

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773


Peanut proteins in selective sensing of BiIII at trace concentrations

Sumanta Kumar Ghataka, Dipanwita Majumdarb, Achintya Singhab and Kamalika Sen*a

aDepartment of Chemistry, University of Calcutta, 92, Acharya Prafulla Chandra Road,Kolkata-700 009, India


bDepartment of Physics, Bose Institute, 93/1, Acharya Prafulla Chandra Road, Kolkata-700 009, India


Abstract : Bismuth is an environmentally significant element due to its usefulness in different aspects of lifeespecially in semiconductors, cosmetics and pharmaceutical industries. It is less soluble in aqueous medium so itis poorly absorbed in human cells, however it plays a crucial role in various diseases mainly gastrointestinaldisorders and neurological disorders. In this report selective recognition of trace concentration of bismuth isobserved with peanut proteins conarachin II, conarachin I and arachin, which are available from cheap sourceand their extraction is cost effective too. For avoiding bismuth precipitation, we have optimized the pH of thesolutions and they were subjected to spectrophotometric analysis. Absorption, fluorescence, circular dichroismand Raman spectra support the interaction of bismuth with peanut proteins.

Keywords : Peanut, bismuth, sensing, absorbance, fluorescence, circular dichroism, Raman spectra.

Introduction

Designing new organic molecules for the sensingof biologically important and environmentally hazard-ous metal ions at trace concentration levels is an excit-ing research in recent years1,2. Some metals are ofenvironmental concern as they cause various healthhazards including carcinogenicity while some othershave useful applications. Among transition-metal ions,mercury is the most dangerous cation for the environ-ment. Being highly reactive to biological molecules, itenters the living cells causing prenatal brain damage,serious cognitive disorders and minamata diseases3–6.Lead is known to be a larger problem for childrenthan it is for adults at higher concentrations. Braindamage and nervous disorders are the most commonoutcomes of high level lead exposure7. Studies alsoindicate that lead accumulation inhibits plant growthdue to denaturation of proteinaceous enzymes that,control cellular processes8. Antimony and its com-pounds are however milder in causing acute humanhealth effects, with the exception of antimony potas-sium tartrate, a prodrug used to treat leishmaniasis.

They rather have medical applications and are used inantiprotozoan drugs9,10. Thallium (Tl+) is known tocause isomorphous replacements with potassium inbiological systems and activate some enzymes as aconsequence. Sometimes this poses a threat as thethallium ion may bind more strongly than potassiumto a site and then fail to bind additional sites as re-quired for the biological activity. Thallium activelyconcentrates in mitochondria of the cell and therebyinterferes in oxidative phosphorylation11. 201Tl radio-isotopes are however potential candidates for heart im-aging12. Bismuth is present at trace concentration (8g/kg) in earth’s crust. It is used in our daily lives insemiconductors, cosmetic products, medicines (fortreatment of syphilis, peptic ulcers and dermatologicaldisorders), alloys, catalyst in the chemical industry,metallurgical additives, and preparation and recyclingof uranium in nuclear fuels13. 212Bi and 213Bi isotopeshave been used for cancer treatment14,15. Howeverbismuth causes several toxic effects in human bodysuch as nephropathy, osteoarthropathy, hepatitis andneuropathology when present at high concentrations16.


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Spectroscopic techniques e.g. atomic absorptionspectrometry (AAS)17, hydride generation-atomic fluo-rescence spectrometry (HG-AFS)18, inductively coupledplasma atomic emission spectrometry (ICP-AES)19,inductively coupled plasma mass spectrometry (ICP-MS)20 and electrochemical methods21 have been usedfor detecting bismuth in several samples at trace con-centrations. These methods however need costly in-strumentation and rigorous sample preparation tech-niques.

To the best of our knowledge reports on metal ionsensing using natural bioactive polymers like plantproteins are still lacking in the literature. Peanuts rep-resent cheap and widely available source of plant pro-teins growing all over the world. In this report wehave developed a spectrophotometric method of bis-muth sensing using the peanut protein fractionsconarachin II, conarachin I and arachin selectively outof other heavy metals like HgII, PbII, SbIII and TlIII ina very convenient and cost effective way.

Experimental

Materials : Sodium citrate and citric acid were ob-tained from Fischer Scientific for buffer preparation.Bi(NO3)3, Tl(NO3)3, Pb(NO3)2, HgCl2, K2Sb2-(C4H2O6)2 salts were taken from Merck. All the solu-tions were prepared in triple distilled water. Peanutswere purchased from the local market. For the deter-mination of protein concentration, the components ofBradford reagent i.e. CH3CH2OH, H3PO4, NaOH,CH3OH were obtained from Merck. Coomassie bril-liant blue G was obtained from HIMEDIA and all theother chemicals in this experiment were of analyticalgrade.

Apparatus : The pH of the solutions was main-tained using the pH/ion meter (S 220-K) of Mettler.The absorption spectra were measured using Agilent8453 diode array spectrophotometer and fluorescencespectra were obtained on a Perkin-Elmer LS-55spectrofluorimeter equipped with a quartz cell with a1.0 cm optical path length. The excitation and emis-sion slits of the monochromator were adjusted to 5nm. RAM HR, Jobin Yvon spectrometer and a Peltier-

cooled charge-coupled-device (CCD) detector was usedfor the determination of the Raman vibrational fre-quencies of the experimental samples. The far-UV CDspectra were obtained using JASCO J-815 spectropo-larimeter.

Methods

The extraction, purification and characterization ofthe peanut protein fractions conarachin II, conarachinI and arachin were done using the methods describedin the earlier report22. The obtained protein fractionswere characterized using the absorption spectra andSDS-PAGE pattern. The maximum absorption inten-sity of conarachin II at 278 nm, conarachin I at 265nm and arachin at 280 nm, agrees with the literaturedata. The SDS-PAGE pattern of conarachin II, andarachin show molecular weight bands at 39800, 33100,26900, 24000, 21900, 18600, 15800 Da and 72400,60300, 39800, 33100, 26900, 21900 Da and conarachinI shows a single band at 18600 Da. The concentrationof the proteins was measured using Bradford reagents23.The concentration of conarachin II, conarachin I andarachin were 0.9, 0.8 and 0.76 mg/mL respectively.

Absorption and fluorescence spectra :

Absorption and fluorescence study were used todetermine the selectivity and sensitivity of peanut pro-teins in the presence of various heavy metal ions, suchas BiIII, TlIII, PbII, HgII and SbIII. The salt solutions(1 mM each) of Bi(NO3)3, Tl(NO3)3, Pb(NO3)2, HgCl2,K2Sb2(C4H2O6)2 were prepared with triple distilledwater. For preparation of Bi(NO3)3 solution a weighedamount of the salt was first dissolved in a drop ofHNO3 and then diluted with water to required vol-ume. This was done to avoid precipitation of Bi as itsoxynitrate directly in water medium. To identify thepossible complexation, 0.3 mL each of these metal ionsolutions was mixed with 0.2 mL of protein solutionsand volume was made up to 1.5 mL with 0.1 M so-dium citrate-citric acid buffer of pH 3. To optimizethe Bi-protein complexation, 1 mM bismuth(III) solu-tion was added to the protein solution in different stoi-chiometric ratios. The solutions were subjected to ab-sorption and fluorescence spectral studies. The experi-

Ghatak et al. : Peanut proteins in selective sensing of BiIII at trace concentrations

775

ment was also studied at the pHs 5.6, 6.7 and 7.9 of0.1 mM phosphate buffer medium. But due to the metalhydroxide precipitation, the experiment was done atpH 3 of 0.1 mM citrate buffer medium.

Circular dichroism (CD) spectra :

To obtain CD spectra, 1 mM BiIII solution wasadded to the 50 L of protein solution in a quartzcylindrical cuvette of 0.1 cm path-length at 25 ºC indifferent stoichiometric ratios. The final spectra havebeen taken after three consecutive scans and base linecorrection.

Raman spectra :

The Raman spectra of the different peanut proteinfractions were compared with the protein-BiIII com-plexes. 20 L of different peanut proteins and the so-lutions of protein-BiIII complexes were taken on glassplate coated with Al foil. The solutions were dried invacuum desiccator over night. Then the samples werethen taken for Raman spectroscopy.


Absorption spectra : Conarachin II, conarachin Iand arachin, show the absorption maxima at 278, 265and 280 nm respectively. The absorption data (Fig. 1)suggest the selective interaction of BiIII ions with allthe three proteins. With increasing Bi concentrationsin the protein solutions a gradual increase in the absor-bance of the three peanut proteins were observed. Fromthe absorbance data we have calculated the bindingconstant and free energy changes upon complexationusing Benesi-Hildebrand equation22.

1 1 1——— = ———— + ——————— (1)A – A0 A1 – A0 (A1 – A0)K[M]

Y = A + BX (2)

where A is the absorbance of the experimental solutioncontaining metal and protein. A0 is the absorbance ofthe protein only. A1 is the absorbance when protein iscompletely bound with the metal. [M] is the metal ionconcentration. K is the binding/association constant.

Eq. (1) can be represented as eq. (2) which is a

Fig. 1. Plot of absorbance of conarachin II, conarachin I, arachinwith different metal ions at pH 3 in citrate buffer (0.1M) medium.

Fig. 2. Benesi-Hildebrand plot of conarachin II, conarachin Iand arachin-BiIII complexes at pH 3.

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straight line having intercept 1/(A1 – A0) and slope 1/(A1 – A0)K. From the Benesi-Hildebrand (BH) plot(Fig. 2), we have calculated the binding constant val-ues. Corresponding free energy change (G) valueswere calculated at a temperature (T) of 298 K usingthe equation

G = –RT ln K (3)

Binding constant, free energy change and stoichiomet-ric ratio (obtained from the BH plot) of the three pea-nut protein-bismuth complexes are tabulated in Table 1.

The binding constants of the conarachin II,conarachin I and arachin-Bi complexes are, 2.4737×103

M–1, 3.7614×103 M–1 and 0.92623×103 M–1 respec-tively. The G values upon complexation of conarachinII, conarachin I and arachin with Bi are –19.488 kJ/mol, –20.533 kJ/mol and –17.038 kJ/mol respectively.The greater value of binding constant and more nega-tive value of free energy change indicate the highestinteraction of conarachin I with BiIII amongst the threeproteins.

Fluorescence spectra : The emission maxima forconarachin II, conarachin I and arachin, were observedat 365 nm, 425 nm and 340 nm respectively. Fluores-cence spectra also support the selective interaction ofBiIII with the three peanut proteins as compared toother heavy metals like TlIII, PbII, HgII and SbIII. Figs.3, 4 and 5 reflect this selective behavior of conarachinII, conarachin I and arachin respectively. With increas-ing bismuth concentration emission intensity decreaseswhich, indicates the interaction of BiIII with three pro-teins. The plot (Fig. 6) F0/F versus [BiIII] shows acurved nature which suggests that the fluorescencequenching is both static and dynamic type indicatingboth ground state and excited state complexations ofthe proteins with BiIII.

CD spectra : The far UV CD spectra (Figs. 7, 8and 9) of the three peanut proteins showed two minimaat 208 and 222 nm which indicates helical structuresof conarachin II, conarachin I and arachin. With in-

Table 1. Some physical parameters of BiIII complexes of the protein fractions

Complex Binding Free energy change Stoichiometric

constant (M–1) (kJ/mol) ratios

Conarachin II-BiIII 2.4737×103 –19.488 1 : 1

Conarachin I-BiIII 3.7614×103 –20.533 1 : 1

Arachin-BiIII 0.92623×103 –17.038 1 : 1

Fig. 3. Fluorescence spectra of conarachin II in presence ofdifferent heavy metals at pH 3 (ex = 278 nm).

Fig. 4. Fluorescence spectra of conarachin I in presence ofdifferent heavy metals at pH 3 (ex = 265 nm).

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creasing bismuth concentration, the gradual decreaseof ellipticity indicates the lower value of helicalcontent in the protein molecules which leads to theformation of sheet structures in all the three pro-teins. The change in the circular dichroism spectra ofthe three proteins upon interaction with bismuth clearlyindicates the modification of the secondary structureof the proteins.

Raman study : Upon interaction with Bi, a largenumber of vibrational frequencies were observed (Fig.10 and Table 2) to appear or disappear for the threeproteins conarachin II, conarachin I and arachin. Thevibrational frequency near 1000 cm–1 indicates the pres-

Fig. 5. Fluorescence spectra of arachin in presence of differentheavy metals at pH 3 (ex = 280 nm).

Fig. 6. Stern-Volmer plot of conarachin II, conarachin I andarachin-BiIII complexes at pH 3.

Fig. 7. CD spectra of conarachin II with varying concentra-tions of BiIII.

Fig. 8. CD spectra of conarachin I with varying concentrationsof BiIII.

Fig. 9. CD spectra of arachin with varying concentrations ofBiIII.

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Table 2. Details of the Raman spectral data

Raman modes (cm–1)

Conarachin II Conarachin II- Conarachin I Conarachin I- Arachin Arachin-

BiIII BiIII BiIII

C-C-C out of plane bending 323

C-C-C out of plane skeletal deformation – 380 400 390 – 371

C-C-C out of plane bending 457 455 455 – 475 –

C-C-C out of plane bending 466 – – – – –

C-C-C inplane skeletal deformation – 520 538 530 557 570

N-H2 wagging 615 620 625 620 619 –

Tyr 636 630 – – – –

Tyr – 830 – 822 829 811

Trp – 900 884 897 – –

C-H out of plane bending – 945 – 939 920 930

C-N-H2 stretching 976 989 975 – 990 –

Phe 995 1000 1000 – 1000 –

C-C, C-N and C-O stretching mode 1075 1075 1091 1050 – –

C-C stretching mode 1130 – – – – –

Tyr, Phe 1197 – – 1190 1171 –

Amide III – 1260 1264 1260 1260 –

CH2 deformation – 1427 – 1425 1451 –

Phe – – – 1617 1607 –

Trp – – – – 1630 –

Amide 1, -sheet – – – – 1666 –

Fig. 10. Raman spectra of peanut proteins with BiIII.

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ence of phenylalanine in the three proteins. C-C-C outof plane bending and C-N-H2 stretching were shiftedfor conarachin II, but were disappeared for conarachinI and arachin. Shifting of the peak positions near 630cm–1 and 830 cm–1 for tyrosine and 900 cm–1 fortryptophan24–27 in the three proteins indicates the in-teraction of bismuth with peanut proteins. Raman spec-tra also suggest that the interaction of the three peanutproteins with bismuth is due to the presence of ty-rosine, tryptophan and phenylalanine in the side chainof the protein molecules. This observation is also inagreement with individual interactions of BiIII with thethree aromatic amino acids, tyrosine, tryptophan andphenylalanine28.

Conclusion

Peanut proteins were subjected to interaction withdifferent heavy metal ions and the solutions were ana-lyzed using different spectral techniques. Absorption,fluorescence, circular dichroism and Raman spectraindicate the preferential interaction of peanut proteinswith bismuth amongst the heavy metals mercury, thal-lium, lead, bismuth and antimony. The results revealthe stoichiometric ratio of peanut protein-BiIII com-plex is 1 : 1 for all the three cases. The greater valueof binding constant and free energy change supportsthe highest interaction of conarachin I with bismuth.The Raman spectra also indicate the presence of threeamino acids tyrosine, tryptophan and phenylalanine inthe side chain of the three peanut proteins. The modi-fication in the secondary structure of conarachin II,conarachin I and arachin obtained from the circulardichroism spectra are due to the interaction of bismuthwith three amino acids tyrosine, tryptophan and phe-nylalanine within the protein molecules. The resultsstrongly support a selective sensing of BiIII amongst aseries of heavy metals at their trace concentrations.

Acknowledgement

One of the authors, Sumanta Kumar Ghatak ex-presses sincere thanks to Council of Scientific & In-dustrial Research, India [Sanc. No. 09/028(0929)/2014-EMR-I dated 11/08/2014] for providing fellowship.

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An efficient pyrimidine-based colorimetric chemosensor for naked-eyerecognition of copper in aqueous medium

Anamika Dharaa, Nikhil Guchhait*b and Subash Chandra Bhattacharya*a

aDepartment of Chemistry, Jadavpur University, Kolkata-700 032, India

bDepartment of Chemistry, University of Calcutta, 92, Acharya Prafulla Chandra Road,Kolkata-700 009, India



Abstract : A pyrimidine-based receptor 1-(9H-fluoren-9-ylidene)-2-(4,6-dimethylpyrimidin-2-yl)hydrazine (HPF)was designed for naked-eye detection of Cu2+ with high selectivity in the presence of other competitive metalions. In aqueous solution, upon addition of Cu2+ to HPF a color change from yellow to wine-red due to com-plex formation. The detection limit (5.03×10–8 M) of HPF for Cu2+ ions is much lower than that recommendedby WHO in drinking water (31.5 M). The sensor shows consistent performance in a pH value range of 4 to11. Moreover, test strips based on receptor HPF were fabricated, which could act as a handy and efficient Cu2+

test for “in-the-field” measurement of Cu2+.

Keywords : Colorimetric chemosensor, hydrazino pyrimidine, Cu2+ recognition, DFT study, test kits.

1. Introduction

The metal copper plays a significant role in theareas of biological, environmental, and chemical sys-tems1. It is the third most abundant metal after ironand zinc in the human body and it plays an importantrole in a number of biological processes, includingiron absorption, haemopoiesis, various enzyme-cata-lyzed and redox reactions2–5. Thus, daily ingestion ofcopper is indispensable for our good health6. How-ever, copper is extremely toxic to some organismssuch as many bacteria and viruses. Owing to its toxi-city to bacteria, elevated concentrations of copper im-peded self-purification capability of the sea or riversand destroy the biological reprocessing systems inwater. On the other hand, unregulated overloading ofcopper can induce severe neurological diseases, in-cluding Alzheimer’s, Parkinson’s, Menkes’, andWilson’s diseases7. The World Health Organization(WHO) has set the safe limit of copper in drinkingwater at 2 ppm (31.5 M)8. Accordingly, facile tech-

niques, enabling professionals to monitor the concen-tration of copper ions in environmental water samplesand visualize its subcellular distribution in physiologi-cal processes, are of considerable significance for en-vironment protection and human health.

For effortlessness, convenience and low cost, thefabrication of small molecular chemosensors into colo-rimetric test kits is highly challenging9. Even thoughsome colorimetric/fluorescent chemosensors have beendeveloped for the detection of Cu2+ so far, the sens-ing methods for fast detection of Cu2+ in aqueoussolution, especially using colorimetric sensors withoutresorting to instruments, are relatively rare. Currentapproaches for environmental and clinical samples relyon costly, time-consuming methods like atomic ab-sorption/emission spectroscopy10 or inductively coupledplasma mass spectrometry11 which are not very con-venient and handy for “in-field” detection. Moreover,in most cases the sensing experiments are carried outusing metal solutions in water and organic solvent


782

mixture12 and hence are of greater academic interest

than practical. Therefore, to develop simple-to-use and

naked-eye diagnostic tool for selective detection of

copper in aqueous medium is a demanding and attrac-

tive topic. Keeping in mind, we have chosen to design

a fluorenone-appended pyrimidine based scaffold HPFas a receptor. Because of chemosensors based on pyri-

midine are being widely studied in recent years owing

to their great variety of biological activity ranging from

antimalarial, antibacterial, antitumoral, antiviral activi-

ties, etc.13,14 which have often been associated to their

chelating capability with trace metal ions. The higher

() acidity of pyrimidine and presence of more than

one hetero atom in the same ring take part in an im-

portant role in its coordination chemistry15–17. On the

other hand, fluorenone family compounds are base

materials for the production of dyes and optical bright-

ening agents. Fluorenones have been used extensively

as catalyst precursors for electro-catalytic oxidation18,

inhibition of DNA tumor viruses19, light-emitting

materials20, etc., but have rarely been used as chemi-

cal sensors. Fluorenone has been investigated as an

attractive element in organic solar cells and display

devices. This has led us to design and synthesize a

colorimetric sensor containing a fluorenone framework

that is particularly suitable for environmental and bio-

logical applications.

In the current paper, we report the synthesis, char-

acterization, and sensing properties of HPF as a selec-

tive colorimetric receptor for detection of Cu2+. The

receptor HPF displayed a highly selective and sensi-

tive (detection limit 5.03×10–8 M) colorimetric rec-

ognition toward Cu2+ by a change in the color from

yellow to wine red in an aqueous solution with the

formation ligand metal complex. Moreover, HPF can

also be used as a practical, visible colorimetric detec-

tion kit for Cu2+. So far our knowledge is concerned,

this is one of the rare examples of the pyrimidine de-

rivative employed for the detection of Cu2+.

2. Experimental

2.1. Materials

9H-Fluoren-9-one was purchased from Aldrich. 2-Hydrazinyl-4,6-dimethyl pyrimidine was previouslyprepared in the laboratory21. Other commercially avail-able chemicals and solvents were used and purified bystandard procedure. Perchlorate salts of some cationssuch as Na+, K+, Mg2+, Li+, Ag+, Al3+, Pb2+,Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+,Cd2+and Hg2+ were purchased from Merck.

2.2. Apparatus

Elemental analyses (carbon, hydrogen and nitro-gen) were performed with a Perkin-Elmer CHN ana-lyzer 2400. Melting points were determined using aBuchi 530 melting apparatus. NMR spectra were re-corded on a Bruker spectrometer at 500 (1H NMR)MHz and 300 (13C NMR) in DMSO-d6. Chemicalshifts ( values) were reported in ppm down field frominternal Me4Si. The electronic spectra were recordedin MeOH-water solution on a Hitachi model U-3501spectrophotometer. IR spectra (KBr pellet, 400–4000cm–1) were recorded on a Perkin-Elmer model 883infrared spectrophotometer.

2.3. Synthesis

2.3.1. Synthesis of reference compound REF

9H-Fluoren-9-one (200 mg, 1.109 mmol) was addedto a methanol solution (5 mL) of 1-phenylhydrazine(0.109 mL, 1.109 mmol). The above mixture was re-fluxed at 80 ºC for 2 h, then the resulting mixture wascooled to ambient temperature. Crude product wasobtained and filtered off, then washed with methanolseveral times and dried under vacuum. The final com-pound REF was obtained as yellow crystalline solid(Scheme 1). Yield : 0.147 g, 48.5%; 1H NMR (300MHz, DMSO-d6) : 10.77 (1H, s), 8.24 (1H, d, J=7.65Hz), 7.94 (1H, d, J=7.45 Hz), 7.87 (1H, d, J=7.40Hz), 7.81 (1H, d, J=7.45 Hz), 7.45 (1H, t, J=0.75Hz, 6.8 Hz), 7.44–7.39 (2H, m), 7.37 (1H, d, J=0.80Hz), 6.82 (1H, s), 2.40 (6H, s); 13C NMR (300 MHz,

Dhara et al. : An efficient pyrimidine-based colorimetric chemosensor for naked-eye recognition etc.

783

DMSO-d6) (ppm) : 31.14, 114.58, 120.91, 121.58,126.26, 128.47, 129.79, 138.23, 140.42,145.75; Anal.Calcd. for C19H14N2 : C, 84.43; H, 5.23; N, 10.48%.Found : C, 84.42; H, 5.22; N, 10.36%.

2.3.2. Synthesis of receptor HPF

The compound 3 was synthesized following a lit-erature method21. To a solution of 9H-fluoren-9-one(1 g, 5.55 mmol) in 10 mL of methanol, then previ-ously prepared 1-(4,6-dimethylpyrimidin-2-yl)hydrazine(0.77 g, 5.55 mmol) was added to the mixture andcooled in ice water. Then glacial acetic acid (5 drops)was added to the mixture drop by drop with continu-ous stirring. Finally the total reaction mixture was re-fluxed at 90 ºC for 12 h. The reaction mixture wascooled to room temperature. The yellow colored crys-talline compound was separated out (Scheme 1). Thesolution was filtered and the compound was collectedand dried in vacuum desiccators. Bright yellow solidof HPF was obtained. Yield : 0.72 g, 43.37%; 1HNMR (500 MHz, DMSO-d6) : 10.77 (1H, s), 8.24(1H, d, J=7.65 Hz), 7.94 (1H, d, J=7.45 Hz), 7.87(1H, d, J=7.40 Hz), 7.81 (1H, d, J=7.45 Hz), 7.45(1H, t, J=0.75 Hz, 6.8 Hz), 7.44–7.39 (2H, m), 7.37(1H, d, J=0.80 Hz), 6.82 (1H, s), 2.40 (6H, s); 13C

NMR (300 MHz, DMSO-d6) (ppm) : 23.87, 31.06,40.75, 113.75, 120.47, 120.84, 121.73, 127.17,128.38, 129.62, 129.89, 130.69, 137.59, 139.09,141.05, 146.03, 160.69, 168.10; IR (KBr) : 1527,1437, 1336, 1183, 1122, 1085, 1029, 953 cm–1. Anal.Calcd. for C19H16N4 : C, 76.10; H, 5.42; N, 18.66%.Found : C, 75.98; H, 5.37; N, 18.65%.

2.4. General procedure for UV-Vis titration

Stock solution of the receptor, HPF was preparedin 10 mM HEPES buffer-CH3OH (99 : 1, v/v) atroom temperature in the concentration range of 10–5

mol/L. 2 ml of the receptor (HPF) solution was takenin the cuvette. Stock solutions of guests in the concen-tration range of 10–5 M, were prepared in the samesolvents and were individually added in differentamounts to the receptor solution. The same stock solu-tion of receptor and guests were used to perform theUV-Vis at 25 ºC.

2.5. Job plot measurements

Copper perchlorate and HPF were dissolved in 10mM HEPES buffer-CH3OH (99 : 1, v/v) to make theconcentrations 20 M. Volumes of 5.0, 4.5, 4.0, 3.5,3.0, 2.5, 2.0, 1.5, 1.0, 0.5 and 0 mL of the solution

Scheme 1. Synthesis of receptor HPF and reference compound REF.

JICS-16


784

of HPF were taken and transferred to vials. Volumesof 0, 0.5, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5 and5.0 mL of the copper solutions were added to separatesolutions of HPF. Each vial had a total volume of 5mL. After shaking the vials for a few seconds, UV-Vis spectra were taken at room temperature.

2.6. Association/binding constant

The binding constant22 of the [Cu2+-HPF] com-plex was determined using the Benesi-Hildebrand (B-H) plot.

1/(A – A0) = 1/{K (Amax – A0) C}

+ 1/(Amax – A0) (1)

A0 is the absorbance of HPF at absorbance maxima (= 428 nm), A is the observed absorbance at that par-ticular wavelength in the presence of a certain concen-tration of the metal ion (C), Amax is the maximumabsorbance value that was obtained at = 428 nmduring titration with varying [C], K is the associationconstant (M–1) and was determined from the slope ofthe linear plot, and [C] is the concentration of theCu2+ ion added during titration studies. The goodnessof the linear fit of the B-H plot of 1/(A – A0) vs 1/[Cu2+] for 1 : 1 complex formation confirms the bind-ing stoichiometry between HPF and Cu2+.

2.7. Detection limit

The detection limit23 (DL) of HPF for Cu2+ wasdetermined using the following equation :

DL = K* Sb/S (2)

where K* = 2 or 3 (we take 3 in this case), Sb is thestandard deviation of the blank solution and S is theslope of the calibration curve.

2.8. Crystallographic measurements

Measurements were done on a Bruker SMARTAPEX II CCD area detector equipped with graphitemonochromated Mo K radiation (k = 0.71073 Å)source in scan mode at 296 K. The structure of thereceptor HPF were solved using the SHELXS-97 pack-

age of programs and refined by the full-matrix leastsquare technique based on F2 in SHELXL-9724. Allnon-hydrogen atoms were refined anisotropically. Po-sitions of hydrogen atoms attached to carbon atomswere fixed at their ideal position.


The compound REF (reference compound) wassynthesized by refluxing 1-phenylhydrazine and 9H-fluoren-9-one in methanol and characterized by 1HNMR, 13C NMR and CHN elemental analysis (Figs.S1-S2, in Supplementary material). The compound 3was synthesized following a literature method and char-acterized by 1H NMR spectra, Mass data, and FT-IRspectra21. We have synthesized pyrimidine-based re-ceptor HPF (Scheme 1) by condensing hydrazino py-rimidine (3) and 9-fluorenone in methanol. The struc-ture HPF was established by 1H NMR, 13C NMR,FT-IR, CHN elemental analysis and Mass spectrom-etry techniques (Figs. S3-S6 in Supplementary mate-rial) and also X-ray crystal structure analysis (Fig. 1).We obtained single crystal for receptor HPF by slowevaporation of DMF/ether (1 : 1 v/v) over a period ofone week and crystallized in a monoclinic P21/c spacegroup (Tables S1 and S2 in Supplementary material).The crystal structure of HPF (CCDC 1050465) is shownin Fig. 1. The C-N and N-N distances for HPF are inthe range of 1.30–1.38 Å. Two adjacent HPF recep-

Fig. 1. Molecular structure of the receptor HPF.

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785

3.1. Visual sensing of Cu2+

Visual color change of receptor HPF and referencecompound REF (20 M) were investigated in an aque-ous medium [10 mM HEPES buffer-CH3OH (99 : 1,v/v) at room temperature]. Upon addition of Cu2+ ionto HPF, a detectable naked-eye color change was ob-served from yellow to wine red (Fig. 3). Simulta-neously, no naked-eye color change was observed uponaddition of Cu2+ to the REF (inset of Fig. 7). Com-plexation does not occur between REF and Cu2+ dueto the absence of pyrimidine ‘N’ in REF. Hence it canbe said that pyrimidine ‘N’ is responsible for com-plexation. Other ions did not exhibit any detectablecolor change.

The sensing abilities of HPF (20 M) toward vari-ous cations (Na+, K+, Li+, Mg2+, Ag+, Al3+, Pb2+,Cr3+, Mn2+, Fe2+, Fe3+, Co2+, Ni2+, Cu2+, Zn2+,Cd2+ and Hg2+) were investigated by UV-Vis spec-troscopy in an aqueous medium [10 mM HEPESbuffer-CH3OH (99 : 1, v/v) at room temperature].Upon the addition of 1.2 equiv. of each cation, onlyCu2+ induced distinct spectral change is observed,while the aforementioned other metal ions did not showcolour change or slight change in the absorption spec-tra relative to the free receptor HPF (Fig. 4). Forcomparison, their optical densities at 428 nm uponaddition of the above mentioned cations to HPF solu-tion are charted in Fig. 5.

The absorption spectrum of receptor HPF (20 M)exhibits strong absorption bands at 360 nm, which isattributed * transition of fluorenone unit of HPF.Upon concomitant addition of Cu2+ to receptor HPF,a new red shifted absorption band centered at 428 nmappears with increasing intensity which is due to com-

Fig. 2(a). H-bonding network within the dimeric unit of re-ceptor HPF.

Fig. 2(b). 1D polymer through intermolecular H-bonding.

Fig. 3. Color changes of HPF (20 M) after addition of 1.2 equiv. of various cations in 10 mM HEPES buffer-CH3OH(99 : 1, v/v) at room temperature.

tors were connected via two types intermolecular hy-drogen bonding interactions with N1-H---O1 and O1-H---O2 having distances of 2.991 Å and 2.839 Å,forming a 1D polymeric structure (Fig. 2a and 2b).

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786

plexation of pyrimidine of HPF with CuII (Fig. 6).Meanwhile, the original absorption band centered at360 nm decreases gradually, generating an isosbesticpoints at 389 nm, which indicates the formation of anew complex between HPF and Cu2+. As shown inthe inset of Fig. 6, a nearly linear dependence of theabsorbance at 428 nm band as a function of Cu2+

concentration from 0 to 1.2 equiv. was observed.

Again, the absorption behaviour of REF (20 M)exhibits initially two absorption bands at 388 and 244nm. The compound REF neither observed any UV-Vis absorption spectral change nor naked-eye detec-tion of color upon addition of Cu2+ (Fig. 7). Fromabove two experiments we have concluded the indis-

Fig. 4. Changes in the UV-Vis spectra of HPF (20 M) afteraddition of 1.2 equiv. of various cations in 10 mMHEPES buffer-CH3OH (99 : 1, v/v) at room tempera-ture.

Fig. 5. Optical density of HPF (20 M) at 428 nm upon theaddition of different cations in 10 mM HEPES buffer-CH3OH (99 : 1, v/v) at room temperature.

Fig. 6. Absorption spectral change of HPF (20 M) after addi-tion of increasing amounts of Cu2+ ions (0.1, 0.2, 0.3,0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1 and 1.2 equiv.) in10 mM HEPES buffer-CH3OH (99 : 1, v/v) at roomtemperature. Inset : Absorption at 428 nm versus thenumber of equivalents of Cu2+ added.

Fig. 7. Changes in the UV-Vis spectra of REF (20 M) afteraddition of increasing amounts of Cu2+ ions in 10 mMHEPES buffer-CH3OH (99 : 1, v/v) at room tempera-ture. Inset : Color change of REF (20 M) after addi-tion Cu2+.

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787

pensable roles of the pyrimidine ‘N’ within receptorHPF and Cu2+ for complexation and naked-eye colourchange.

We have studied the better selectivity of HPF as acolorimetric receptor for the recognition of Cu2+ inthe presence of various competing metal ions. Forcompetition studies, receptor HPF (20 M) was treatedwith 1.2 equiv. of Cu2+ in the presence of 1.2 equiv.of other metal ions, as indicated in Fig. 8. There wasno interference in the detection of Cu2+ from variouscompetitive cations. Thus, HPF could be used as aselective colorimetric sensor for Cu2+ in the presenceof most of the competing metal ions.

Fig. 8. The absorbance of HPF (20 M) at 428 nm in thepresence of different metal ions (1.2 equiv., dark yel-low bars), and in the above solution upon addition ofCu2+ (1.2 equiv., navy bars) in 10 mM HEPES buffer-CH3OH (99 : 1, v/v) at room temperature.

For further determination the stoichiometry betweenHPF and Cu2+, Job’s plot25 analyses were also used.When molar fraction of Cu2+ was 0.5, the absorbanceat 428 nm reaches to maximum (Fig. 9), demonstrat-ing the formation of 1 : 1 complex between HPF andCu2+.For further information on the coordination modeof the HPF-Cu2+ complex, we performed 1H NMRtitration of HPF with Cu2+, but it was not doing wellowing to the paramagnetic nature of Cu2+ ions. Based

on the Job’s plot study, we propose the structure ofthe HPF-Cu2+ complex as shown in Scheme 2.

Based on UV-Vis titration, the association constant22

(K) of HPF with Cu2+ ion was calculated using theBenesi-Hildebrand equation (Fig. S7 in Supplemen-tary material). The K value turned out to be 4.69×103

M–1. The detection limit23 of receptor HPF for theanalysis of Cu2+ ions was calculated to be 5.03×10–8 M (Fig. S8 in Supplementary material). The de-

Fig. 9. Job’s plot for the binding of receptor HPF and Cu2+,where the intensity at 428 nm was plotted against themole fraction of Cu2+.

Scheme 2.A possible sensing mechanism of the receptor HPFto Cu2+.

tection limit of HPF for Cu2+ was much lower thanthat recommended by the US EPA in drinking water(1–4 ppm). Therefore, HPF could be a good indicatorfor the detection of copper ions in the drinking water.

We investigated the effect of pH on the absorptionresponse of receptor HPF to the Cu2+ ion in samemedia with pH values ranging from 2 to 12 (Fig. 10).The color of the Cu2+-HPF complex remained in the

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788

wine red region between pH 4 and 11, while its colorchanged to the original yellow at pH 2, 3 and 12.These results indicate that Cu2+ could be clearly de-tected by the naked-eye or UV-Vis absorption mea-surements using HPF over the wide pH range of 4.0–11.0. The color change of the Cu2+-HPF complexfrom wine red to yellow at very low pH (2 and 3) andhigh pH (>11) might be due to demetallation of thecomplex, thus regenerating the receptor HPF with itsyellow color.

To ensure the practical application of receptor HPF,test strips were ready by immersing filter papers into amethanol solution of HPF (20 M) and then drying inair. These test strips were applied for sensing differentCu2+ concentrations (0, 10 M, 30 M), exhibiting

distributions and orbital energies of the highest occu-pied molecular orbital (HOMO) and the lowest unoc-cupied molecular orbital (LUMO) of HPF and thecorresponding Cu2+ complex were also generated us-ing this calculations (Fig. 12b). The HOMO is spreadover the whole molecule, whereas LUMO is distrib-uted on the -C=N bond and pyrimidine N in HPF,and the imine N and pyrimidine N are favorably coor-dinated with Cu2+. In HPF-Cu2+ complex, the elec-trons of HOMO are mainly located on the C=N andthe LUMO is mostly spread over the pyrimidine ring.The energy gaps between the HOMO and LUMO ofthe probe and corresponding Cu2+ complex were foundto be 2.094 eV and 0.184 eV, respectively (Table S3in Supplementary material). This calculated resultsexhibit the red shifting absorption band compared tothe free ligand which corroborates with the observedabsorption spectral band position. Calculation also pre-dicts better stabilized of HPF-Cu2+ complex than thebare HPF ligand.

4. Conclusions

In summary, we have developed a pyrimidine basedsensor HPF, which could detect Cu2+ in aqueous so-lution with specific selectivity and high sensitivity in avery short time. Our method is simple, rapid, cost-effective and could find potential application as a ‘na-

Fig. 10. Variation of absorbance intensity (428 nm) of free HPF(20 M) and in the presence of Cu2+ ion in 10 mMHEPES buffer-CH3OH (99 : 1, v/v) at room tempera-ture, solution with different pH conditions.

Fig. 11. Photographs of the test kits with HPF for detectingCu2+ ion in methanol solution with different concen-trations. Left to right : 0, 10 M, 30 M.

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colorimetric changes differentiable by bare eyes (Fig.11). Development of such test strips is helpful as in-stantaneous qualitative information without resortingto the instrumental analysis.

To better understand the nature of the coordinationof Cu2+ with HPF, energy-optimized structures ofHPF and HPF-Cu2+ (Fig. 12a) were obtained onDensity Functional Theory (DFT) calculations at theB3LYP level using 6-311G** basis set for simple re-ceptor (HPF) and LANL2DZ basis set for metal com-plex using the Gaussian 09 program26. The spatial


789

ked-eye’ indicator for Cu2+ ions in aqueous solution.In particular, there was no interference response fordetection of Cu2+ in the presence of competing metalions. The detection limit of Cu2+ was found to be5.03×10–8 M. On the other hand, the decrease in cal-culated HOMO-LUMO band gap energy evidences redshifted absorption band of the complex than the baremolecule which corroborates well with experimentalfindings and calculation also predicts stabilization ofcomplex of receptor HPF with Cu2+. Moreover, teststrips based on sensor HPF were fabricated, whichcould serve as a practical colorimetric sensor for “in-field” measurement of Cu2+ and does not require anylabor-intensive synthetic protocols. We anticipate that

this report of a colorimetric pyrimidine-based sensorfor Cu2+ ion could prompt the development of moresensitive and cost-effective methods for Cu2+ ion de-tection from the scientific community.

Supporting Information

Supporting Information is available in the Website :indianchemicalsociety.com.

Acknowledgement

AD acknowledges the financial support providedby University Grants Commission, India (Award Let-ter No. F.4-2/2006(BSR)/CH/14-15/0163, dated May,2015) through the Dr. D. S. Kothari Post DoctoralFellowship (DSKPDF).

Fig. 12(b). HOMO-LUMO energy gaps for respective compounds and interfacial plots of the orbitals of HPF (left) and HPF-Cu2+

(right).

Fig. 12(a). Stereoscopic view of the energy optimized structures of HPF (left) and HPF-Cu2+ (right).

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790

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A thienyl-pyridine-based Hantzsch ester fluorescent probe for the selectivedetection of nitric oxide and its bio-imaging applications

Syed Samim Ali, Ajoy Kumar Pramanik, Sandip Kumar Samanta, Uday Narayan Guria andAjit Kumar Mahapatra*

Department of Chemistry, Indian Institute of Engineering Science and Technology, Shibpur,Howrah-711 103, West Bengal, India



Abstract : A new Hantzsch ester fluorescent probe (TPC) containing thienyl-pyridine group appended to thedihydropyridine ring was synthesized and characterized and successfully applied in the fluorescent sensing ni-tric oxide (NO) in aqueous solution. The fluorescence of probe TPC shows extremely strong blue fluorescent,while its fluorescence was greatly switched off upon the addition of NO solution and showed high selectivityand sensitivity to NO. The limit of the detection was calculated to be ~0.4 M. A reaction mechanism fordihydropyridine with NO is proposed in this study. The probe shows good stability over a broad pH range (pH> 4). The structure of the TPC probe has been established by single-crystal XRD. DFT and TDDFT calcula-tions were done to demonstrate the electronic properties of the probe and the corresponding aromatic product.Moreover, the utility of the TPC probe in detecting NO in live cells has also been demonstrated using Raw 264.7cells as monitored by fluorescence imaging.

Keywords : Nitric oxide, single crystal X-ray, DFT, live cell imaging.

Introduction

Nitric oxide (NO) is one of the smallest gaseousmolecules which exists in the atmosphere. It is a highlyreactive and multifunctional free radical, and was pri-marily accepted as an air pollutants known as nitrogenoxides (NOx)

1. NO is produced from the reaction ofN2 and O2 during high-temperature combustion pro-cesses in car engines, power plants or industrial pro-cesses and naturally by lightning during a thunder-storm. High NOx levels in the atmosphere can causeserious diseases such as carcinogenesis and asthma2.NO is also a ubiquitous bioactive signaling molecule3.Recently, researchers have discovered that NO alsoserves as an indicator for many biological processes inthe human immune system, nervous system, epithe-lium system and its pivotal role in cardiovascular sys-tem4. Several analytical methods for NO detection havebeen developed such as electrophoresis, electron para-magnetic resonance (EPR) or GC-mass spectroscopies,chemiluminescence and electrochemical methods5. In

comparison to these protocols, colorimetric or fluo-rescence-based techniques present a large number ofadvantages, such as simple detection in situ or at sitewithout any sample pre-treatment or use of low-cost,widely available equipment. Thus, the construction ofsmall fluorescent sensors suitable for specific NO de-tection in living systems has received great attention.A number of fluorescent probes have been reported todate6. The most common approach for NO detectioninvolves the use of o-diamino aromatics under aerobicconditions. These molecules react with N2O3 (formedfrom NO and O2), to yield fluorescent triazole deriva-tives7. Another family of probes, in this case reactingdirectly with NO, is based on transition-metal com-plexes8. An increasing number of other strategies havebeen described in the literature9.

A widely adopted approach involves a two-compo-nent “fluorophore-modulator” strategy for designingreaction-based fluorogenic NO probes. The reactionbetween o-phenylenediaminophenyl and NO is the most

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well known and widely used modulating mechanismof fluorogenic NO probes. It is believed that an aro-matic vicinal diamine can efficiently quench afluorophore subunit through the photoinduced electrontransfer (PET) mechanism. The reaction of an aro-matic vicinal diamine with NO in the presence of oxy-gen leads to the formation of triazole that blocks thenonradiative PET relaxation pathway and restores fluo-rescence emission of the fluorophore10. However, thisdeveloped strategy still has its limitations as follows :(i) Self-oxidation-sensitive electron-abundant o-phe-nylenediamine groups makes it easily react with otherreactive oxygen/nitrogen species, lighting up its fluo-rescence and interfering with the sensing process ofNO; (ii) oxidized species rather than NO itself aredemanded to react with the vicinal aromatic diamines.To solve those problems, our group recently reporteda highly selective fluorogenic probe for NO sensingbased on the 4-substituted Hantzsch 1,4-dihydropyridines (DHP)11. According to the formerwork, DHP is not only able to quantitatively reactwith NO to give the corresponding pyridine, but alsoexhibits a good selectivity for NO against other inter-fering reactive oxidative species (ROS), a reaction thatis independent of oxygen12.

Taking the above information into consideration,we report herein the rational design and experimentalvalidation of a highly selective fluorogenic probe forNO sensing based on a fluorescence switching-on-offmechanism. It is known that 1,4-dihydropyridine(Hantzsch ester) is the key switching unit, its oxida-tion to pyridine by NO would result in the formationof corresponding aromatic pyridine ring. A dramaticelectron structure change (from nonaromatic to aro-matic) accompanies this chemical transformation, whichmakes the Hantzsch ester a likely candidate as a pen-dant group for developing PET based fluorescence NOprobes. Herein, we report the PET-based fluorescentNO probe, TPC, for the detection of NO with highselectivity over other reactive oxygen species with highsensitivity for NO.


The probe molecule, TPC, was synthesized viaknown Hantzsch pyridine synthesis procedure starting

from 6-(2-thienyl)-2-pyridinecarboxaldehyde (1) asshown in Scheme 1. The structure of TPC was con-firmed by 1H NMR and LCMS spectra. The molecu-lar structure of TPC has been further established onthe basis of single-crystal XRD data. Subsequently,we examined their reactivity toward NO through UV-Vis and fluorescence spectra in HEPES buffers (15mM, pH 7.4, containing 40% DMSO) at 25 ºC.Isoamyl nitrite was used as the source of NO, which iscommercially available with a half life of 30 min.

Scheme 1.Reagents and conditions : *Ethylacetoacetate, NH3(1), reflux for 8 h.

Crystal structure of TPC

The fine and X-ray quality single crystal of pale-yellow colored TPC was grown by slow evaporationof the solvent from a solution in MeOH-CH3CN (1 : 1)at room temperature with ambient condition over aweek. The probe crystallizes as monoclinic system withspace group P21 and CCDC entry no. 1547182. Asummary of the crystallographic data and structurerefinement parameters of the probe TPC are given inExperimental section. The X-ray bond lengths andangles well reproduced with the standard parameters.

The unit cell structure of TPC exhibits a regulartwinned aggregation of the same species joined to-gether in some definite mutual orientation. Twinningmay occur when a unit cell has higher symmetry thanimplied by the space group of the crystal structure.The molecular structure constitutes of two moietiesi.e. thienyl-pyridine moiety and Hantzsch ester moi-ety. Now, two planes between the two thienyl-pyri-dine moieties of the twin are almost parallel to eachother having angle of 177.11º and distance of 7.457 Å.Similarly, planes between the two Hantzsch ester moi-eties of the twin are almost parallel having angle of177.58º and distance of 2.551 Å.

The crystal structure of TPC is stabilized by inter-molecular hydrogen bonding which forms a 1-dimen-

1

Samim Ali et al. : A thienyl-pyridine-based Hantzsch ester fluorescent probe for the selective detection etc.

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sional supramolecular network. Interesting feature ofcrystal packing (Fig. 2) is the two types of face-to-face intermolecular H-bonding [N2–H2…..O3] and[N4–H4…..O5] with donor–H.....Acceptor distance of2.983(2) and 3.004(4) Å to a series of adjacent paral-lel unit viewed along b-axis (Fig. 2).

spectra of a mixture of probe TPC with different con-centrations of NO in aqueous-DMSO solution. Whenincreasing the concentration of NO, a new absorptionband at ~338 nm was gradually enhanced, while theintensity of other two bands at ~265 and ~305 nmwere decreased correspondingly. The color of the so-

Fig. 1. ORTEP structure of TPC, showing 35% thermal ellipsoids probability level for non-H atoms with atom numbering scheme.

Fig. 2. The crystal packing diagram of TPC shows 1-D supramolecular network involving face-to-face H···bonding viewed alongthe b-axis (H atoms not involved are omitted in for clarity).

Spectroscopic properties and response to TPC

The absorption spectra of free probe TPC and thecolorimetric sensing ability with NO in HEPES buffer(15 mM, pH 7.4, containing 40% DMSO) was moni-tored at 25 ºC and exhibited different bands from 200to 400 nm. These spectra are typical of an aromaticcompound. Fig. 3 shows that the UV-Vis absorption

lution of probe TPC was changed from colorless tolight yellow. Two clear isosbestic points were observedat ~273 and ~319 nm. This result demonstrates thata complex formation of probe TPC with NO is takingplace via radical interactions. The formation of theseradicals affects the electronic properties of thefluorophore, resulting the change between the non-

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aromatic and the aromatic species. The spectra hereare very similar to those of the probe we reportedpreviously. On the account of the quantitatively NO-induced conversion of Hantzsch ester into the pyridineform, the change of the fluorescence herein inspiredus to utilize the probe TPC as effective NO fluores-cent probe.

To check the fluorescence response of probe TPCto NO, the fluorescence spectra of TPC and its titra-tion experiments upon the addition of various equiva-lents of NO in HEPES buffer (50 mM, pH 7.4, con-taining 40% DMSO) was monitored at 25 ºC. The

probe TPC exhibits strong fluorescence emission at~439 nm owing to the inhibition of photoelectron trans-fer (PET) process from the thienyl-pyridine aromaticpart to dihydropyridine moiety due to geometrical con-strained (orthogonal arrangement), when excited at~338 nm in HEPES buffer. Titration of probe TPCwith NO results in the decrease of the fluorescenceintensity as a function of the added NO concentration(Fig. 4) and the intensity saturates at 3 equiv.

The switch off fluorescence emission is expected tothe photoelectron transfer (PET) upon the aromatiza-tion of the Hantzsch ester, which caused the attain-

Fig. 3. (a) Absorbance spectra of TPC (4×10–5 M) at ex = 338 nm, upon addition of NO (4×10–4 M) in DMSO-H2O (15.0 mMHEPES buffer, 4 : 6 v/v, pH 7.4, at 25 ºC). (b) Plot of absorption intensity vs concentration of NO (M).

Fig. 4. (a) Fluorescence spectra of TPC (4×10–5 M) at ex = 338 nm, upon addition of NO (4×10–4 M) in DMSO-H2O (15.0 mMHEPES buffer, 4 : 6 v/v, pH 7.4, at 25 ºC). (b) Plot of fluorescence intensity vs concentration of NO (M).

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ment of planarity. In addition, a linear relationshipbetween the fluorescent intensity and the concentra-tions of NO was constructed. Meanwhile, the calcu-lated lowest limit detection (LOD, 3/slope) was de-termined to be about 0.40 M, which is similar withthe values in literatures13, suggesting that probe TPCis a highly sensitive agent for detecting NO.

In order to check whether probe TPC is sensitiveto only NO or even to the other reactive oxygen spe-cies (ROS), fluorescence titrations were carried out inthe same medium with different ROS, viz. NO, HNO,OH, NO3

–, H2O2, NO2–, ClO– and ONOO– no sig-

nificant fluorescence change was found in the pres-ence of these ROS (Fig. 5a).

Moreover, the interfering experiment was conductedby adding 10 equiv. of the above mentioned reactivespecies to the solutions of TPC (Fig. 5b) in the pres-ence of 3.0 equiv. of NO. Noticing that the addedinterfering species were largely excessive, it demon-strated that TPC can serve as a highly selective fluo-rescence probe for NO.

Under UV light, the solution of probe TPC is in-tense blue fluorescent, whereas in the presence of NO

Scheme 2. Proposed reaction mechanism between TPC and NO.

Fig. 5. (a) Fluorescence spectral changes of TPC (4×10–5) in the presence of 10 equiv. of different analytes. (b) Competitiveselectivity of TPC (4×10–5) toward NO (3 equiv.) in the presence of other analytes (10 equiv.).

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non-fluorescent was observed (Fig. 6) and no suchfluorescent color change was observed in case of theother ROS. Thus, NO can easily be differentiated byits fluorescent color change from the other ROS.

Next, we studied the influence of pH on the fluo-rescence of probe TPC during NO titration. The titra-tions carried out at different pH of the medium exhi-bited no variation in the fluorescence intensity in thepH range 2.0–10.0. These measured results provedthat probe TPC was a potential fluorometric candidatefor monitoring NO in the environment and living cells.

Considering the importance of the responsive timeto the analyte for a reactive probe, the time dependentfluorescence spectra were investigated after additionof 3.0 equiv. of NO to the solution of probe TPC(Fig. 7). After addition of NO into the solution of

probe TPC, the fluorescence emission decreased ra-pidly, and it became stable in 30 min.

Computational studies

Here NO transformed a nonaromatic speciesdihydropyridine (TPC) derivative to its correspondingaromatic pyridine (TPC-Ar) moiety, which makes adramatic electronic and structural changes occurs inthis chemical transformation.

To gain insights into the nature of this fluorescenceswitching-off mechanism based on NO-induced aro-matization of the Hantzsch ester, computational stud-

Fig. 6. The visible color (top) and fluorescence changes (buttom) of receptor TPC in aq. DMSO (DMSO : H2O = 4 : 6 v/v, 15mM HEPES buffer, pH = 7.4) upon addition of various analytes.

Fig. 7. Change of fluorescence intensity in presence of NO atdifferent pH.

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Fig. 8. Energy minimization structure of (a) TPC and (b) TPC-Ar.


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ies based on density functional theory (DFT) and time-dependent DFT (TDDFT) were performed at theB3LYP/6-31G(d,p) level of the Guassian 09 program.

Results from calculation revealed that the ELUMOlevel of the dihydropyridine Hantzsch ester (Fig. 9)was about –1.899 eV, higher than that of the ELUMOlevel (–2.483 eV) of the thienyl pyridine moiety. UponNO-induced aromatization of the Hantzsch ester, theELUMO level (–3.17 eV) of the pyridine was belowthat of the ELUMO level (–2.483 eV) of the thienyl-pyridine moiety, which drive PET process and there-fore turn off fluorescence of the probe.

The TDDFT calculation revealed the absorptionbands in the region of max, 250–450 nm. The verticaltransitions were found to have a good aggrement withthe experimentally found data.

Fig. 9. Molecular orbital plots for the HOMO-1, HOMO, LUMO and LUMO+1 of TPC and TPC-Ar.

TDDFT calculations showed absorption band at~357 and ~339 nm belonging to the HOMOLUMO(84.31%) (f = 0.1302) and HOMOLUMO+5(74.41%) (f = 0.034) energy states respectively forprobe TPC.

Bioimaging in living cells

We then assessed the potential utility of TPC forthe specific imaging of NO in living cells. For thispurpose, the cytotoxicity of probe TPC toward Raw264.7 cells were evaluated by using a standard MTT(3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) assay.

The results in Fig. 10e showed that compound TPCdisplayed low cytotoxicity in the adoptive concentra-tion ranges, indicating the high feasibility in biologi-

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Fig. 10. Images of Raw 264.7 cells : (a) bright field, (b) confocal images of Raw 264.7 cells incubated with TPC (20.0 M) for4 h, (c) bright field, (d ) confocal images of 264.7 cells incubated with 1 (20.0 M) for 3 h, and then incubated with NO(3 equiv.) for 15 min and (e) MTT assay to determine the cytotoxic effect of TPC on Raw 264.7 cells.


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cal measurement. Probe TPC was then applied fordetecting endogenously generated NO by incubatingRaw 264.7 cells in the presence or absence of bacte-rial lipopolysaccharide (LPS)14. Raw 264.7 cells wereincubated in PBS buffer (pH 7.4) containing 15 M ofthe probe TPC for 20 min at 37 ºC, followed bywashing the cells with the same buffer to remove theexcess of the probes. At this stage, the fluorescencemicroscopy image of Raw 264.7 cells displayed in-tense blue fluorescence. However, upon the additionof isoamylnitrite (source of NO) into the cells via in-cubation with LPS solution for 20 min at 37 ºC, thecells exhibited non-fluorescent (Fig. 10d). These re-sults demonstrate that the probe TPC is capable ofdetecting NO in living cells.

Conclusion

We have synthesized and characterized thienyl-py-ridine-based Hantzsch ester, TPC fluorescent probefor detecting nitric oxide (NO) in HEPES buffer me-dium. The selectivity and sensitivity was demonstratedon the basis of fluorescence, absorption, LC-mass spec-trometry, and visual fluorescent color changes. Fluo-rescence titrations showed the probe is sensitively andlinear-dependent to the NO concentration and can bedetect up to ~0.4 M. The probe displayed a highspecificity for NO when compared with other N-, O-sensitive species. NO-induced aromatization of theHantzsch ester is responsible for the fluorescencechange mechanism in the sensing process. The struc-ture of TPC has been established by single-crystalXRD. Theoretical calculations were performed to un-derstand the photo-physical behaviors for probe TPCand its aromatized compound, and the obtained resultsalso supported the sensing mechanism. The probe TPCdemonstrated good photophysical stability in the pHrange of 4.0–9.0 and low cytotoxicity. The probe wassuccessfully applied in the field of cell imaging fordetecting NO in the cell. Strong fluorescent imageswere observed when Raw cells were incubated withthe probe TPC alone. However, non-fluorescence wasobserved in Raw cells in the presence of NO. Furtherwork to improve the sensitivity and put into practicalapplications is in progress.

Experimental

Chemicals, method and measurements : UV gradedimethyl sulphoxide (DMSO) was purchased fromspectrochem. Isoamyl nitrite was purchased fromSigma-Aldrich Pvt. Ltd. (India). Other chemicals wereobtained from commercial suppliers and were usedwithout further purification. Mass spectra was carriedout using a Waters QTOF Micro YA 263 mass spec-trometer. 1H spectra was recorded on a Brucker 400MHz instrument. For NMR spectra, DMSO-d6 andCDCl3 were used as solvent using TMS as an internalstandard. Chemical shifts are expressed in ppm unitsand 1H–1H and 1H–C coupling constants in Hz. UVspectra were recorded on a JASCO V-530 spectropho-tometer. Fluorescence spectra were recorded on aPerkin-Elmer Model LS 55 spectrophotometer.

Cell culture procedure : Raw 264.7 cell lines wereprepared from continuous culture in Dulbecco’s modi-fied Eagle’s medium (DMEM, Sigma Chemical Co.,St. Louis, MO) supplemented with 10% fetal bovineserum (Invitrogen), penicillin (100 g/mL), and strep-tomycin (100 g/mL). The Raw 264.7 were acquiredfrom the American Type Culture Collection (Rockville,MD) and maintained in DMEM containing 10% (v/v)fetal bovine serum and antibiotics in a CO2 incubator.At first cells were initially cultivated in 75 cm2 poly-styrene, filter-capped tissue culture flask in an atmo-sphere of 5% CO2 and 95% air at 37 ºC in CO2incubator. When the cells almost reached the logarith-mic phase, the cell density was adjusted to 1.0×105

per/well in culture media. Then the cells were used toinoculate in a glass bottom dish, with 1.0 mL (1.0×104

cells) of cell suspension in each dish. Culture mediumwas removed just after cell adhesion. The cell layerwas rinsed thrice with phosphate buffered saline (PBS),and then used according to the experimental need.

Confocal imaging procedure : For confocal imag-ing studies Raw 264.7 cell, 1×104 cells in 1000 L ofmedium, were seeded on sterile 35 mm covered glassbottom, Petridis, culture dish (ibidi GmbH, Germany),and incubated at 37 ºC in a CO2 incubator for 10 h.Then cells were washed with 500 L DMEM and thenincubation with (8 M) isoamyl nitrite dissolved in500 L DMEM at 37 ºC for 1 h in a CO2 incubatorand analyzed under an Olympus IX81 microscope


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equipped with a FV1000 confocal system using 1003oil immersion Plan Apo (N.A. 1.45) objectives. Allimages obtained through section scanning were ana-lyzed by Olympus Fluoview (version 3.1a; Tokyo,Japan) with excitation at 338 nm monochromatic laserbeam, and emission spectra were integrated over therange 400–650 nm (single channel). The cells wereagain washed thrice with phosphate buffered salinePBS (pH 7.4) to remove any free isoamyl nitrite andincubated in PBS containing probe TPC to a final con-centrations of 4.0×10–6 M, incubated for 10 min fol-lowed by washing with PBS three times to removeexcess probe outside the cells and images were cap-tured. The confocal microscope settings for all im-ages, such as scan speed and transmission density wereheld constant for comparing the relative intensity ofintracellular fluorescence.

General computational method : Geometries havebeen optimized using the B3LYP/6-31G (d,p) level oftheory. The geometries are verified as proper minimaby frequency calculations. Time-dependent densityfunctional theory (TDDFT) calculation has also beenperformed at the same level of theory. All calculationshave been carried out using Gaussian 09 program.

Determination of the detection limit : The detectionlimit was calculated based on the fluorescence titra-tion. The fluorescence emission spectra of TPC weremeasured, and the standard deviation of blank mea-surements was acquired. To obtain information aboutthe slope, the fluorescence intensity at Io/I was plottedas a concentration of NO. Then, the detection limitwas calculated using the following equation : Detec-tion limit = 3/k. In which is the standard deviationof 10 blank measurements, k is the slope between thefluorescence intensity versus NO concentration.

General procedure for preparation of solution forUV-Vis titration : A stock solution of the probe TPC(4×10–5 M) was prepared in DMSO/H2O (4 : 6 v/v).All experiments were carried out in DMSO/H2O solu-tion (4 : 6 v/v, 15 mM HEPES buffer, pH = 7.4).Titration experiment was carried out by adding (4×10–5 M) solution TPC in a quartz optical cell of 1 cmoptical path length, and the analyte stock solutions

(4×10–4 M) were added into the quartz optical cellslowly by using a micropipet.

General procedure for preparation of solution forfluorescence titration : A stock solution of the probeTPC (4×10–5 M) was prepared in DMSO/H2O (4 : 6v/v). All experiments were carried out in DMSO/H2Osolution (DMSO/H2O = 4 : 6 v/v, 15 mM HEPESbuffer, pH = 7.4). Titration experiment was carriedout by adding (4×10–5 M) solution TPC in a quartzoptical cell of 1 cm optical path length, and the analytestock solutions (4×10–4 M) were added into the quartzoptical cell slowly by using a micropipet. In selecti-vity experiments, the test samples were prepared byplacing appropriate amounts of the analyte, stock into2 mL of solution of probes (4×10–5 M).

Synthesis of TPC : 6-(2-Thienyl)-2-pyridinecarboxal-dehyde (1) (0.3 g, 1.58 mmol), ethyl acetoacetate (1.1g, 9.2 mmol) and ammonia solution (0.2 g, 9.2 mmol)were dissolved in 10 ml ethanol and refluxed for 10 h.After cooling to room temperature, the reaction mix-ture was concentrated in vacuo to get crude productwhich was purified by column chromatography usingethyl acetate in hexane (1 : 9) and finally get purecompound TPC. Yellow solid, (260 mg, 44%), m.p.190–195 ºC, MS (LCMS) : (m/z, %) : 413.2 [(TPC+H+), 100 %]; Calculated for C12H24N2O4S : 412.15.1H NMR (DMSO-d6, 400 MHz) (ppm) : 7.47 (2H,m), 7.38 (1H, d, J=7.6 Hz ), 7.26 (1H, t J=13.16),7.04 (2H, m), 5.70 (1H, s), 5.12 (1H, s), 4.11 (4H,m), 2.35 (6H, s), 1.23 (6H, m).

X-Ray crystal structure determination

The X-ray quality single crystal of TPC was grownby slow diffusion in MeOH-CH3CN (1 : 1) under am-bient condition for a week. Block shaped pale-yellowcrystal of dimension 0.18×0.14×0.09 mm. Unit cellparameters were determined from least-squares methodwith the data in the range of –9 h 9, –22 k 22,–23 l 23 and angle varies 1.197 < < 28.85º.Other unit cell parameters are a = 7.3479(6), b =16.3371(13), c = 17.2146(12) (Å) and = = 90(°), = 98.693(4). Out of total reflections 68151unique data 6125 (I > 2(I)) were used for structuredetermination.

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Acknowledgement

The authors would like to acknowledge DAE-BRNS[Project No. 36(1)/14/12/2016-BRNS/36012] for fi-nancial support. SSA and AKP thanks to the UGC-MANF and SERB-NPDF, New Delhi for the financialsupport.

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Toward dithia-aza based malachite green probe : Colorimetric chemosensorfor Hg2+ ions

Samaresh Ghosh* and Rajkumar Manna

Department of Chemistry, Bankura Sammilani College, Kenduadihi, Bankura-722 102,West Bengal, India


Manuscript received 07 June 2017, accepted 12 June 2017

Abstract : A new probe 1 based on a malachite green derivative bearing two dithia-aza subunits was prepared,and studied as colorimetric chemosensor for the sensitive detection of Hg2+ ions over other representative metalions.

Keywords : Hg-sensor, dithia-aza, malachite green, naked-eye.

Introduction

In recent years, there has been growing interest inthe development of chemosensors for the sensitivedetection of mercuric (Hg2+) ion because of its toxi-city causing serious problems for public health andecology1–5. Although a number of molecular probesfor the detection of Hg2+ ions have been developed,colorimetric chemosensors for the selective detectionof Hg2+ are quite attractive because of their excellentsensitivity, rapid response, and ability to do the so-called “naked-eye” detection in a straightforward andinexpensive manner6–14. Thus, developing simple andpractical colorimetric chemosensors for Hg2+ ions isstill a challenge. However, dithia-aza binding motif iscurrently used15,16 in the design of chemosensors ow-ing to its ability to coordinate specific metal ions throughN and S-donors. Furthermore, the malachite green en-tity is one of the most attractive chromogenic unitsdue to its well characterized UV-Vis absorption andeasy synthesis. To continue our interest in chemosensordeve-lopment for transition and heavy metal ion moni-toring17, we report herein a simple malachite green(MG) derivative 1 as colorimetric chemosensor bear-ing dithia-aza chelating units for the rapid detection ofHg2+ ions in aqueous media. Malachite green (MG) 2was taken as reference compound. However, to the bestof our knowledge, there is no report on the design ofMG-derivatives for “naked-eye” detection of Hg2+ ions.


N-Phenyl-5,11-dithia-8-azapentadecane 1a was syn-thesized according to the Scheme 1. 1a was then con-densed with benzaldehyde to give 1 in good yield.Titled molecule 1 was characterized by FT-IR, UV-Vis, NMR, MS, and elemental analyses. The UV-Visabsorption spectrum of 1 in THF-water (4 : 1, v/v)solution ([1] = ~4.9×10–5 M) exhibited three ab-sorption bands at 307, 435 and 625 nm (weak) ( =1.01×104, 6.42×102, and 1.42×103 M–1 cm–1, re-spectively) typical of MG subunit. Absorption titra-


802

tion experiments were carried out by using the set ofrepresentative metal ions, such as Ni2+, Cu2+, Zn2+

(all as sulphates), Pb2+, Mg2+, Mn2+ (all as perchlo-rates) and Hg2+(as nitrate) to evaluate the metal ionbinding selectivity and sensitivity of 1.

Upon interaction with this set of metal ions, onlyHg2+ induced a progressive and sizable increase inabsorption band at 626 nm. Fig. 1 gives detailed UV-Vis spectral changes of 1 upon titration with Hg2+

ions. Remarkable feature pertaining to this molecularprobe is the change of color from light yellow to gree-nish blue (Fig. 2) which can be used for a “naked-eye” detection of Hg2+ ions in aqueous solution.

No significant color/absorption changes were ob-served upon interaction with other potentially compet-ing metal ions mentioned (Figs. 2 and 3) demonstrat-ing its unique selectivity and sensitivity toward Hg2+

Scheme 1. (i) p-TsCl, pyridine, 0–5 ºC (80%); (ii) (a) Thiourea/EtOH, reflux, (b) NaHCO3/H2O, 80–90 ºC; (iii) (a) dry THF/NaH,reflux, (b) n-BuBr, rt, 65%; (vi) (a) PhCHO, ZnCl2 (anh.), THF (dry), reflux, (b) PbO2, conc. HCl, water, rt, 70%.

Fig. 1. UV-Vis spectral changes of 1 (4.9×10–5 M) upon theincremental addition of 14 equivalents of Hg2+ ion inTHF-water (4 : 1, v/v).

Fig. 2. Color changes of 1 (4.9×10–5 M) upon the addition oftested metal ions in THF-water (4 : 1, v/v).

ions. The stoichiometry of the complex (1.Hg2+) inthe interaction process was determined from the titra-tion curve (Fig. 4). The break of the titration curve atHg2+/[1] = 2 indicates the formation of 1 : 2 com-plexes (sensor 1 : Hg2+ ion).

Fig. 3. Plot of absorption intensity at 625 nm as a function ofmetal ion concentration.

Ghosh et al. : Toward dithia-aza based malachite green probe : Colorimetric chemosensor for Hg2+ ions

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1H NMR spectrum of 1 in the presence of Hg(NO3)2was broadened to some extent and resulted in downfieldshifting of the MG-protons, especially in the dithia-aza moiety. These shifts suggest that dithia-aza moi-eties are involved in the coordination with Hg2+ ions.LCMS measurement provided additional evidence forthe formation of 1 : 2 stoichiometry of the complex 1-Hg2+.

Conclusion

In summary, dithia-aza based malachite greenchemosensor 1 has been synthesized for the first time.A distinct color change was observed when 1 wastreated with Hg2+ ions. This is presumably attributedto the perturbation of the -system upon complexationof Hg2+ ions at the binding domains. Such impressivecolor change as well as the high reproducibility andthe large persistence (days) of the coloration of probe1 permit a “naked-eye” detection of Hg2+ ions in aque-ous media. Work in this direction is currently underprogress in our laboratory.

Experimental

Synthesis of 1a : A solution of aniline (0.5 g, 5.38mmol), 2-chloroethanol (4.3 g, 53.76 mmol), and cal-cium carbonate (2.5 g, 25.0 mmol) in 50 ml waterwas refluxed for 18 h, and the filtrate was extractedwith dichloromethane. The organic phase was driedover anhyd. Na2SO4 and filtered. The solvent wasremoved in vacuo to get the crude product, which waspurified by column chromatography on silica geleluating with petroleum ether gradually increasing to1 : 1 petroleum ether/EtOAc to obtain pure product ofN-phenylated diethanolamine (70%). Conversion of N-phenylated diethanolamine to the corresponding dithiolwas achieved by following the reported procedure15.

Fig. 4. Plot of absorption change of 1 at 625 nm as a functionof [Hg2+]/[1].

Scheme 2. Suggested co-ordination induced sensing mechanism of 1 for Hg2+ ions.

The signaling mechanism might be attributed to theHg2+ chelation induced perturbation of the -cloudsof non-planar propeller-like twisted structure (A) to-ward the corresponding more conjugated quinoid-likecationic forms (B and C) as illustrated in Scheme 2.Both nitrogen and sulfur atoms of dithia-aza subunitparticipate in Hg2+-ion binding cooperatively.

In order to look into the thiophilic speciality in thechelation induced absorption effect in 1, UV-Vis titra-tion experiments of well known reference compound 2lacking S-donors were carried out. We noticed thatadding Hg2+ to the solution of 2 induced no change incolor/absorption features. This observation revealedthat the presence of two proximal S-donors is a pre-requisite for promotion of Mx+-N interaction inducedsignaling mechanism.

The complexation induced signaling phenomenonof 1 with, for instance, Hg2+ ions was further evi-denced by LCMS and 1H NMR measurements. The


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Under nitrogen atmosphere, to a solution of the corre-sponding dithiol (0.154 g, 0.723 mmol) in dry THFwas added NaH (0.0696 g, 2.90 mmol) and refluxedfor 2 h. Then n-butylbromide (0.397 g, 2.90 mmol)was added to the reaction mixture and stirred over-night at room temperature. The reaction mixture wasevaporated to dryness under reduced pressure. Theresidue was redissolved in EtOAc, washed with brinesolution, dried over anhyd. Na2SO4 and filtered. Thesolvent was removed in vacuo to get the crude pro-duct, which was purified by column chromatography(silica gel 60–120, petroleum ether/EtOAc 1 : 50) toobtain pure product 1a (65%). FT-IR (KBr) max :2957, 2929, 2862, 1591, 1505, 1458, 1343, 1277,1181, 1143, 743, 686 cm–1; 1H NMR (CDCl3, 500MHz) : 7.23 (2H, t, J=7Hz), 6.70 (1H, t, J=7Hz),6.66 (2H, d, J=8Hz), 3.52 (4H, t, J=7.5Hz), 2.71(4H, t, J=7.5Hz), 2.57 (4H, t, J=7.5Hz), 1.62–1.56(4H, m), 1.46–1.38 (4H, m), 0.92 (6H, t, J= 7.5Hz).Anal. Calcd. for C18H31NS2 : C, 66.40; H, 9.60; N,4.30. Found : C, 66.38; H, 9.59; N, 4.28.

Synthesis of 1 : Compound 1a (0.02 g, 0.061 mmol)was dissolved in minimum volume of dry THF. Ben-zaldehyde (0.0016 g, 0.0153 mmol) was then addedinto the solution of 1a and the resulting solution wasrefluxed with anhyd. ZnCl2 for 24 h. After solventevaporation, the residue was treated with water, PbO2and few drops of conc. HCl. The reaction mixturewas stirred at room temperature overnight. Then themixture was treated with water and extracted withCH2Cl2. The organic phase was dried over anhyd.Na2SO4 and filtered. The crude product obtained afterremoval of solvent was purified by preparative thinlayer chromatography using 6% EtOAc/petroleum etherto yield 1 (70%) as a green semisolid. FT-IR (KBr)max : 2930, 2859, 1726, 1596, 1510, 1453, 1386,1348, 1281, 1167, 1119, 1024, 891, 814, 691, 614cm–1; 1H NMR (CDCl3, 500 MHz) : 7.28 (4H, d,J=10Hz, ArH), 7.12–7.06 (5H, m, ArH), 6.96–6.90(2H, m, ArH), 6.54 (2H, d, J=10Hz, ArH), 3.94–3.91 (4H, m), 3.55–3.51 (4H, m), 3.07–3.05 (4H,m), 2.90–2.80 (4H, m), 2.73–2.71 (6H, m), 2.62–2.60 (2H, m), 1.73–1.70 (4H, m), 1.42–1.40 (4H,m), 1.29–1.21 (8H, m), 0.94–0.86 (12H, m); 13C(CDCl3, 125 MHz) : 141.5, 130.3, 129.7, 129.5,129.3, 128.9, 127.8, 120.4, 116.6, 116.0, 110.4, 81.0,

70.0, 52.3, 51.8, 51.5, 44.7, 41.1, 32.1, 29.7, 26.7,14.1, 13.7. Anal. Calcd. for C43H65N2S4.Cl : C, 66.75;H, 8.47; N, 3.62. Found : C, 66.80; H, 8.53; N,3.60; m/z (ES+) 772.4 (M+), 737.7 (M–Cl–)+.

Acknowledgement

Funding from Department of Science and Techno-logy (SR/FTP/CS-88/2007), Government of India, isgratefully acknowledged.

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July-M- pmd


Fluorescent sensing of H2PO4– by in situ prepared Cu2+ complex in aqueous

medium via displacement approach

Soma Mukherjee*, Palash Mal and Shrabani Talukder

Department of Environmental Science, University of Kalyani, Kalyani-741 235, Nadia,West Bengal, India

E-mail : [email protected], [email protected]


Abstract : The in situ prepared Cu2+ chelate of luminescent ligand, pyridine-2-carboxaldehyde-benzoic acid hy-drazone (1) was investigated for fluorescent sensing of H2PO4

– in aqueous medium via restoration of the fluo-rescence intensity of the receptor through Cu2+ displacement approach. The sensing behavior of this ligand to-wards Cu2+ via fluorescence quenching has already been reported in our previous work. The present study de-picts in situ prepared Cu2+ chelate as a turn-on fluorescent chemosensor of H2PO4

– detecting in the micro mo-lar range. The Cu2+ chelate possesses excellent tolerance to other interfering anions Cl–, Br–, I–, OAc–, NO3

–,SO4

2–.

Keywords : Luminescent, hydrazone, fluorescent chemosensor, anion recognition, H2PO4–.

Introduction

The development of anion selective sensors hasreceived considerable attention in recent years as an-ions play a potential role in several environmental pro-cesses. Especially, chemosensors based on the anioninduced luminescence changes are attractive due tothe simplicity, selectivity and high sensitivity1–3.

Among the significant anions, phosphate and itsderivatives act as essential ingredients in biologicalsystems and are responsible for different biologicaland environmental functions4–12. Therefore, a num-ber of phosphate recognition methods have been in-vestigated13–18. At physiological pH phosphate existsas both H2PO4

– and HPO42– 19 and the selective de-

tection of dihydrogen phosphate (H2PO4–) gets more

attraction to the researchers in phosphate recognition.Due to the very high free energy of hydration (G0 =–465 mol–1), it is difficult to design a chemosensor forH2PO4

– sensing in water20. The receptors having theability of detection of H2PO4

– in an aqueous mediumat neutral pH is highly desirable and of special signifi-cance due to their potential applications in biologicalsciences21,22. A number of chemosensors are devel-oped that rely on hydrogen bonding via the amide23,

urea and thiourea subunits24. However, a serious dis-advantage of such receptors is that they are not able tosense anions in aqueous solvents because the aqueousmedium strongly competes for hydrogen bondingsites25. To overcome this problem, sensory systemswere developed in which an electrostatic affinity fortransition metal ions facilitates a strong interaction26.In the context of metal complexes as anion sensors,the paradigm has shifted toward the metal ion dis-placement approach where, a metal ion quenches thefluorescent intensity of the receptor and the treatmentof such a metal complex with an anion displaces themetal ion from the coordination sphere of the originalorganic receptor, restoring the fluorescence intensityof the receptor27–29. However, a lot of works havealready been reported and most of the receptors aredeveloped using tedious synthetic protocols and some-times shows poor compatibility in water30–36.

Earlier the selective response of the receptor 1 (py-ridine-2-carboxaldehyde-benzoic acid hydrazone) to-wards Cu2+ was investigated and reported37. In con-tinuation to the previous work herein we have exploredthe sensing behavior of the in situ prepared Cu2+ che-late, 1-Cu2+, towards H2PO4

– by turn-on mechanismvia Cu2+ displacement approach.


806

Table 1. Some reported receptors used for H2PO4– sensing via metal displacement approach

Sl. Structure Metal Quantum yield Limit of detection of Reference

No. displaced of receptor receptor-metal chelate

1. Zn2+ – – 30

2. Zn2+ – – 31

3. Cu2+ – 1.41 M 32

4. Zn2+ 2.0×10–3 – 33

Mukherjee et al. : Fluorescent sensing of H2PO4– by in situ prepared Cu2+ complex in aqueous etc.

807

5. Fe3+ 0.89 1.0×10–9 mol/L 34

6. Cu2+ – – 35

7. Fe3+ – 1.69×10–8 mol/L 36

8. Cu2+ 0.0167 2.53×10–6 M 37

and the

present work

Table-1 (contd.)

Experimental

Reagents :

All starting materials and solvents were purchasedfrom Sigma Aldrich and Merck chemical company andused without further purification unless otherwise stated.

Physical measurements :

Spectroscopic studies were carried out with aHitachi-F7000 Luminescence Spectrometer at ambienttemperature in aqueous medium (CH3CN/H2O; 1 : 4,v/v) at fixed concentration of 1 (1.0×10–5 M). Titra-tion experiments were carried out in 10-mm quartzcuvettes at 25 ºC. Copper(II) salt and anions as tetra-butylammonium salts (1 M; except for NO3

– andSO4

2–) in aqueous medium (CH3CN/H2O; 1 : 4, v/v)were added to the host solution and used for the titra-

tion experiment. 1-Cu2+ solution for H2PO4– detec-

tion was prepared by addition of 1.0 equiv. of Cu2+ tocompound 1 solution in aqueous medium (CH3CN/H2O; 1 : 4, v/v). Mass spectra were recorded usingXevo G2-SQT.

Synthesis and characterization of receptors :

The receptor (1) was synthesized following the re-ported method37. The characterization data and thesingle crystal X-ray structure of the receptor 1 werealready reported in our previous work37.


Spectral studies :

The binding stoichiometry of the receptor 1 withCu2+ ion was found to be 1 : 1 as reported in our

JICS-19


808

previous work37. The positive ion mass spectrum of 1upon addition of Cu2+ indicated that a peak at m/z =401.10 which was assignable to [1+Cu2++Br–+H2O](Calcd. 401.65). Thus, the 1 : 1 stoichiometry of the1-Cu2+chelate was also supported by the ESI-massspectrometry analysis.

The sensing of 1 towards Cu2+ was reported inour earlier study37. The 1-Cu2+ chelate exhibits strongabsorption band in the visible region (max = 435 nm)in aqueous medium. The emission peak of 1 (em =405 nm), undergoes quenching upon complexation withCu2+ 37. In the present work, the in situ preparedCu2+ chelate was investigated as a turn-on fluorescentchemosensor for H2PO4

– ions in aqueous medium. Thesuccessive addition of H2PO4

– (0–100 M) to the insitu prepared solution of 1-Cu2+(10 M) resulted in astep-wise enhancement of emission intensity at 405nm and gradually restored to the original emission stateof 1 (Fig. 1). The proposed binding mode of receptor1 involving Cu2+ and H2PO4

– ions is illustrated inScheme 1. Fluorescence-ON receptor 1 selectively bindsto Cu2+ to form a Fluorescence-OFF 1-Cu2+ chelate.This is accompanied by the loss of a proton as re-vealed from mass spectroscopic studies. The 1-Cu2+chelate selectively interacts with H2PO4

– therebyresulted in the restoration of the Fluorescence-ONemission spectra of receptor 1.

The detection limit of the 1-Cu2+ chelate as a fluo-rescent sensor for the analysis of H2PO4

– was deter-mined from a plot of emission intensity as a functionof concentration of the added anion. Based on theemission titration curve it was found that the fluores-

cence enhancement factor (I – I0)/I0 was proportionalto the concentration of H2PO4

– ions (Fig. 2). The lin-ear range of fluorescence response of 1-Cu2+ forH2PO4

– ions was 0–10 M (R2 = 0.9818) and detec-tion limit for H2PO4

– ions was calculated as 2.53×10–6

M following the reported method38–40.

The response of 1-Cu2+ (10 M) with selectedanions (H2PO4

–, Cl–, Br–, I–, AcO–, NO3–, SO4

2–)was examined through specrofluorimetric study in aque-ous medium (CH3CN/H2O; 1 : 4, v/v) at 298 K. Theintensity of 1-Cu2+ solution containing other competi-tive anions (Cl–, Br–, I–, OAc–, NO3

–, SO42–) and

H2PO4– ions is shown in Fig. 3, indicating negligible

interference of other anions for selective sensing ofH2PO4

– via Cu2+ displacement approach.

A plot of I/I0 (where, I and I0 represent the fluo-

Scheme 1. Proposed mechanism of fluorescence sensing of H2PO4– by 1-Cu2+.

Fig. 1. Emission spectra (ex = 290 nm) of 1-Cu2+ (10 M)upon successive addition of H2PO4

– (0–100 M) in aque-ous medium (CH3CN/H2O; 1 : 4, v/v) at 298 K.

Mukherjee et al. : Fluorescent sensing of H2PO4– by in situ prepared Cu2+ complex in aqueous etc.

809

rescence intensity of various metal ions and that offree metal respectively) at 405 nm (Fig. 4), shows aratio 1 for most of the metal ions with the notableenhancement in emission intensity (> 8-fold) forH2PO4

– ions.

Conclusion

The in situ prepared 1-Cu2+ chelate was investi-gated as a fluorescent sensor for H2PO4

– by the Cu2+

displacement approach in aqueous medium. No sig-nificant interference was observed in presence of se-lected anions. The sensor 1-Cu2+ was highly sensitiveand selective for H2PO4

– with detection limit in themicro molar range.

Fig. 3. (a) Changes in the fluorescence spectra (ex = 290 nm) of 1-Cu2+ (10 M) upon addition of tetrabutylammonium salts ofanions (100 M) in aqueous medium (CH3CN/H2O; 1 : 4, v/v). (b) Changes in the emission intensity of 1-Cu2+ (10 M)on addition of H2PO4

– (100 M) over selected anions (100 M) in aqueous medium (CH3CN/H2O; 1 : 4, v/v) at 405 nmat 298 K. The blue bars represent the emission intensity of 1-Cu2+ in the presence of selected anions; the red bars representthe emission intensity of the above solution on addition of H2PO4

–.

Fig. 2. (a) Plot of the fluorescence enhancement factor (I – I0)/I0 at 405 nm versus the concentration of H2PO4– ions added. (b)

Representation of linear range.

Fig. 4. Emission intensity ratios (I/I0) for 1-Cu2+ (10 M) at405 nm in the presence of selected anions in aqueousmedium (CH3CN/H2O; 1 : 4, v/v) at 298 K.


810

Acknowledgement

Financial supports received from DST-PURSE andDST-FIST, New Delhi, India, are gratefully acknowl-edged for financial support and instrumental facilities.We are also grateful to the University of Kalyani forproviding infrastructural facilities.

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38. N. Zhang, F. Qu, H. Q. Luo and N. B. Li, Anal.Chim. Acta, 2013, 791, 46.

39. S. Mukherjee, P. Mal and H. Stoeckli-Evans, J.Luminescence, 2016, 172, 124.

40. Y.-J. Chang, P.-J. Hung, C.-F. Wan and A.-T.Wu, Inorg. Chem. Commun., 2014, 39, 122 andreferences therein.

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July-N- pmd


A novel compartmental Schiff base ligand as a fluorescence chemosensor :Microscopic to macroscopic detection of ZnII acetate

Bhaskar Biswas

Department of Chemistry, Raghunathpur College, Purulia-723 133, West Bengal, India

E-mail : [email protected], [email protected]


Abstract : A novel compartmental Schiff base ligand, (L = N,N-bis(salicyaldehydene)-1,3-diaminopropan-2-ol)serves as a fluorescence chemosensor for the detection of zinc(II) acetate salt. The fluorescent probe (L) under-goes a remarkable fluorescence enhancement upon binding to zinc acetate in pure acetonitrile (MeCN). Inter-estingly, addition of other 3d-metal acetate salts to the probe, exhibit usual fluorescence quenching phenom-enon in MeCN solution. The fluorescent probe (L) can selectively detect ZnII acetate salt in a concentration rangeof 1.0×10–6 to 1.0×10–4 mol/L. Further, trinuclear ZnII-Schiff base complex has been characterized by singlecrystal X-ray diffraction study supports the selectivity and binding ability of L with Zn2+ ion.

Keywords : Molecular recognition, zinc(II), Schiff base, X-ray structure, fluorescence probe.

Introduction

The creation of organic molecular species havingthe ability to sense analytes selectively, especially cat-ions, are of biologically and environmentally impor-tant and have been emerged as a significant goal in thefield of chemical sensors in recent years1–3. Chemicalfluoro-sensors which are based on ion-induced fluo-rescence changes have created huge interests to chem-ists in terms of sensitivity, selectivity, response time,simplicity, high degree of specificity and low detec-tion limit1–4. Fluorescent sensors remain an indispens-able tool for visualizing or monitoring metal ions inreal-time and real-space at a molecular level withoutany special instrumentation, and are applicable in manyfields such as medical diagnostics, environmental con-trol, living cells, and electronics1,4–7. Zinc is a veryimportant bio-essential element and plays significantroles in the living system effecting, DNA synthesis,microtubule polymerization, gene expression, apoptosis,immune system function, and the activity of differentmetallo-enzymes such as carbonic anhydrase and ma-trix metalloproteinase8. Moreover, zinc ion is also a

contributory factor in neurological disorders such asepilepsy and Alzheimer’s diseases9.

Over the past few decades, many fluorescent chemo-sensors of different class for Zn2+ have been deve-loped, using quinoline10, anthracene11 and coumarin12

as fluorophores. However, some of them require com-plicated syntheses and are insoluble in aqueous me-dium. Further, various fluorescence-based zinc probeshave been recently developed – some of these are fluo-rescent adducts of Zn-chelating peptides13–15 and pro-teins16, while others are dye adducts of Zn-chelatingmacrocyclic compounds17–19. Although some of thesefluorescent probes can be utilized to detect zinc ions inenvironmental or biological samples, they have seri-ous disadvantages like insufficient selectivity or sensi-tivity. Schiff bases (SBs) have the potential to formstable complexes of varied nuclearities with transitionmetal ions and act as ion carriers. The feature of SBsgives geometric and cavity control of host-guest com-plexation and modulation of its lipophilicity, and pro-duces remarkable selectivity, sensitivity and stabilityfor a specific ion. Thus, in this present report a novel


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compartmental Schiff base ligand, (L = N,N-bis(salicyaldehydene)-1,3-diaminopropan-2-ol), Scheme1, is used as an ionophore with N and O donor atomsfor construction of a ZnII chemosensor. Further, atrinuclear ZnII-Schiff base complex is characterized inthe form of single crystals in MeCN medium with anexternal addition of little amount of DMF, and thisphenomenon supports and consolidates the selectivityand efficient binding ability of L with Zn2+ ion in a2 : 3 binding manner.


Syntheses and formulation of the Schiff base (L)

The Schiff base ligand, L, N,N-bis(salicyaldehy-dene)-1,3-diaminopropan-2-ol was synthesized by con-densing 1,3-diaminopropan-2-ol with 2-salisaldehydein 1 : 2 molar ratio in dry ethanol following a reportedprocedure20. The formulation was confirmed by ele-mental analysis, IR, UV-Vis, 1H NMR, and ESI-MSspectral analysis. The schematic presentation of syn-thesis is given below :

characteristic absorption bands at 316, 363 and 414nm in acetonitrile solvent (10–4 M; Fig. 1). The highlyintense band at 316 nm is assigned to -* transitionof the C=N chromophore, whereas the band with lowintensity at 363 nm is due to n-* transition and abroad band ~414 nm is appeared for intraligand chargetransfer21 (Fig. 1). The fluorescence spectral measure-ment for receptor, L in the absence of any divalentmetal ions was carried out in acetonitrile using 10–4 Mat room temperature and the spectrum is also given inFig. 1. The ligand, excited at 363 nm, have shownemission band around 424 nm. The lower fluorescenceintensity of L may be attributed for losing of co-pla-narity.

In order to probe the solution stability of L, ESI-MS spectrum in acetonitrile solution at room tempera-ture is taken. It consolidates the solution stability of Lwhich exhibits the molecular ion peak as base peak atm/z 299.17 in acetonitrile medium. ESI-MS (MeCN)m/z 299.17 [L+H+] (Calcd. 299.13).

Scheme 1. Preparative procedure of the Schiff base (L).

Photophysical and solution properties of L

The photophysical study and the structural integ-rity in solution state for the Schiff base ligand (L) hasbeen examined by proton NMR, UV-Vis spectroscopy,fluorescence study and ESI mass spectral studies. Theligand is soluble in common organic solvents like metha-nol, acetonitrile, dichloromethane etc. The 1H NMRspectral data for this Schiff base compound is pro-vided in Experimental section and accounts in favourof the formulation of L. The UV-Vis spectrum for Lshows high intensity transitions in the range of 200 to500 nm (Fig. 1). The Schiff base solution shows the

Fluorogenic ZnII-acetate recognition

To establish the practical applications, the fluores-cence response behavior of the ligand was examinedupon addition of 3d divalent metal acetate in ace-tonitrilemedium. Fig. 2 shows the fluorescence intensity of Lin the presence of different metal acetates. Only Zn-salts resulted in a pronounced fluorescence enhance-ment, whereas other transition metal salts includingCu2+, Ni2+, Co2+, and Mn2+ did not change fluo-rescence (Fig. 3) even at concentrations 10-fold higherthan the corresponding Zn2+ ion concentration as thereis a probability of an electron and energy transfer be-

Biswas : A novel compartmental Schiff base ligand as a fluorescence chemosensor etc.

813

tween the metal ion and ligands22 or quenching offluorescence intensity of a ligand by transition metalions during complexation is a rather common phenom-enon which is explained by processes such as mag-netic perturbation, redox-activity, electronic energytransfer, etc.23. When the experiment was carried outwith ubiquitous intracellular metal ions such as K+,

Na+ and Ca2+, which exist at very high concentra-tions inside the cell, no significant fluorescence wasobserved, even at concentrations that were 100-foldhigher than Zn2+ ion concentration. However, treat-ment of various zinc(II) salts like chloride, nitrate,sulphate, bisulphate, phosphate with L exhibit similarkind of enhancement of fluorescence intensity. En-hancement of fluorescence through complexation is,

Fig. 1. Left : UV-Vis spectrum of L in MeCN; Right : Fluorescence spectrum of L in MeCN (ex = 363 nm).

Fig. 2. Fluorescence titration of L (1×10–6 M) in MeCN me-dium upon addition of different divalent late 3d acetatesolution of 1×10–6 M concentration (ex = 363 nm).Inset : Visual fluorescence changes of sensor L in thepresence of zinc acetate. The photo was taken under ahand held UV (365 nm) lamp.

Fig. 3. 3D bar diagram of fluorescence intensity of L (1×10–6

M) in MeCN with different 3d acetate solution.


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however, of much interest as it opens up the windowfor photochemical applications of the metal com-plexes24,25. Factors like a simple binding of ligand tothe d10 metal ions25, an increased rigidity in structureof the complexes26, a restriction in the photoinducedelectron transfer (PET)24,27, etc. are assigned to theincrease in the photoluminescence. For this case, theenhancement of fluorescence intensity may be accountedby considering the imposed rigidity through complex-ation and decrement of the non-radiative decay of theexcited-state with increasing radiative decay.

The photophysical responses of the Schiff baseligands are modulated when the fluorophore and theguest binding units are covalently integrated, resultingin a possible excited-state electron transfer pheno-menon between the two. This can be termed as a Zn2+

ion selective chelation enhanced fluorescence. The ex-periment shows that there is a significant increase influorescence intensity for L upon addition of zinc(II)acetate salt. The emission peak intensity of L at 424nm upon successive addition of zinc acetate concomi-tantly increases (Fig. 4) whether excited at 363 nm.Other salts of zinc(II) ion can enhance the fluorescenceintensity of the L but the extent of enhancement is

significantly small compared to the acetate salt. Atleast 2–3 fold more enhancement of fluorescence in-tensity is observed for the acetate salt compared toother zinc(II) salts. Thus, it is concluded that the Zn2+

ion selectively in a concentration range from 10–6 (M)to 10–4 (M) in acetonitrile medium using L as a fluo-rescence chemosensor.

Isolation of zinc-L complex in macroscopic scale

In order to evaluate the coordination nature of L toa Zn2+ ion, Zn2+-L complex is isolated and crystal-lized. Simple mixing of zinc acetate and L, instantlyproduced crystalline colourless compounds. But suc-cessful isolation of single crystals, external addition oflittle amount of DMF is very much essential. Differ-ent ratios of zinc acetate and L are used (10–2 M each)to isolate monomeric or dimeric or trimeric or poly-meric Zn-L complex, but obtained beautiful colourlesscrystals of rectangular shape when 3 : 2 Zn-acetateand L molar proportion is used. In this synthesis, ac-etate ions have contributed as bridging units to bindadjacent zinc ions together. The coordination geom-etry of trinuclear zinc(II) complex, [Zn3(L)2(OAc)2].

2DMF was determined by mainly single crystal X-raydiffraction study along with different spectroscopic andanalytical techniques.

Description of crystal structure

The X-ray structural determination of[Zn3(L)2(OAc)2].2DMF reveals a trinuclear neutralzinc(II) complex with the space group P1–. The com-pound has been found to have a trinuclear moleculewith a central ZnII ion lying on a center of inversion.An ORTEP view28 of -acetato--phenoxo-trizinc(II)complex of [Zn3(L)2(OAc)2].2DMF with an atom la-belling scheme is shown in Fig. 5. The crystallographicparameter for the compound is given in Table 1. Thetrinuclear complex is built up of two mononuclear Zn-Lmoieties linked through bridging acetate and 2-phenolato groups to the central Zn atom. The coordi-nation geometry around the terminal Zn centers (Zn1and Zn1i) may be regarded as square pyramidal geom-

Fig. 4. Fluorescence titration of L (1×10–6 M) in MeCN me-dium upon successive addition of Zn-acetate solution of1×10–6 M concentration (Excitation wavelength, 316nm or 363 nm).


815

etry where the equatorial plane of a terminal Zn1/Zn1i

atom is formed by phenoxo-bridged oxygen (O2/O2i),phenoxo oxygen (O1/O1i), imine nitrogen (N3/N3i),imine nitrogen (N4/N4i) and the axial positions areoccupied by acetato bridged oxygen (O3/O3i). How-ever, the coordination geometry of the central zinc ion(Zn2) may also be best described as distorted octahe-dral geometry, formed by six oxygen atoms from thesame Schiff base ligands and bridging acetate ions whichcoordinate the terminal zinc ions. Thus four phenoxo-oxygen (O1/O1i, O2/O2i) from two Schiff base ligandsand two acetate ions bridge the two terminal zinc ions(Zn1 and Zn1i) and the central zinc ion (Zn2). Thefour equatorial positions for terminal zinc ions (Zn1and Zn1i) are occupied by two phenoxo oxygen atoms(O2/O2i, O1/O1i) and two imine nitrogen atoms (N3/N3i, N4/N4i) where as the central zinc ion is sur-rounded by four phenoxo-bridged oxygen atom (O1,O1i, O2, O2i) from the two Schiff base ligands. Axialpositions for all three zinc ions are coordinated byacetate bridged oxygen atom (O3, O4, O4i, O3i). Thethree zinc ions are in a perfect linear arrangement(Zn1-Zn2-Zn1i = 180.0º). The distances betweenthe central zinc ion (Zn2) and the two terminal zincions (Zn1/Zn1i) are 3.061(1) Å.

Conclusion

Herein, I report a novel compartmental Schiff baseligand as a fluorescence chemosensor for the detectionof zinc(II) acetate salt. The fluorescent probe (L) un-dergoes enhancement of fluorescence emission inten-sity upon binding to zinc acetate or any zincII salts inMeCN while addition of other 3d-metal acetate saltsto the probe, exhibit usual fluorescence quenching phe-nomenon in MeCN solution. The fluorescent probe(L) can selectively detect ZnII acetate salt in a concen-tration range of 1.0×10–6 to 1.0×10–4 mol/L. Fur-ther, isolation of trinuclear zinc-Schiff base complex,[Zn3(L)2(OAc)2].2DMF in the form of single crystals

Fig. 5. Bond line view of the X-ray structure of the trinuclearzinc(II)-Schiff base complex.

Table 1. Crystallographic data of [Zn3L2(-O2CCH3)2]

Crystal parameters Zn-L complex

Empirical formula C46H50N6O12Zn3

Formula weight 1049.87

Temperature 293(2) K

Wavelength 0.71073 Å

Crystal system Triclinic

Space group P1–

Unit cell dimensions a = 9.5022(12) Å

= 66.820(18)º,

b = 10.5453(15) Å

= 78.47(3)º,

c = 13.0143(8) Å

= 89.67(3)º

Volume 1170.8(3) Å3

Z 1

Density (Calcd.) 1.433 Mg/m3

Absorption coefficient 1.586 mm–1

F(000) 520

Theta range for data collection 1.7 to 27.50º

Index ranges –12<=h<=12,

–13<=k<=13,

–16<=l<=16

Reflections collected 12711

Independent reflections 10199

R(int) 0.106

Goodness-of-fit on F2 0.99

Final R indices [I>2(I)] R1 = 0.1093,

wR2 = 0.3635

Largest diff. peak and hole 1.26 and –0.98 e. Å–3

JICS-20


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in MeCN medium with an external addition of DMFsupports and consolidates the selectivity and bindingability of L with Zn2+ ion in 2 : 3 ratio. Thus thiscompartmental Schiff base, L can effectively and se-lectively recognize Zn2+ ion in a mixture of otherbivalent 3d ions and behaves an efficient fluorescencechemosensor from micro to macro scale of concentra-tion.

Experimental

Materials and methods :

High purity salisaldehyde (E. Merck, India), 1,3-diaminopropan-2-ol (Lancaster, UK), methylene blue(Aldrich, UK), zinc(II) acetate dihydrate (E. Merck,India), copper(II) acetate dihydrate (E. Merck, India),nickel(II) acetate (E. Merck, India), cobalt(II) acetatedihydrate (E. Merck, India), manganese(II) acetate di-hydrate (E. Merck, India) and iron(II) acetate dihy-drate (Sigma-Aldrich, USA) were purchased from re-spective concerns and used as received. All the otherreagents and solvents are of analytical grade (A.R.grade) and were purchased from commercial sourcesand used as received.

Physical measurements :

Infrared spectrum (KBr) was recorded with a FTIR-8400S Shimadzu spectrophotometer in the range 400–3600 cm–1. 1H NMR spectrum in DMSO-d6 was ob-tained on a Bruker Avance 300 MHz spectrometer at25 ºC and was recorded at 299.948 MHz. Chemicalshifts are reported with reference to SiMe4. Groundstate absorption was measured with a JASCO V-730UV-Vis spectrophotometer. Fluorescence spectra wererecorded on a Hitachi F-7000 fluorescence spectro-photometer. Elemental analyses were performed on aPerkin-Elmer 2400 CHN microanalyser.

Preparation of L and zinc(II) complex :

Salisaldehyde (0.244 g, 2 mmol) was heated underreflux with 1,3-diaminopropan-2-ol (0.089 g, 1 mmol)in 30 ml dehydrated alcohol. After 10 h the reactionsolution was evaporated under reduced pressure to yielda gummy mass, which was dried under vacuum and

stored over CaCl2 for subsequent use. Yield : 0.278 g(82.8%). Anal. Calcd. for C17H18N2O3 (L) : C, 68.48;H, 6.08; N, 9.39. Found : C, 68.40; H, 6.02; N,9.35; 1H NMR (CDCl3) : 3.68 (2H, dd, J=12.4,6.8 Hz), 3.84 (2H, dd, J=12.4, 4.0 Hz), 4.23–4.25(1H, m), 6.88 (2H, t, J=7.2 Hz), 6.96 (2H, d, J=8.4Hz), 7.25 (2H, dd, J=7.6, 1.6 Hz), 7.32 (2H, td,J=8.8, 1.6 Hz), 8.36 (2H, s) ppm; 13C NMR :62.9, 70.2, 117.0, 118.5, 118.6, 131.5, 132.5, 161.1,167.3 ppm; IR (KBr, cm–1) : 1634, 1611 (C=N),3412 (OH); UV-Vis (max, nm) : ~316, 363, 424nm.

An acetonitrile solution of Zn(OAc)2.2H2O (0.657g, 3 mmol) (10 ml) was added dropwise to a solutionof L (0.596 g, 2 mmol) in the same solvent (15 ml).The yellow solution of the ligand immediately becamecolourless with quick precipitation of white crystallinecompound. It was filtered and recrystallized from ace-tonitrile-dimethyl formamide (DMF) solvent mixture.The solvent mixture produced colourless crystals.Yield : 0.453 g (69% based on metal salt). Anal. Calcd.for C38H38N4O10Zn3 (1) : C, 50.32; H, 4.22; N, 6.18.Found : C, 50.40; H, 4.27; N, 6.27%; IR (KBr,cm–1) : 3436 (OH), 1634, 1605 (C=N), 1497, 1460,1420 (OAc), 1276 (PhO); UV-Vis (max, nm) : 266,361.

X-Ray diffraction study :

Single crystal X-ray diffraction data were collectedusing a Rigaku XtaLABmini diffractometer equippedwith Merury CCD detector. The data were collectedwith graphite monochromated Mo-K radiation (=0.71073 Å) at 295(2) K using scans. The data werereduced using Crystal Clear suite and the space groupdetermination was done using Olex2. The structurewas resolved by direct method and refined by full-matrix least-squares procedures using the SHELXL-97 software package using Olex2 suite28,29.

Zn2+ sensing experiments :

The spectral analyses display an excellent chemosen-sory response both in absorption and fluorescence of


817

the Schiff base ligand upon addition of trace amountsof zinc acetate in acetonitrile solution. The selectivityof receptor for Zn2+ ion over other 3d cations wasstudied in detail.

Acknowledgement

Financial support from the Science and Engineer-ing Research Board (SB/FT/CS-088 dated 21/5/2014),New Delhi, India is gratefully acknowledged by BB.The author is greatly indebted to Dr. Tapas Majumdar,Department of Chemistry, University of Kalyani,Nadia, West Bengal, India for his kind co-operationthroughout the fluorescence measurements. Thanks toDr. Arabinda Mallick, Department of Chemistry,Kashipur Michael Madhusudan Mahavidyalaya,Purulia, West Bengal for valuable discussions. Fur-thermore, BB sincerely acknowledges Dr. AngshumanRoy Choudhury of IISER, Mohali for collecting X-ray diffraction data for the complex.

References

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July-O- pmd


A rhodamine-based fluorescent sensor for rapid detection of Hg2+ exhibitingaggregation induced enhancement of emission (AIEE) in aqueous surfactantmedium

Dipankar Dasa, Rahul Bhowmicka, Atul Katarkarb, Keya Chaudhurib and Mahammad Ali*a

aDepartment of Chemistry, Jadavpur University, Jadavpur, Kolkata-700 032, India

E-mail : [email protected] Fax : 91-33-24146223

bDepartment of Molecular & Human Genetics Division, CSIR-Indian Institute of Chemical Biology,4, Raja S. C. Mallick Road, Kolkata-700 032, India


Abstract : An easily synthesizable rhodamine-based chemosensor, L2, selectively recognizes Hg2+ and Al3+ ionsin the presence of all biologically relevant and toxic heavy metal ions. Very low detection limit (47 nM for Hg2+)along with cell permeability and negligible cytotoxicity provides a good opportunity towards cell imaging of Hg2+.SEM studies reveal rod-like microstructure for L2 in water, which changes to a porous microstructure in thepresence of Hg2+. It was interesting to note that the presence of SDS solubilized the otherwise insoluble probein pure aqueous medium. In case of Al3+ it was astonishingly observed a fluorescence quenching on increasingthe SDS concentration, while a ~33-fold enhancement of FI of [L2-Hg2+] complex was observed compared tothat in the absence of SDS, making the prove selective towards Hg2+ over Al3+ in the aqueous SDS medium.In SDS/water system, there is a steep rise in FI, reaches a maximum at ~7 mM of SDS and then fluorescenceintensity decreases gradually with the increase in [SDS] up to 28 mM. These observations clearly signify theSDS-assisted formation of polymer aggregates of the complex on the surface of monolayer of SDS formed inpre-micellar concentrations with higher FI, which is converted to the monomer being trapped inside the micel-lar cavity beyond the critical micellar concentration (cmc) with comparatively lower FI, indicating an interest-ing AIEE phenomenon. This proposition is further supported by the dependence of fluorescence anisotropy (r)on [SDS].

Keywords : Rhodamine-based turn-on Hg2+ sensor, aggregation induced enhancement of emission (AIEE),fluorescence anisotropy (r), microstructure formation, live cell imaging.

Introduction

The fabrication of appealing supramolecular assem-blies are achieved through bottom-up approach by anelegant use of noncovalent interactions like electro-static, hydrophobic, van der Waals, hydrogen bond-ing etc.1–4. Out of these, ionic self-assembly by vari-ous combinations between peptides, polyelectrolytes,surfactants and extended rigid organic scaffolds hasattracted considerable attention of the researchers dueto its application towards the fabrication of optical ma-terials and advanced nano-devices. Another interest-ing feature of these supramolecular assemblies is theirdifferent behaviour in solution and solid state exhibit-

ing either aggregation induced emission enhancement(AIEE) or aggregation caused quenching(ACQ)5–9. Till date, the AIEE mechanism has beenobserved in silole derivative10, 1,1,2,2-tetraphenyl-ethene (TPE)11–13, 1-cyano-trans-1,2-bis-(4-methyl-phenyl)ethylene (CN-MBE) etc.14,15.

The amphiphilic nature of surfactants readily pro-duces various supramolecular aggregates like micellesand vesicles in aqueous solution16,17. It could be usedas a coupling unit to induce structural changes by ionicself-assembly. Recently surfactants have been suitablyexploited to generate AIEE18–20.

Heavy metals like mercury, lead, cadmium and


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semimetal arsenic21 are widely distributed, extensivelyused, and highly toxic, and pose the greatest environ-mental threat; as soils and sediments are the ultimatesink for them. The water-soluble Hg2+ ion is highlytoxic and can damage the brain, nervous system, kid-neys, and endocrine system22,23. Again, due to veryhigh thiophilicity Hg2+ can deactivate many thiol-con-taining enzymes, thereby stopping or altering the meta-bolic processes24–26. Over the past decade, increasingattention has been paid to the development of efficientchromo- and fluorogenic sensors for Hg2+ ions forreal-time monitoring of environmental, biological andindustrial samples27–35.

Here, a rhodamine-based probe with potential NO3donor atoms have been synthesized and used success-fully for the selective and rapid recognition of toxicHg2+ ion (Scheme 1) exhibiting chromo- andfluorogenic metal-induced OFF-ON responses throughthe opening of the spirolactam ring.

In addition, although the current probe is poorlysoluble in 100% aqueous medium, the presence of SDSmakes it soluble in this medium, thereby making ituseful for monitoring Hg2+ ion in the purely aqueous

medium in the presence of SDS with enhanced sensi-tivity and selectivity, even in the presence of Al3+

which otherwise gives fluorescence response with theprobe in the absence of SDS19,20.

Experimental

Materials and reagents :

All solvents used for the synthetic purposes wereof reagent grade (Merck). For spectroscopic (UV/Visand fluorescence) studies double-distilled water andHPLC-grade MeCN were used. Rhodamine 6G hy-drochloride, ethylene diamine, methyl acrylate and per-chlorate salts of Na+, K+, Ca2+, Ni2+, Zn2+, Pb2+,Cd2+, Fe2+, Co2+, Hg2+ and Cu2+ were purchasedfrom Sigma-Aldrich and used as received. Sodium saltsof anions like SO4

2–, NO3–, PO4

3–, S2–, Cl–, F–, Br–,I–, OAc–, H2AsO4

– and N3– were of reagent grade

and used as received.

Steady-state fluorescence studies were carried outwith a PTI (QM-40) spectrofluorimeter. UV/Vis ab-sorption spectra were recorded with an Agilent 8453diode array spectrophotometer. Bruker spectrometersof 300 and 500 MHz were used for 1H and 13C NMR

Scheme 1. Synthetic steps leading the formation of [L2-Hg2+] complex.

Das et al. : A rhodamine-based fluorescent sensor for rapid detection of Hg2+ exhibiting aggregation etc.

821

studies. The ESI-MS+ spectra were recorded on aWaters XEVO G2QTof (Micro YA263) mass spec-trometer.

Synthesis of rhodamine 6G hydrazide (L1) :

It was synthesized by the literature method36.

Synthesis of rhodamine 6G probe (L2) :

To a suspension of 30 mL ice-cooled (0 ºC)methanolic solution of L1 (0.456 g, 1.0 mM), a 4 mLmethanol solution of methylacrylate (0.36 mL, 10mmol) was added dropwise over a period of 30 min.The reaction mixture was allowed to warm slowly toroom temperature at which it was stirred for 3 days.The final product was obtained after evaporation ofmethylacrylate and methanol under vacuum as whitecrystals (yield 0.55 g, 88%) with m.p. 134–136 ºC,Rf = 0.66 in a toluene/ethanol = 2 : 1 solvent system.

FT-IR (KBr) cm–1 : 3324 (-NH); 2942, 2896(-CH); 1723 (-C=O); 1670 (-COOMe); 1622, 1518and 1450 (-ArCH).

1H NMR (DMSO-d6, 500 MHz), ppm : 7.75–7.77 (1H, t, -Ar-H), 7.48–7.53 (2H, m, Ph-H), 6.98–7.00 (1H, t, -Ar-H), 6.25 (2H, s, Ph-H), 6.03 (2H, s,-Ar-H), 5.0 (2H, t, ArNHCH2), 3.33 (6H, s,-COOCH3), 3.09–3.17 (4H, m, ArNHCH2), 2.89–2.92(2H, t, -CH2NCOAr), 2.42–2.44 (4H, t, -CH2-CH2COOMe), 2.11–2.14 (4H, t, -CH2-COOMe),2.01–2.05 (2H, t, -CH2-N), 1.88 (6H, s, -ArCH3),1.18–1.20 (6H, t, -CH2CH3); ES+ MS = 629.3890[L2+H+].

Methods of characterization :

A scanning electron microscope (SEM) (ZEOL,JSM 8360) operating at an accelerating voltage of 5kV was used for the study of morphologies of the freeprobe (L2) in aqueous medium and also in the pre-sence of Hg2+ (L2-Hg2+). Before SEM, the sampleswere vacuum dried and then gold coated to minimizethe sample charging.

Fluorescence anisotropies (r), defined by eq. (1),were measured on a PTI QM-40 spectrofluorometer.

r = (IVV – G·IVH)/(IVV + 2G·IVH) (1)

where, IVV and IVH indicate the emission intensities

with the excitation polarizer oriented vertically andemission polarizer oriented vertically and horizontally,respectively, and corresponding G factor is calculatedas in eq. (2)19;

G = IHV/IHH (2)

where, IHV and IHH refer to the intensities correspond-ing to the vertical and horizontal positions of the emis-sion polarizer, with the excitation polarizer being hori-zontal.

Cell culture and cell cytotoxicity assay :

HepG2 (Human hepatocellular liver carcinoma) celllines (NCCS, Pune, India) grown in DMEM wassupplemented with 10% FBS and antibiotics (penicil-lin –100 g/ml; streptomycin – 50 g/ml). Conditionsfor the culture of cells are : 37 ºC in 95% air, 5%CO2 incubator. To test the cytotoxicity of L2, the 3-(4,5-di-methylthiazol-2-yl)-2,5-diphenyl-tetrazoliumbromide (MTT) assay was performed following theprocedure described previously36–39.

Cell imaging studies :

Cell imaging studies were performed by using theprotocol as described previously36–39.


A simple reaction between L1 and methyl acrylatein methanol leads to the formation of L2 in quantita-tive yield (Scheme 1) which was thoroughly characte-rized by 1H NMR (Fig. S1), 13C NMR (Fig. S2),ESI-MS+ (Fig. S3) and IR studies (Fig. S4a).

Steady-state absorption and emission studies :

The UV-Vis titrations reveals that on gradual addi-tion of Hg2+ to a 50 M solution of L2 there occurs agradual growing of two peaks at 350 nm and 528 nm(Fig. S5) clearly manifesting the chelation inducedopening of the spirolactam ring of the probe. The prob-able coordination mode of L2 towards Hg2+ is demo-nstrated in Scheme 1. When absorbances were plottedagainst [Hg2+] it gives a non-linear curve of decreas-ing slope (Fig. S5). Eq. (3)19 was employed to solvesuch dependence with a and b as the absorbance in theabsence and presence of excess metal ions, c (= Kf) isthe apparent formation constant and n is the stoichio-


822

metry of the reaction. The evaluated apparent associa-tion constant Kf is (3.08±0.53)×103 M–1 with n =1.0.

a+bxn

y = ————— (3)1+cxn

Job’s method also gives 1 : 1 (Fig. S6) complexationbetween L2 and Hg2+ which was further supported bymass spectrometric analysis (m/z = 440.2579)

[Hg(L2)(MeOH)(H2O)]2+ (see Fig. S3a in the Sup-porting Information).

The fluorescence titration was carried out by gradualaddition of Hg2+ (0–130 M) to a fixed concentrationof L2 (20 M) in MeCN/water (1 : 1, v/v, HEPESbuffer, pH 7.2) which yielded ~126-fold enhance-ment in fluorescence intensity at 558 nm on excitationat 502 nm (Fig. 1). The titration data were again solvedby employing eq. (3) under the condition 1>>c×xn

with n = 1 prevailing a linear form. A linear least-square fitting of data gives the apparent associationconstant Kf = (1.01±0.01)×104 M–1 (see Fig. 1 in-set). Al3+ also induces an opening of spirolactam ringof the probe leading to enhancement of fluorescenceintensity. So, analogously the fluorescence titration datafor L2-Al3+ complexation was solved and the appa-rent formation constant was calculated to be (1.45±0.02)×104 M–1( Fig. 2 inset)

Selectivity of the probe :

The probe was found to be sensitive towards Hg2+,but interfered by the presence of Al3+. However, inthe presence of SDS in aqueous medium the fluores-cence of L2-Al3+ was completely quenched, but notthe L2-Hg2+ complex, instead there was an increasein FI (vide infra). In case of L2-Al3+ complexationthe quenching of fluorescence intensity may arise dueto abstraction of Al3+ from the [L2-Al3+] complex bySDS arising out of strong hard-hard interaction be-tween sulfonic-O and Al3+ ion; which is absent forL2-Hg2+ complex. Again, the detection of Hg2+ wasnot perturbed by 5 equivalents of metal ions like Na+,K+, Ca2+, Mg2+, Fe3+, Co2+, Cu2+, Cr3+, Mn2+,Fe2+, Ni2+, Zn2+, Cd2+ and Pb2+ (Fig. 3) under theidentical reaction conditions. Also, the introduction of5 equivalents of anions like SO4

2–, NO3–,

PO43–, S2–, CN–, Cl–, F–, Br–, I–, OAc–, H2AsO4

–

and N3– into the solution of L2 (Fig. S7) did not show

any appreciable fluorescence change. However, I– hasa strong affinity towards Hg2+. As a result I– ab-stracts Hg2+ ion from the [L2-Hg2+] complex result-ing the disappearance of emission band at 558 nmthrough the re-establishment of the spirolactam ring(Fig. S8). The quantum yield () of the [L2-Hg2+]complex and ligand were determined to be 0.8609 and

Fig. 1. Fluorescence titration of L2 (20 M) in MeCN-H2O(1 : 1, v/v) in HEPES buffer at pH 7.2 by the gradualaddition of Hg2+ (0–130 M) with ex = 502 nm.Inset : linear curve-fit of FI vs [Hg2+] plot.

Fig. 2. Fluorescence titration of L2 (20 M) in MeCN-H2O(1 : 1, v/v) in HEPES buffer at pH 7.2 by the gradualaddition of Al3+ (0–130 M) with ex = 502 nm.Inset : linear curve-fit of FI vs [Al3+] plot.


823

0.011 respectively (rhodamine 6G as a standard). Thelimits of detection (LOD) of Hg2+ and Al3+ werefound to be as low as 47 and 73 nM, respectively(Fig. S9) as delineated by 3 method. The increasedquantum yield () and lifetime () of [L2-Hg2+] overthe free ligand L2 clearly indicate the enhanced stabi-lity of the formed complex in the excited state.

pH-stability of the probe was checked over a widerange of pH (2–12). There is no obvious fluorescenceemission of L2 in the range of pH 4–12, establishingthe fact that the spirolactam form of L2 is stable overthis wide pH range (Fig. S10). However, the presenceHg2+ ion induces the opening of spirolactam ring atpH 7.0 resulting a fluorescence enhancement andhence seems to be compatible for biological applica-tions under physiological conditions.

IR studies showed a characteristic amidic “C=O”stretching frequency of the rhodamine moiety at 1723cm–1 which is shifted to a lower wave number (1657cm–1) in the presence of 1.2 equivalent of Hg2+ (Fig.S4b). Thus a strong binding of L2 to the Hg2+ ion andthe cleavage of N-C bond in spirolactam ring is appa-rent. The 1H NMR spectra showed the ring proton“b” (Fig. 4 and Fig. S1) of the rhodamine moiety isshifted downfield in the presence of 1.2 equivalents ofHg2+ ions. The “f” proton of -NH+ group vanishes as

this group possesses a positive charge due to ring open-ing upon binding with Hg2+ ion. The “a” proton alsoshows a down-field shift. The down-field shift of “a”and “b” protons in the presence of Hg2+ arises mainlydue to decrease in electron density on opening of thespirolactam ring. The signal pattern of the other aro-matic protons in [L2-Hg2+] also indicates the involve-ment of the receptor unit of L2 in the binding to Hg2+.

Time resolved fluorescence studies :

The fluorescence decay behaviour of the L2 and[L2-Hg2+] were studied in aqueous medium (Fig. S11)both in the absence and presence of SDS. The bi-exponential decay of L2 resulted life times of 1.53 ns(1) and 6.11 ns (2). But in the presence of SDS itprevails a mono-exponential decay with = 3.63 ns.In the presence of Hg2+ the decay processes are mono-exponential both in the absence and presence of SDSwith respective values of 4.47 and 5.44 ns (Fig.S11). Bi-exponential decay of free ligand may arisedue to stacking interactions between the probemolecules. The enhanced life time of L2 and [L2-Hg2+]complex in the presence of SDS may arise due to en-hanced stability of the probe and its complex in the

Fig. 3. (a) Histogram of the fluorescence responses of differentmetal ions (100 mM) towards L2 (20 mM) in 1 : 1 v/vMeCN/water in HEPES buffer at pH 7.2 with ex =502 nm, em = 558 nm. (b) Fluorescence responses ofdifferent cations (100 mM) towards L2 (20 mM) in 1 : 1v/v MeCN/water in HEPES buffer at pH 7.2.

Fig. 4. 1H NMR spectra of (a) L2 and (b) L2 in presence of 1.2equivalent of Hg2+. Both spectra were recorded on aBruker 500 MHz spectrometer in DMSO-d6 (For atomnumbering, please see Fig. S1).

JICS-21


824

excited state in the presence of SDS. Thus this obser-vation clearly indicates the fact that SDS imposes morerestriction on the movement of the probe in micro-heterogeneous environments through the formationpolymeric aggregates.

Steady-state fluorescence studies in aqueous SDS :

In purely aqueous solution the L2-Hg2+ complex isweakly fluorescent, however, in the presence of SDSenhanced fluorescence was observed. Thus, steady statefluorescence studies were also carried out in the pres-ence of SDS in two separate experiments. In one case,the SDS concentration was kept fixed at 7 mM and[Hg2+] was varied in the range 0–40 M giving anon-linear curve of decreasing slope which was solvedby adopting eq. (3) (Fig. 5b) and evaluated apparentformation constant Kf = (1.00±0 0.02)×105 M–1 wasfound to be an order of magnitude higher than thatobtained in the absence of SDS. This enhanced stabi-lity constant value may be due to the restricted move-ment of the doubly positively charged L2-Hg2+ com-plex, which were held fixed in position by the strongelectrostatic interaction with the negatively chargedsulphonic acid head groups of SDS in the form oflayer structure. This causes the formation of aggre-gates of L2-Hg2+ complex through strong cooperative

interactions among the complexes held sidewise(Scheme 2).

In another experiment both [L2] and [Hg2+] waskept fixed at 20 and 150 M, respectively and [SDS]was varied between 0–28 mM. A plot of FI vs [SDS]showed a gradual increase in FI with the increase in[SDS], reaches a maximum at ~7 mM and then gradu-ally decreases with the increase in [SDS] (Fig. 6b).The fluorescence maximum at [SDS] ~7 mM clearlypoints out a critical micellar concentration (CMC) ofSDS as ~7 mM under the experimental conditions.The decrease in FI with [SDS] beyond 7 mM may beattributed to a change in polymeric aggregates of thecomplex to a monomer arising due to the formation ofspherical micelle on increasing the [SDS] (Scheme 2)in which the complex is trapped. The increase in FIwith [SDS] manifests the fact of aggregation inducedenhancement (AIE) of fluorescence. In the case of Al3+

ion, fluorescence quenching was observed on increas-ing the concentration of SDS (Fig. 6c) and may beexplained by considering the fact that sulphonic acidgroup abstract Al3+ ion from [L2-Al3+] complex bystrong electrostatic interaction. So in aqueous SDS theprobe becomes more selective towards Hg2+. The fluo-rescence quenching experiment by iodide ion in thepresence of [SDS] = 7 mM was carried out to verifysuch a proposition and was evaluated to be KSV =(7.23±4.3)×106 indicating an easy accessibility of the[L2-Hg2+] complex located on the laminar surface ofthe SDS to I– ion to form HgI2.

Fig. 5. (a) Florescence titration of L2 (20 M) by Hg2+ (0–40M) in presence of [SDS] = 7 mM with ex = 502nm. (b) Plot of fluorescence intensity as a function of[SDS].

Scheme 2. Schematic presentation of the formation of polymericaggregates and monomer in presences of SDS be-fore and after the cmc. Adopted from http://www.ecoboss.com.au/img/micelle.jpg.


825

Determination of steady-state fluorescence anisot-ropy :

Steady-state fluorescence anisotropy is usually takenas a measure of the extent of restriction imposed bythe micro heterogeneous environments on the dynamicproperties of the probe. An increase in rigidity of thefluorophore results in an increase in the fluorescenceanisotropy19. We have monitored the fluorescenceanisotropy as a function of SDS concentration at afixed concentration of L2 and Hg2+ (20 and 150 M,respectively) at 558 nm which showed a marked in-crease in anisotropy on increasing SDS concentrationup to 3.5 mM, then gradually decreases with SDSconcentration reaches a plateau at ~5 mM and main-tains steady value up to 12 mM. In the range 1–3.5mm concentration, the SDS arranges them in a layeredfashion. Now, the doubly charged [L2-Hg2+] com-plexes are held firmly by the strong electrostatic inter-actions between negatively charged sulfonic acid headgroups and doubly positively charged complexes; whichare again held together by strong interactionsthereby restricting their free movement. As a result,there occurs a sharp increase in anisotropy in the [SDS]~1–3.5 mM. Further increase in the SDS concentra-tion, a phase transition occurs through the formationof micelle. The slight drop in r values with [SDS]beyond 3.5 mM may be rationalized by consideringthe formation of a monomer of [L2-Hg2+] complex

which is again trapped inside the cavity of the micelle.The higher values of r in case of polymeric aggregatesarise due to cooperative interactions among the [L2-Hg2+] complexes which is absent in monomer trappedinside the cavity of the micelle. The variation of fluo-rescence anisotropy (r) as a function of SDS concen-tration is presented in Fig. 7.

Fig. 6. (a) Fluorescence titration of L2 (20 M) by [SDS] in presence of [Hg2+] (130 M) with ex = 502 nm; (b) plot offluorescence intensity as a function of [SDS]; (c) fluorescence titration of L2 (20 M) by [SDS] in presence of [Al3+] (130M) with ex = 502 nm; (d) plot of fluorescence intensity as a function of [SDS].

Fig. 7. Plot of fluorescence anisotropy (r) as a function of [SDS]in purely aqueous medium at 25 ºC and [L2] = [Hg2+]= 20 M, ex = 502 nm, em = 558 nm.

SEM study :

The SEM micrographs of L2 (0.50 mM) prevailsrod-like microstructures which interestingly changesto porous like architecture in presence of Hg2+ (0.50mM) (Fig. 8). In case of pure ligand in water the


826

structures are similar to the hexagonal prisms that arisesdue to the presence of two different polar ends (xan-thine moiety and carboxylic ester moiety) favoring thestacking of L2 one over another. However, in pres-ence of Hg2+ these stacking interactions are disruptedleading to the formation of porous microstructurescentering Hg2+ with the ligands at the periphery.

Cell imaging applications :

Hg2+ capturing capability of L2 was assessed byperforming the fluorescence imaging of L2 with Hg2+

into the live HepG2 cells (Fig. 9). The cytotoxicityeffects of L2 determined by MMT assay indicate nosignificant cell cytotoxicity for HepG2 cells up to 60M (<30% cytotoxicity) of L2 (Fig. S13). Interest-ingly, up to 10 M of L2 there was more than 90% ofcell viability and fluorescence imaging were carriedout at 1 M, 5 M and 10 m of L2. Significantly, anexcellent red intracellular cytoplasmic fluorescence wasobserved inside the live HepG2 cells pre-incubated with10 M of Hg2+ followed by washing with 1X PBSand subsequent incubation with 1 M, 5 M and 10M of L2. Interestingly, we observed that L2 has ex-cellent Hg2+ capturing capability even at low concen-tration likely at 1 M and 5 M at cytoplasmic levelof Hg2+ ions (Fig. 9). Moreover, the concentrationdependent binding of the L2 with Hg2+ ions was ob-served (Fig. 9). Parallel staining of cells were carriedout with DAPI and superimposed with the correspond-ingly treated cells with Hg2+ (10 M) followed by L2(10, 1, 5, 10 M) to show the cytoplasmic staining ofL2 with HepG2 cells.

Fig. 8. SEM images of microstructures conditions : (i) L2 0.5mM and (ii) L2+Hg2+ (0.5 mM each) in aqueous me-dium.

Fig. 9. Cell imaging study of Hg2+ ions with L2. The fluores-cence images of HepG2 cells were captured (40X and100X) after incubated with 10 M of L2 for 30 min at37 ºC, also in pre-incubated 10 M of Hg2+ for 3 h at37 ºC followed by washing twice with 1X PBS and,subsequent incubation with 1 M, 5 M and 10 M ofligand L2 for 30 min at 37 ºC. The imaging studiesshowed the strong red fluorescence when L2 binds withcytoplasmic Hg2+ ions. The merge images show thecytoplasmic Hg2+-L2 fluorescence.


827

Conclusions

In summary, we present herein a rhodamine-basedchemosensor with potential NO3 donor atoms for theselective and rapid recognition of toxic Hg2+ ions.The binding stoichiometry of the sensor with Hg2+

was established by the combined Job’s and HRMS (m/z) methods. All biologically relevant as well as toxicheavy metal ions did not interfere with the detection ofHg2+ ion. The detection limit of Hg2+ calculated by3 method gives a value of 1.52 nM. Its exhibits livecell imaging application of Hg2+ with no or negligiblecytotoxicity. SEM studies reveal a rod-like microstruc-ture for L2 which changes to a porous microstructurein presence of Hg2+ (0.50 mM). The presence of SDScauses enhanced quantum yield (), life time (), andstability constant (Kf) by an order of magnitude com-pared to those in the absence of SDS. Again, the FI of[L2-Hg2+] complex is enhanced by 33-fold in the pres-ence of 7 mM SDS to that in the absence of SDS. InSDS/water system, there is a steep rise in FI with theincrease in [SDS], reaches a maximum at ~7 mM andthen FI decreases gradually with the increase in [SDS]up to 28 mM, indicating the formation of polymericaggregates of [L2-Hg2+] complex on layers of the SDSat pre-miceller concentrations with higher FI values –a phenomenon reminiscent with the aggregation in-duced emission enhancement (AIEE). However, it turnsinto monomer and trapped inside the cavity of themicelle beyond CMC with comparatively lower FI.This proposition is further supported by the depen-dence of fluorescence anisotropy (r) with [SDS].

Acknowledgement

Financial supports from DST, New Delhi (Ref. SR/S1/IC-20/2012) and UGC CAS-II are gratefully ac-knowledged.

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intracellular detection of toxic hypochlorite anion by ...rhodamine b (1 g, 2.88 mmol) in dry...

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